Challenge of human synovialis with rheumatoidlymphocytes within diffusion chambers

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Authors McNutt, Neil Scott, 1940-

Publisher The University of Arizona.

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Neil. Scott McNutt

A Thesis Submitted to the Faculty of the

DEPARTMENT OF AGRICULTURAL' BIOCHEMISTRY

In P artia l Fulfillment of the Requirements

For the Degree of

MASTER OF .SCIENCE

In the Graduate College

■ THE UNIVERSITY OF ARIZONA

1962

STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of req u ire ­ments for an advanced degree at The University of Arizona and is deposited in The University Library to be made available to borrowers under ru les of the L ibrary.

Brief quotations from this thesis are allowable without special perm ission, provided that accurate acknowledgment of source is made. Requests for perm ission for extended quotation from or reproduction of this m anuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in their judgment the proposed use of the m aterial is in the in terests of scho lar­ship. In all other instances, however, perm ission must be obtained from the author.

SIGNED:

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

C.A.L. Stephens^/Tr., M.

ii

\ : ACE^OWLEDGMENTS ' :

Grateful thanks are due Associate P rofessor Alice B„ Stanfield

and Charles A. L. Stephens, F.A.C.P. for the ir untiring

efforts in thought, encouragement, and editing of this worki Without

their aid this study would never have been carried out.

", >' Acknowledgment is especially given to Dr. A. R. Kem m erer '

and the United States Public Health Service for financial backing and

direction for this .investigation* The author is also deeply indebted to

the Southwestern Clinic and Research Institute, Inc. for financial and

technical assistance. A mention? here has been well earned by M rs .

Julia P. Bailey for her help in preparation of slides and photographs.

Of course,,. without the enthusiastic cooperation of the volunteer

donors of cells and tissues, any study of this nature is impossible.

TABLE OF CONTENTS'

List of Tables'..........................

List of Figures.........................

'■' EiliAPTER' . ;;

x'T-F-; INTRODUCTION.....

n EXPERIMENTAL PROCEDURES

M : ■ RESULTS. ^ ............

1"F" DISCUSSION, o o ... o o . ...ooo.ooo. ... . .. . . o .

IZ" ■ iSii i ............... . 0 0 . . . . . . . . . . . . .

. ' LITERATURE C IT E D ...............

v y v ; - ;:v ' i i s T0 F.mBLES - ■ : v - ; ' ■■'■ •Table : : y . - y - ' / r :v' ■ ;:>: V,. ; ; Page;

■ :: Composition of Earle’s Balanced Salt. Solution....... ' '24 ’, ’"

, 2 o ' Sample iE%peirinxental iPIan® <> < > » e ® o # » o ® o » e . ®» e ® ® # o o » ? > » # <> <> o o « » ® » o 25

. 3.;: Composition of Blended Bat D iet.. , » . , . . . . . . - > . . . . . . . . 2 5

4 . „ Sources of Synovial T i s s u e S o " « V p o . i , . . . . . . o . . o . ? . . . O . o . . . < , = , , 0 . .30

5 „ Sources of Blood Lymphocytes, „ » . . . » . 3 1

6„ Effect of Recentrifucation Technic on Two BloodSamples , p o , p , , m , * , o P , , o o o o p m p , , p o @ p o o o o .o o p p p p o » o o o o p p p p o p p e 'o e e o o a e o 32

7p Lymphocyte Inocula per Cham ber. . . a . . a a a a » a . a . o , . a a « » : . . o . 33

8 a Destruction of Lymphocytes Shown by F ilter,S45C9,nrXn3tlOn, a a a a a o a o a p a a a a p a a o a a a a a a a a a a a a a a a a a a a a a a a p a a a o a a a o a a a a a , 3 4 .

' ::9. . 'Destruction of Tissue Fragm ents Shown by Paraffin • y •5eCtions a a a a a a a a a a a a a a. a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a ■ ■ ‘3 5

1 0 a ■ Effect of Double Implantation on Chamber N ecrosis., 3 6 ■

I ! . . Challenge N ecrosis - -Lymphocytes and Tissue y.; '0/ Ond )S r e la a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a 5 1

LIST OF FIGURES

' Figure v . ' ./. Page

'''lV : ■ Rlieumatoid Syiiovialis cultured alone. . . . . . . . . . . . . . . . . . .. ... . 62./

2 ; lElheumatoid synovialis cultured with nonrheumatpid' . ■ l ^ ^ m ^ ^ I l O C ^ ^ t O S ^ e o ''o O' e <) o o- o d e . o o- o e-'e o o .e o 0 o o -o e O'Oi o o o » - e e o e o o 0 - 0 o o e tt o o » o o o t> e o o o o o ' 6 6 .

■ ■ 3 o / ■'Rheumatoid synovialis cultured with .rheumatoid :. lymphocyte 8 0 * * -# # * * » » e * " * <>.0 • e - * < • • • «* o * ♦ ♦ o » 6 6

4 o Nohrheumatoid synovialis cultured a l o n e . . . . . . » 64

5. Nonrheumatoid synovialis cultured with nonrheuma-■ ■1 to id ' lymphocyte s .. o.... . . . . . . 0 o o o.. 0 , 0 o o... o... o. o. o. o o. * o. o o..... 6 5

6 0 Nonrheumatoid synovialis cultured with rheumatoid' lymphocytes, o o o o o o o o o o o o o o o o o o . o o o o o o . o o o . o o o o o o o o o o o o o o . 0 0 0 0 0 0 0 0 0 0 . 6 5

7. Rheumatoid lymphocytes cultured alone.. 0 . 0 .p....o....... 6 6

8 . Rheumatoid lymphocytes cultured near rheumatoids y n o v i a l i a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................ 67

9. Rheumatoid lymphocytes cultured near nonrheuma-- toid synovialis.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

10. . Nonrheumatoid lymphocytes cultured alone.. . . . . . . . . . . . . . 6 8

11. Nonrheumatoid lymphocytes cultured near rheum a-, 'toicl synovialis. . . . . . . . . . . . . . . . . o o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

12. Nonrheumatoid lymphocytes cultured near nonrheu­matoid. synovialis o . . . . . 6 9

VI

; v. ■chajpte'e r i

' INTRODUCTION ;- ; v. • - 1: : .

Rheumatoid a rth ritis is a "constitutional d isease which mani- ‘

fests itself as a profound system ic disturbance ahd by chronic, deform- •

ing, progressive polyarthritis. It has no known single cause and no

known single specific cure." It was so described by Holbrook and ; 1

mu (i). ■ V - - ' > v ^

The anatomical structure which shows very early manifestations

of the disease is the synovialis, the membrane lining the joint capsule (2).

The synovialis and capsule, being attached to the articu lar cartilage :

surfaces, form a seal for the joint cavity, which contains a viscous,

lubricating fluid—the synovial fluid. Normal synovialis is a m oderately -

cellular connective tissue which contains a capillary network near the : ■

joint cavity. The relatively smooth surface of the synovialis may be

thrown into folds either tem porarily due to the position of the joint or

permanently due to the form ation of villi which project into the joint .

cavity. The increase in size.' and number of norm al v illi is directly

proportionate to age. Normal villi consist of a collagenous fiber network

2

containing blood vessels and lym phatics, fibrocytes, macrophages,

fat cells, and a few leukocytes (3, 4). v ; , . ' •

, Rheumatoid a rth ritis may exhibit periods of g reat activity and

periods of natural rem ission (5, 10) - In the active disease, the syno-

vialis develops many folds and, enlarged villi, which protrude into the

joint cavity. The outer portion of these villi is formed by a dense p ro ­

liferation of surface synovialis cells. The central portion of a villus

is highly vascularized and consists mainly of large, medium, and sm all

lymphocytes, both diffusely distributed and present in follicles. Also,

plasm a cells are diffusely distributed throughout the cores of the

The main antibody forming organs, lymph nodes and spleen,

are composed mainly of lymphocytes arid, plasma .cells. The., p resen ce :

of these cells in the cores of the v illi suggests that an immunologic

reaction is occurring in the synovialis. The plasm a ce ll has been

associated with the formation and secretion of the serum gamma glob­

ulins, which eontairi the Serum antibodies (7, 8). The specific role of

the lymphocyte in immunologic reactions is as yet undefined (9). It is

known,, however, that in certain immunologic reactions, the lymphocyte

is necessary and plays a central role (33).

■ A histological picture sim ila r to that of the rheumatoid synovialis

is encountered in studies of tissue transplantation from one Vertebrate

animal to another. Of utmost importance are genetic differences not

the specific type of tissue transferred . If tissues are tran sferred from

one identical twin to the other, an isograft, the relationship between

the recipient and the graft is the same as that occurring in 'an individual

with tissues tran sferred from one place to another on his own body, an

autograft. If a graft is made between two members of the same species

who are not identical twins, the recipient will destroy an initial homo- ,

graft by the f irs t-s e t homograft reaction. Rejection will not occur if

the recipient has been made tolerant due to prenatal or neonatal contact

with nucleated cells from the same graft donor (11, 12) or has been

made unresponsive by various s tre s so rs (11). h

. The f irs t-s e t homograft reaction is an invasion of the graft by

hematogenous mononuclear cells. This occurs after vascular flow in

the graft has been re-established from the host granulation tissue in

the graft bed. The invasion form s perivascular cuffs of proliferatihg

mononuclear cells with associated destruction. When the graft is of

skin, destruction is usually described in term s of the readily observed

changes in the graft epithelium: vacuolation of epithelial cell cyto­

plasm containing invading m ononuclears,: sometimes associated with v

pycnotic epithelial nuclei. Often epiderm al regeneration processes

compete with the invasion-destruction process, resulting in excessive

epiderm al thickening. P lasm a cell nests occur in the inflammatory

infiltrate but make up no m ore than five percent of the invading cells (13).

Connective tissue damage is histologically identified with loss of ■

nuclear staining or pycnosis and fragmentation of nuclei-(M). However,

in skin graft rejection the collagen fib rils rem ain unaffected (13).

The involvement of hematogenous mononuclear c ells (mainly

lymphocytes), plasm a cell nests, and marked thickening of the epithe­

lium in the f irs t-s e t homdgraft reaction suggest the histological picture

seen in rheumatoid synovial tissues. Despite this s im ila rity ,. there are

a number of marked differences. In rheumatoid synoyialis, the follic- : ;

ular arrangem ents of the lymphocytes and other reticulo-endothelial

cells a re independent of adjoining blood vessels and therefore are not

perivascular cuffs of hematogenous mononuclear cells (15). Since this

fact was not derived from a study of the initial lesion of rheumatoid

a rth ritis , it does not exclude the possibility that this follicular arrange­

ment la ter replaced the perivascular cuffs of hematogenous mononuclear

cells. ■, \

Another difference should be noted concerning the plasm a cell

involvement, although the picture is less clear in rheumatoid. a rth ritis ; '

than in the homograft reactions. In the homograft reaction pMsma cells N.

appear to be stim ulated to develop by antigens released in the invasion-

destruction process. This conelusioh is based on the following observa­

tions: plasm a cells' are not with the invasive cells initially; piasm a cells

’ never com prise m o re than fiVe percent of the invading cells; plasm a : • .d }

cells may .be formed in 'areas not in immediate, contact-with th e .g ra ft (IS) '

The serum gamma globulins--hemagglutihins: (17) and leukoagglutinins (18) —

produced by these plasm a cells are immunologically specific for the

graft donOr (19). In the rheumatoid synovialis, however^ the plasm a

cell nests have been associated with a high m olecular weight gamma

globulin--the rheumatoid factor. Two facts suggest that the rheum a­

toid factor might represen t a reaction to abnormal gamma globulins.

F irs t, rheumatoid factor has a sedimentation coefficient of 22S. This

22S complex has been separated into two components—one 19S and one

S,. The 19S component is a. member of the macroglobulin class and

has the characteristic reactivity to gamma globulin shown by the other

mem bers of this class. The 7S component does not exhibit specificity

(20, 21, 22). Secondly, the rheumatoid factor reac ts with human and

, certain other species' gamma globulins which have been modified

either by being in an antibody-antigen complex, or by being adsorbed

onto a la rger particle such as latex (23). That the rheumatoid factor

does hot play the significant role in this d isease is suggested by: the

rheumatoid factor is p resent in only ten percent of the cases of child-

: - hood rheumatoid a rtb ritis (2%); the incidence of rheumatoid a rth ritis is

higher among patients with agammaglobuliriemia than among the general

population (25); patients with lupus erythem atosus, syphilis, and

sarcoidosis often give a positive rheumatoid factor te s t (24); tra n s ­

fusions of rheumatoid plasm a into nOnarthritic patients maintained a

high tite r of rheumatoid factor for s ix weeks without producing

manifestations of the disease ( 2 6 ) Here, the sim ilarity to homograft

hypersensitivity re tu rns. Serum antibodies are not the important fac­

to rs in homograft rejection because they are not able by them selves to

destroy tissue homografts (31, 32, 33) but, can destroy these same

cells in suspension (34), In homograft rejection, as contrasted to

rheumatoid a rth ritis , a difference is suggested by homograft tolerance

in cases of agammaglubulinemia (22) since in this condition there is a

higher than norm al incidence of rheumatoid a rth ritis (8). Medawar has

suggested that the few reported cases of homograft tolerance in

agammaglobulinemia represen t two manifestations of an underlying

phenomenon (28).

A number of studies have indicated that the lymphocyte is the

cell responsible for the rejection of the homograft. Passive transfer

of hypersensitivity to homografts by means of lymphocytes has suc­

ceeded but by means of serum has failed (29, 30). The work of Algire,

Weaver, and Prehn has firm ly established that lymphocytes, or less

specifically lymphoid cells,, are the active agents of the destruction of

tissue homografts (31, 32, 33), .

The initial lesion of the synbvialis in rheumatoid a rth ritis may

involve the same meehahismms homograft rejection. An essential link

in drawing such a comparison would be the demonstration that the

lymphocyte is the agent of chronic destruction of the rheumatoid syno-

vialis, ... ■ . ■ , ^ ; " ' ' . :

1 Si nce rheumatoid a rth ritis is a Human disease which has yet to ■

. ;;-be reproduced , in anim als, the available technics' for such a dem onstra­

tion of destructive agents are lim ited. Tissue culture of human cells

■ represents one of the most widely used technics by which such destrue- ,

tive agents might be demonstrated.

Six frequently quoted investigators have attempted to demon­

stra te in tissue culture that the lymphocyte is the agent of destruction ,

in the homograft reaction, an otherwise w ell-established principle.

Trowell used organ culture of lymph node fragments in contact with

fragm ents of u re te r. The target u re te r epithelium m igrated over the

sensitized lymph node but resulted only in tissue fusion, with no lympho­

cytic infiltration or specific destruction (35). The two main questions

about Trow ell's experiments concern the degree of cellu lar contact and

the use of the completely synthetic T8 medium. Scothorne and Nagy

also used T8 medium for the organ culture of lymph node from a skin

graft donor in contact with regional lymph node from the homologous

Skin g raft recipient. They failed to observe any cell destruction in

sensitized system s that did not occur in controls (36), Again, cellular

contact and the use of protein-free T8 medium are factors to be con-

sidered, Medawar cultured fragm ents of sensitized lymph node and

spleen with skin from the sensitizing graft donor and found the lymphoid

cells did no damage to the mitotic or niigratory activity of the cultured;

epiderm is (37). The cellular contact ‘may have been adequate since

some lymph nodes were completely perm eated by epiderm al cells.

However, m ost of his experiments used spleen which plays a minor

role in orthotopic skin graft hypersensitivity (36). When using lymph

node only one experiment survived beyond four days. Weaver, A lgire,

and Frehn also combined spleen and epiderm al cells in tissue culture

and sim ilarly found no clear destruction of the epiderm al cells (33). In

contrast to these four failures, two of the six investigators reported

cytolytic action by lymphocytes. Govaerts reported that living lympho­

cytes from graft recipients produced specific cytotoxic lesions in renal

cell culture from corresponding donors. He found that complement

increased the cytolytic properties of the lymphocytes (38) . RoSenau

and Moon added sensitized homologous lymphocytes to tissue cultures

of mouse fibrocytes and reported clustering of lymphocytes about the

fibrocytes, which exhibited marked cytopathogenic. changes. They

observed that addition of complement and protein enrichm ent to their

completely synthetic, medium were without effect on this phenomenon (39).

W eaver, A lgire, and Frehn had reported failure in the use Of

in yitro technic in demonstrating the cytolytic properties of sensitized

lymphocytes in contact with their target cells. They did repdrt success

when this, system was enclosed'within- a diffusion..chamber implanted in

an animal (33). Their success was repeated by Amos , (61). ■ . ■

A diffusion chamber cdnsists; essentially of a plastic tube with

M illipore filte rs sealed on both ends. The pore size of; the filte rs may

be regulated within very fine lim its. Usually, the pore size is sm all

enough to prevent cellular exchange between the chamber contents and

the host animal, but is large enough to allow the perfusion of host

in tercellu lar fluid through the chamber. A construction so simple has

many variations to fit the needs of the u se r. Algire, Weaver, and Pretm

have laid the foundation for the use of the diffusion chamber both in

general and specifically in the study of the homograft reaction (31, 32,

33, 40, 41, 42, 43, 44). Previously, the same principles had been

applied in the use Of collodion bags (45, 46) . Since its introduction, the

diffusion chamber has been used to delineate the effects of cellular

contact in contrast to humoral factors (33, 47, 52). It has been used

to study the effects of the inflammatory environment on the morpholog­

ical and behavioral characteristics of lymphoid cells (48, 56, 57, 58)

and the effect of nutritional factors on any such morphological change

(49, 50). The morphological differentiation of human mononuclear

leukocytes into fibroeytes in diffusion chambers has been studied in

individuals with rheumatoid a rth ritis (50, 51) and without this disease

(48, 50). No qualitative growth or. differentiation differences were

found between norm al volunteers and persons with rheumatoid a r th r it is .

It was found that cortisone prevented the differentiation into fibro-

cytes (50). Cells and tissues in diffusion cham bers have been shown

by the atioye reports to maiirtain fo^ ability. In addition, the '

viability is assured both by studies made on endocrine transplants in

'diifusion: chambers as therapy for endocrinological d isorders (53) and

' particularly by immunologicaliy com petent'cells within diffusion, t

chambers participating in the immunologic reactions of the h o stan i-

■ m ai:(54','55). : ■ v/ ; V V';

It is the purpose of the experim ental portion of this study to ;

compare the homograft reaction with the joint inflammation in rheum a­

toid a rth ritis . The approach taken involyes the technic of challenge

studies within diffusion cham bers implanted subcutaneously in ra ts .

Using this technic in the study of homograft reaction, Weaver, A lgire,

and Prehn found that combination or challenge of sensitized lympho­

cytes with their specific target, of sensitizing cells resulted in faster

and g rea ter destruction of the contents of the chamber than combina­

tions of these same target cells with nonsensitized lymphocytes. This

pattern was not affected by the species of the host animal (33).

. The heterograft nature of the chamber contents in relation to

the host did not affect the growth characteristics for a period of fifteen

days (44) or the immunologic reactiv ity of the chamber contents for at

least six days (33) i Therefore, this technic represented a method for

the detection of sensitized lymphocytes apd their particu lar target cells .

This investigation recognized the above method as applicable to the ,

: study ofthe human .disease^ rheumatoid' arthritis. Thus, .rheumatoid:;: : -: pi

: : , : ^ ^ \ f ; : : \ ; : ; . ^y ::; :,;Y ' - / ; : , , ; : - : ' ' : ; % : . .11;

and; nonrheumatoid synovial tissues were combined in diffusion .ctiaih-

bers with blood lymphocytes from patients with ahd without the d isease»:

Histological c r ite ria were employed during micrbscopic examination of

the chamber contents to determine the extent of cellular interaction.

CHAPTER ;i l '

' EXPERIMENTAL PROCEDURE :

On the basis of resu lts obtained from six pilot experim ents, the

following procedure of choice was adopted„ ■ The diffusion chamber

technic employed was eclectically chosen from that of A lgire et aL (44)

Weaver et aL (33) , and Najarian and Feldman (54) „ After the title of

each subsection, an estim ate is included of the time required for one

person to process th irty cham bers. Manipulation tim e is extremely

c ritical, since the degree of necrosis of the cells not only increases

with tim e but also is the crite rion of damage due to cellu lar interaction

• P reassem bly of diffusion \chamber components (5-6 hours)

Each diffusion chamber consisted of one Plexiglas ring mounted

between two M illipore f ilte rs with MF cement, formulation No. 1 (Milli

pore F ilte r Corporation, Bedford, M assachusetts),,}

Plexiglas tubing was machined to ,produce rings: one-half inch

outside diam eter, three-eighths inch inside diam eter, one-eighth- inch

in height. In the side of the rings, a ope -sixteenth inch access opening

was drilled.

. . 'Y:: :

Type HA Millipord filte rs (diameter 13 pore size 0.45

m icrons) were used. The ME cement was best applied with a glass tube

drawn put to a fine point in a flame and fire polished to produce a sm all

: behd.'"' ' ' ' . , ' . •

Millipore filte rs were placed severa l inches apart on a sheet of

alumihum foil. A-ring was held with forceps while ME cement was

- applied tp thetop-rim . '■ lt':was inverted on a filter and held firm ly until.

:' The,procedure was repeated;in attaching the ■' .

second filte r to the opposite rim of the ring. Each diffusion chamber .

was. labelled opposite the access opening in the Plexiglas ring by sea l-

■ ing pn a typewritten number with clear cement.

Sterilization of the prefabricated diffusion chambers was

accomplished by filling and c over ing them for one -half hour with 1:750

aqueous Zephiran chloride (Winthrop L aboratories, New York). Fol-

lowing chamber sterilization, all manipulations were ca rried out under

standard s te rile conditions. The chambers were removed from the

Zephiran chloride, rinsed three tim es with E arle ’s balanced salt solu­

tion (BSS, Table 1), and stored until final assemblage in 100 ml. of

BSS plus 10, 000 units of penicillin-streptom ycin m ixture (Microbio- :

logical. A ssociates, Bethesda, Maryland).

: The closure for the side opening in the cham ber was a sm all

Plexiglas cone ground from rod using an ordinary pencil sharpener.

The point was then firm ly inserted in the opening and cut to allow

approximately one -sixteenth of an inch for handling purposes. These

plastic stoppers were stored in 70% ethanol until needed.

•: ' • P reparation of human tissue . - dand lymphocyte specimens (5-9 hours)

: ,' Synoviaitissue specimen&'were obtained at the tim e.of open .-.d:

'su rg e ry ,■ transported within'One. hour to the labora to ry ln :BSSv/ith ; ;’V;

added penicillin-streptom ycin mixture (100 u n its /m l.), and incubated

at 37° C. O rdinarily, 6-9 hours elapsed before use in final chamber .

assembly, : ' ' '."i : : :

* Blood samples were obtained by venipuncture and the lympho­

cytes were separated by a modified Jago (60) technic. Twenty ml. were

divided between two ste rile 15 ml. conical centrifuge tubes, each con­

taining 0.05 ml. of isotonic heparin solution, 1,000 units per ml.

(Liquaemin sodium, Organon Inc., West Orange, New Jersey). The

sam ples w ere mixed very carefully to avoid the presence of bubbles in

the centrifuge tubes. If bubbles were form ed, they were aseptically

drawn off with sm all bore straight Pasteur pipets. The tubes containing

blood from patients with active rheumatoid a rth ritis were centrifuged

at 1,100 r.p .m . (266 tim es gravity, xG) for five minutes. The blood

tubes from norm al volunteers were centrifuged at 1,500 r.p .m . (499 x<3)

for five minutes. The decrease in relative centrifugal force used when

centrifuging rheumatoid Mood sam ples compensated in p art for the

increased sedimentation ra tes characteristic of active rheumatoid

a rth ritis . With s te rile straight Pasteur.p ipets, the supernatant plasm a

and "buffy. coat” were removed and placed.in a clean, s te rile 15 mb

centrifuge tube. The cell suspension was well mixed, avoiding' bubbles,

and recentrifuged at 900 r.p .m . (179 xG) for ten minutes. The super­

natant plasm a from this second centrifugation was removed down to

the top of the sedimented red blood cells, which inevitably contamin­

ated the "buffy coat” in the f irs t separation. This recentrifugation

increased the purity of the lymphocyte suspension. Total white blood

cell and differential counts were made on the suspensions by standard

hemocytometer technic. In order to equalize the lymphocyte concen­

trations in the normal and rheumatoid suspensions, often it was neces­

sary: to Centrifuge again at 900 r.p .m . for ten minutes, to remove

carefully and aseptically a certain amount of Clear plasm a from the top

of the supernatant, and then to resuspend the sedimented lymphocytes

by gentle aspiration in the remaining plasm a volume, Immediate use

followed equalization of the lymphocyte concentrations in the normal

and rheumatoid suspensions. Approximately 2-4 hours were required

for venipunctures, centrifugations, and cell Counts,

: : - : " v f ' ' ; \ y: ' ' ; ' ' ' ' : ' ; ' : . r ■

:: ". ■ Final assembly and sealing v .,, , , -/ v. ; of diffusion cham bers . . :■■ -V’ (2-3 hours)

Small s trip s of synovialis, approximately orie-sixteenth of an

inch wide and one-eighth of an inch long, ' were dissected on a glass -

plate with fractured razo r blades. A typical experim ental plan

required nine such strip s of each tissue (Table 2). The se ries listed

in the squares of Table 2 were the identifying numbers on the randomly

selected diffusion cham bers. Nine deep P e tri dishes were labelled for

each treatm ent and three randomly selected chambers were placed in

each dish. The diffusion chambers were balanced on the ir sides,

access openings up. Using a sm all pointed m etal probe, the appropriate

tissue slices were carefully introduced through the side opening into

the cham bers. After gentle aspiration, 0.2 m l. of the appropriate

lymphocyte suspension was dispensed from a tuberculin syringe to fill

the designated.cham bers. The lymphocyte suspensions in autologous

plasm a readily displaced any BSS inside the chamber. In control

cham bers, 0.2 ml. of BSS was substituted.

The opening in the Plexiglas ring was dried with a heated m etal

probe and a drop of MF cement was applied. Immediately one of the

dry conical Plexiglas Stoppers was inserted firm ly into the cement^ '

filled aperture. The P e tr i dish lid was left a jar for one-half hour to

allow the cement to thoroughly dry. To insure a good seal, additional

MF cement was applied and again allowed- to d ry . P he completed - , :

chambers were stored together in 100 ml. of BSS at 37° C. until time

of implantation in the ra ts .

Subcutaneous implantationof cham bers in ra ts (2-3 hours)

Male Holtzman rats (Holtzmari Co., Houston, Texas) weighing

125 to 375 gms. were used for implantation; all ra ts in a single exper-■ . ■ ' . , ■ i. . ' ■ ■ ‘ 1 ' -*, '... ■ , -

imental run were the sam e weight and age. The ra ts were anesthetized

with ether. An incision approximately one inch in length was made

through the skin in the scapular area. On each side of the incision, a

subcutaneous tunnel was opened by spreading the connective tissue with

as little traum a to the blood vessels as possible. One randomly selected

chamber was inserted in each tunnel to a. depth of approximately one

inch. The incision was securely sutured with cotton thread. Penicillin-

streptom ycin m ixture, 1,000 units, was injected into the operation

a rea after closure of the incision. The: ra ts were placed in separate •

. cages and ra t diet (Table 3) .and water were available adZlibitum.''

. ■ ■■■ Removal of chambers /1 ; from the ra ts - (2-3 hours) >

Six days following implantation, the ra ts were, anesthetized with :

ether. The positions of the chambers were located by ca re M manipu-

Tation o f the skin of the operation area . The stitches w ere cut: and;the ry .

orig inal incision reopened. The host ra ts reacted to the presence; of /.V ;

the diffusion chambers by the foreign-body reaction, which produced

fibrous connective tissue capsules around the cham bers. By carefully

spreading-away the connective tissue organization with the points of

sc is so rs , the edges of the diffusion cham bers were freed of the cap­

sules. Exercising care to avoid puncture of the M illipore filte rs , the

chambers were removed with large blunt forceps and stored immedi­

ately in 10% form alin at 37p G. for approximately eight hours.

Processing and opening ' of sealed cham bers . (5-8 hours)

: The host ra t connective tissue and blood cells were removed

from the outside of each chamber by abrasion with Kimwipes (Kimberly-

Clark Corp., Neenah, Wisconsin). The wipes were used alternately -

dry and wet with 10% •formalin. The- chambers' were opened with a

.scalpel by cutting through the filte rs a t the-inner edge of the. Plexiglas

rings. : : : . . v: /:'' : i ' - ' ' v'-V' '-

Lbosely attached protein m aterial and cells were rinsed away

very gently with., a few drops of.10%.formalin,.dispensed from a wash. ;

" b o ttle .. V/ith .fine-pointed forceps, the 'tissue'fragm ents were-carefully

removed from the M illipore filte rs and stored in labelled test tubes of

• 10% form alin until.tim e of sectioning. :

. • . The M illipore filters, were strung on; a cotton sewing, thread; with

a fine sewing needle, in se rted first"through;the abraded surface. ...An, V

V ■ ■ ■■ ^ . ; ,'' '

accurate ch a rt was kept of the order of the filte rs on the string. .They-;

were spaced approximately one-fourth inch apart by glass beads. The

string of filters, was stored in 10% form alin until tim e of staining.

. . V Paraffin sectioning of cultured 'fragm ents of synovialis ' (16-24 hours)

The fragm ehts of synovialis were each tied in a separate gauze

bag, strung on a length of sewing thread, and identified by paraffined

paper labels between each bag. The fragm ents were transferred from

■one bath of dehydrating1 agent to another aCGbrding to- the folio wing ' ' -

schedule: 80% ethanol (one-half hour) 95% ethanol (one-half hour), a

Second 95% ethanol (one-half hour), n-butanol (one hour), a second

n-butanol (one hour), a large bath of paraffin at the melting point (one .

hour), and another. sim ilar paraffin bath (one -half hour). The use of

n-butanol in place of absolute ethanol: was adopted to give softer m ore

pliable fragm ents, easie r to section. One labelled bag at a time was

removed from the string and cut with warm Scissors. With warmed

forceps, the gauze was opened and the tissue fragm ent was gently

picked up and embedded inside a previously prepared mold of warm

paraffin. The label was inserted in the edge of the mold and the paraffin

was allowed to harden. , :

The paraffin blocks were placed in an ice bath. The ice-cold

blocks were sectioned on a microtome to produce slices 4 microns ;V

thick. These slices were floated on the surface of a warm water bath

and straightened by pulling on the paraffin adjacent to the fragment.

The sections were transferred to clean m icroscope slides and allowed

to dry. : . ' •. ' - - . - . ■ ' ; " : '

Staining of paraffin sections ; ' and M illipore filte rs " ' (2 hours) ''

Hematoxylin and eosin stain was used throughout. Paraffin

sections for one experimental run were stained simultaneously. The

same was true for the M illipore filte rs . . '

To remove the paraffin from the sections, the slides were

placed in a rack and dipped through two baths of xylene for several

minutes each. They were then hydrated by dipping for several minutes

in n-butanol, two baths of 95% ethanol, and finally in w ater. The se c ­

tions were im m ersed for three minutes in H arris Alum Hematoxylin,

transferred to tap water (one minute), to tap water made basic with a

few drops of ammonia (one minute), and then to 0.5% eosin (three m in­

utes). They were dehydrated by dipping quickly through two baths of

95% ethanol, and then into two baths of n-butanol and two baths of xylene

for three minutes each. The slides were removed one by one from the

final xylene bath, a drop of Permount mounting medium (Fisher Scien­

tific Co., Fair Lawn, New Jersey) placed on each, - and a clean cover-

.slip positioned over the sections. Once the Perm ount had dried several

hours, the slides were ready for microscopic examination.

The procedure for staining the M illipore filte rs was more

sim ple„ The string of filte rs needed only to be rinsed in two baths of

water to remove most of the form alin before transfer into hematoxylin.

The time in hematoxylin was decreased to one minute. The time in

eosin was decreased to two seconds. The filte rs were dehydrated and

mounted on slides with Permount. In the xylene, the M illipore filte rs

became .transparent and remained' so when permanently mounted. ,

M icroscopic examination was possible after several hours drying tim e.

" • ■ . . . Examination of slides (24-36.hours)

All examinations were made without knowledge of the identity

of the slides. Descriptions were w ritten for each filte r and for each

slice of the sectioned fragm ents. The degree of necrosis was estim ated

on the basis of a plus system . Undamaged, tissue sections and filte rs

were represented by a rating of less than one plus. Almost totally

necrotic f ilte rs or tissues were designated four plus. The c r ite ria for

necrosis in filte r examinations were two in number: (1) the absence

of added lymphocytes, and (2) the presence of debris from nuclear

fragmentation. The c r ite r ia for necrosis in paraffin section examina­

tions were three: (1) the presence of eosinophilic nuclei, (2) the p res­

ence of hypochromic.nuclei, and (3) a qualitative reduction in number

of normally stained tissue cell nuclei Over comparable areas in the

controls. Any disruption of the fib rilla r s tructu re of the presence of

holes around lymphocytes were considered as possible artefacts. Upon

completion, all descriptions were sorted and compiled with reference

to the original experimental plan chart.

D epartures from the — --------OToeedtire of choice

There were six pilot studies, RI (Rat Implantation) 1 - 6, and

four experimental runs, R1 7-10.

Variations were presen t in the chamber sterilization procedure

with Zephiran chloride. In RI 7, three washes of BSS plus penicillin-

■ streptom ycin m ixture (100 units/m l.) were used. In RI 8 and 9, each ; '

chamber was washed with one ml. of anhydrous ether, dispensed from

a syringe into the side opening. Before the filte rs dried, the chambers

were dropped into ste rile BSS to prevent filte r contraction and the

resultant splitting due to excess drying.

It Was also necessary to modify the cementing procedure. ;

Instead of MF cement, a thick preparation of 4% Plexiglas in dichloro-

methane/'Was utilized. in Rl 7 and RI 8. This cement was la ter aban­

doned since it was necessary to use large amounts to eliminate host

invasion of the implanted cham bers. In RI 8, an extra modification

Occurred when the side opening Was not cemented before receiving, the

Plexiglas stopper.

AvailaMlity of synovial tissues im posed.aii unavoidable varia ­

tion- In RX 7, synovialis from a nonrheumatoid patient was not obtained-

In RI 9, 10, and 11, rheumatoid synovialis was not available -

Finally, in RI 7, ten hours instead of 2-3 hours elapsed before

the rheumatoid lymphocytes were used to challenge the tis su e .

Table 1. Composition of, E a rle ’s Balanced Salt Solution (59)7

Component , . 7 Amount per lite r

' \ NaCl : ■. ' ' : v,';.; ; 6.80 gms, '

. ; 7KCL ■ 1 , - ■ , : ./ ' :::0. 40 ■:n: : 7 '■

MgSG4 .. 7 HgO '-',.7 " / ' 0.20 ' " - '

■ . ' NaHgPCy . HgQ : -; . 7 . . Cl. 1/1 : 7 '

, • -■ ; N a H C o 3 . ■ 7 ; ; - : 2 , 2 0 \ ^ 7

, Glucose : //. : . 7 : . 1 .00\ ■l' ^

, (triple distilled) to volume: • z ■ ; ■ - ; ■ ■■ ■. • ? : . ' .7.7' " . -7- : :

7, phenol red (0,5%) ; • 7 ' \ . - 0.15 mL . .. - -

Table-2o' Sample Experimental Plan.

Tissue-v -

1 ;; . - ; ■ Lymphoeytes’ Rheumatoid

: ; 1 a rth ritis.' Normal '

' ’ controls ' Blank . ;. •

Rheumatoid a rth ritis . 10, 2 3 ,2 5 . ; ; - 7, 15, .26 - 1, 4, 11

O ther' d isorders : . ; 13r 18, -27 6, 17, 20':. ■•5,;9, 19

Blank;' ' ■ : 2,; 12, 24- 16, 21, 22 ; '3, 8, 14

Note: Numbers are labels on cham bers.

Table- 3v Composition; of .Blended S a t Diet.- ^

Compbhent . '■ : : :f ; ■

' ■ ’■ t - v ■'. . ’ i

: . . ■ % by weight- - - ■

Ground whole wheat . ,. - 58.0 „ : ;Dried skini milk . .. . ' 29.0Meat scraps 7.0Wesson oil • 4 . 4 i - . ' ;V i-

SaltCod liver oil ’■ , . 0.5.; - ’ '

CHAPTER M

■ : : . • RESULTS': • . ■ ; ' y / = ':

Specimens of synovialis were obtained from two patients with

rheumatoid a rth ritis and four patients with other orthopedic d isorders

(Table 4)/. : \

The rheumatoid blood sam ples were from volunteers with an

established diagnosis of active rheumatoid a rth ritis , either chronic or

acute, Nonrheumatoid lymphocytes w ere separated from the blood of

individuals with no known joint d isorder or autoimmune disease (Table

The recentrifugation technic for lymphocyte separation increased

the purity of the lymphocyte suspensions. The effect of this procedure

on two blood sam ples is shown by the data in Table 0. Since the lympho­

cytes .for different experim ental runs were from different indiyiduals,

it was necessary to equalize the concentrations at different levels.

Therefore, the inocula varied over a wide range (Table 7). Sim ilarly,

the polymorphonuclear leukocyte contamination varied widely.

In challenge chambers containing rheumatoid lymphocytes,

destruction of lymphocytes averaged twice that seen in contro ls-- •

rheumatoid lymphocytes alone or the sam e tissues challenged with non-

rheumatoid lymphocytes (Table 8) „

The tissues within chambers containing rheumatoid lymphocytes

exhibited an average of three to four tim es more necrosis than tissue

challenged with equal numbers of nonrheumatoid lymphocytes (Table '9)Vv';:;

-An average of RI 10 mid 11 show: control# tissues 0.3, tissues chal-

lenged with nohrheumatoid lymphocytes 1.0, tissues contacted by rheu­

matoid lymphocytes 1.7. This indicates thaty for RI 10 and 11, nonrheu-

matoid lymphocytes caused alm ost as much damage Us rheumatoid lymph

ocytes. However, in RE 7, 8, and 9, this was not true since challenge

with nonrheumatoid lymphocytes produced the same average tissue

necrosis as culture of synovialis alone.

The difference between necrosis in challenges and controls was

not g reat either for the lymphocytes in E l 10 or for the tissues in RI 10

’ and 11., The ' relatively-low lymphocyte: inpcula used in .these two ra t ■ \

implantations might partially explain the ir difference from; E l 7, 8, and

9 (Table 7). Polymorphonuclear leukocyte contamination of the inocula

imposed no conclusive stim ulatofy or inhibitory effect oh observed

necrosis. .

Photographs included.demonstrate the types of changes observed.

Figures 1, 2, and 3 display the changes observed when rheumatoid

synovialis (Figure 1) was challenged with nonrheumatoid lymphocytes .

' : ■ ' ■ 28

(Figure 2) and with rheumatoid lymphocytes (Figure 3} „ Note the

decreased number of undamaged nuclei in Figure. 3. Sim ilarly, this

was observed for challenge of nonrheumatoid synovialis (Figures 4, 5, \

and 6). \ . . - . : - -

On the filte r surfaces, a decreased number of rheumatoid lymph­

ocytes wa^ also an observed consequence of challenge (Figures 7, 8,

and 9). No such destruction was significant for nonrheumatpid lympho­

cytes (Figures 10, 11, and 12) „

Various possibilities were tested for interaction between the two

chambers "a” and !!b?' implanted subcutaneously in a single ra t (Table

10). In El 7, where chambers ”an and r'bn were identical, the lympho­

cyte necrosis resu lts were identical, although the tissues showed some

degree of variation. In RI 8 and 11, the condition of the challenged

lymphocytes or tissue in chamber "a" did not affect the condition of the

chamber "bn contents. In RI 9, four of the chambers ”ipin were identical

and their necrotic indices were more sim ila r to each other than to their

corresponding chamber nafrm atest For El 10, the f i r s t three combina­

tions listed indicate the necrosis due to challenge with rheumatoid lymph­

ocytes in chambers ,fa” did not influence the necrosis in chambers nbn,

which contained the same tissue challenged with nonrheuihatoid lympho-

cytes. Therefore, on the basis of the data presented in TabM lO, no

necrotic interaction between doubly-implanted cham bers occurred.

Variations in technic caused only incidental differences in

resu lts . For example, in RI 8 the ex tra modification of not cementing

the side access opening before inserting the Plexiglas stopper produced

40% loss of chambers due to host invasion. No bacterial contamination

was encountered due to variation of the sterilization method. ,

Table 4, Sources of Synovial Tissues,

Experiment ’ Sex' t T t

l : . ' A g e '? • t

Diagnosis,. "ErythrQcyte .

7 * v. Sedimentation ' RateXrnm,/hr,)-

, v M S r eoperatiye ', • - ‘ Medication

El 7 V ■ 21 ••• - Rheumatoid, a rth ritis

"7 7' Gold - . • -v .

■ F 63 \ Rheumatoidarth ritis

;94"di''vr Steroids

E l 8 m ; 27 ' Degenerativearth ritis

No steroids

.. M 18 B ilateralbunions

No steroids

- E l 10 '■ ■ M 3 7 : Degenerativearth ritis

No steroids "

y W 11 F / ■ 51 Splayfoot 7 No steroids

Table 5„,: Sources; of"Bloed :fcymphdc3rt:es..

Experi­ment

t -i : S e x 'r -

. t• r';v:'4ge;

4-Steroid •iJT'; " "-pcnR < ' -• x: ' ■ •; WBC ;

, (per cu.’ mm.)

Rheumatoid A rthritisRI "7 F \ ' 86 : ; No ' 88 7,400 ; :R I8 ' M . ' ; '56 NO 49 9,500RI 9 M ■v 24 No 52 ; 9,800RI 10 : 6 3 ' No ■■ 41 10,000

. RI 11 M . '5 2 ' Yes 45 8,000

Normal Controls

RI 7 F : .24 No 18 8,800

' RI 8 ' ; , H ; : : 21 ; ■ No 2 ' 7,000VRI 9 F ' 3 3 -. No ; 1? 6,600

RI 10 ; Vm . 40\ No ' : o; . 6, 600

. RI 11 ; ; m . ; ■ :3 ; 4 q 3 No ' 0. ;• ' ' . 6 , 600';

Note; ESR ss Erythrocyte sedimentation ra te . / ; WBG @ Wtiite'blboti^c ' ,

32

Table @ = Effect of Recentrifugation Technic on Two Blood Samples.

Separations; t

Iv ;. ... . 1 . . . ■ ■:'.R m N /m i. /

Rheumatoid A rthritisNo. 1 plasm a

supernatant : 266 : 5 200,000 100,000

buffy coat layer . ' 27/300,000 20,600,000

No. 2 plasm a and buffy coat combined

179 10 900,000 200,000

■ ' No. 3; v concentrated 4 tim es \ ■ V 179 ' IQ ; ;; 3,000, 000. ■ 90,000

Normal Control '' No. 1 : : : 499 ' , 5 2,050,000 / 150,000

' No. 2 concentrated 1.5 tim es 179 10 3,800,000

Note: RGF ^ relative centrifugal fo rce / xG « tim es gravity; t ® tim e .in miiiutes; -SL - sm all lymphocyte; PMN polymorphonuclear

. leukocyte. . : ■ / - • : .

Table 7, Leukocyte Inocula per Chamber .

Experiment ' Rheumatoid A rthritis .. ' Normal Controls :, r . . No. of.SL * . No. Of PMN -' No. of SL ' - No., of PMN: -

' RI '7 • - ■ ••■'z; 250,000;; : 200,000 2;i5O;OO0 V ' 95,000 ' - -

,m 8 : ■ 1, 200,000 : ■■ ■' 0 - ' ■ ' ■ V930, 00d\'f ; :;-v - 18,000 - '

:66o, ooo ; . c"O5B.0OO ■;y: •

; ; ■ Rl 10 000,000:; 1 8 , 0 0 0 v.:; ■ 770,000 ,

RI 11 'iPyOOO: hv-;■ •

/ \ 770,000 . ; ' ' V'v

Note: SL « sm all lymphocyte; PMN polymorphonuclear leukocyte.

03W

34

Table 8. Destruction of Lymphocytes Shown by F ilte r Examination,

Experimentt

. ? .. . , Estim ated N ecrosisf

, L*' 4- t* 'L *4vt , i + t^ :L4-,t': '

m 7 . ' 1. \ 4-4" 4-4-4- :y'<:4.y:2 44- v 4-4-4- ' -<. 4- -3 - 4 - 4 - , 4-4-4-

R1 8 1 4- 4-4- 4-4- < 4- ; . / i - :;v--;;:_ 4-4-2 , :>-■++: 4-4-4- i i . . 4- 4-

3 : .. i - i . 4-4-4-4- ■ " i . .■ i. ’ - I , i -RIB. 1 •• 4-4- 4-4-4- 4- 4- - 4- 4-

2 •4-4- 4-4--K4- - 4- 4- 4- 4-

3 • 4i4-4-4- : 1 'i '

4 i ' 4-4-4-4-RI 10 1 <.+ 4- ' < 4- < 4-

2 > 4- - 4-4- : : 4- 4- ' .

,3 . la ; . 4-4-4- ‘ - r - . 4- 4-

m i i . 1- K < 4- - 4-4--H < 4- : ■-'<■ +'2 .. 4- 4-4-tf < 4- ■ 4-

3 : • . ' i ' - 44-4-4- i • 4- '

Average l: 1,4 ■: 2,6 3.2 0.4. 1,3

Note: Rheumatoid: - lymphocytes, t* r synovialis; nonrheumatoid:L - lymphocytes, t - synovialis; i ss host invasion; LA s labora­tory accident. ' ' ■ -

Table 9. Destruction of Tissue Fragm ents Shown by Paraffin Sections,

i Estim ated Necrosh i r ; - - '' ? . v".;'"'."'

' r & v i

? ■- ;

RI 7 - ‘ '**+*: 2- '; :: W ' ; i . . ■***": .

.’ s ' i-" ft*"M 8; I,. '; : '- ,V:; \ . i - . •< 4- i'

-i-f-; : " : ' ; i i i i ; .+4-+ ■

■ i D i ■ i r, i;;': . y'ii- i . ' _ i■ , . " t t :

RE: 9 \ : ' .;¥■ i . : ; < + . , "< 4-. ' " . 4- . - '

. < 4- '

; 4" : ; ; :.4-4-4-'- 4-44-

RE 10 ' i i - < + : " . <" + • .

’ 2. ' : ; - ^ 4- -. + •.

3 ;;';i "V: ;:4-;- : "■+■ +4- '■ : v, + t * . ;< : 4-. :. 4-4- - - -

/ - < 4- - ; ' :

;-3 .4-4-:'"':. r : :f+ /y . ' .

A verage': ; : i 0*8 :: 0.7; 0 :0 -:

Note: Rheumatoid: lymphocytes ? t* - synovialis; nonrheumatoid:: L- - lymphocytes, \;t': - synovialis; 1. ^ .hpst invasion; DA /«' labor a- v tory accident, ' ...........................

36

Table 10./' Effect of Double Implantation on Chamber N ecrosis.

. ■ : C h a m b e r . .c o m b i n a t i o n s r

-E y m p h o .c y te '■ ■ n e c r o s i s ;

. T" 1 '<

' ' - T i s s u e / ;-;v n e c r o s i s

■ ■ ' / - a Z';.: t • a - ' " b ;

R 1 .7 ; L * ■ < r :. f f . - ; / ' .

t * ; W - h M ' :" ; / / f f f ’ ' / ' f f f ',' f f f f : ' f f f ; / ' - ,

' L • . ' f ■' -■

' ; ' : • L -h ' t • l f t ■: ' ;'< ; + . f f f - .v , , f , , ' ■ : v’

R I 8 . L * > t - " : ~ Z T f f f f < 4 ■

. L * ' + t* /

L + t t* f - f

R i l l - L*' + t L * ■ f f f f

L +. t - L * ; : j < f .4-''

' . L ^ t L : ; . f . < f

H I 9 L L * ' f t ' : f f ■

: . L * - ::L * ' ' f t / : ' f f ; ■,++++'■'

• L -f t ' L * - f t - " ' . ' f f . ++++ 4- '' , ’ . 4-4"4- - ;

L +■■ t . m ;f tV :;;;': ’ , ++*f+ ; < K ' - 4- "

■/ / ';; l > t " ^ 4" . 'C 4*

H I 10 L * -f t ' :/ L > t : •; : : f f ; ,< 4- ' -< 4* ; :

L * -f t L f t ' 'O f f f - ' f • ■ 44*4* ' , ; 4

' ' L * ' - f t L f t ' '• f ■; 4 ; ■' 4 4 •

t ' . ; t . \ v < f : " < f c

Note: Rheumatoid: L*- lymphocytes, t* - synovialis; nohrheumatoid: L - lymphocytes, t - synovialis; i ® host invasion.

CHAPTER IV

DISCUSSION ; - .

This study has dem onstrated that rheumatoid lymphocytes are

agents of destruction to synovialis within diffusion cham bers. There

are three possible explanations for this resu lt. One, the destruction

involves an immunologic mechanism presen t within the human body.

Two, rheumatoid lymphocytes might be m ore readily lysed than non-

rheumatoid by toxic synovial tissue products. In turn, the products of

lymphocytic lysis might have damaged the tissue. 'Three, rheumatoid

lymphocytes might have a g rea ter metabolic requirem ent than nonrheu-

matofd for some nutrient also required for tissue survival but present

only in low concentrations in the chamber fluid. The la tte r two explana­

tions represen t possible, artefacts produced by the s tre s s of the diffusion

chamber environment.

The f irs t and m ost plausible explanation is that necrosis within

the chambers is the resu lt of combination of sensitized lymphocytes

with their specific antigen-containing target tissue. Thus, it would be

implied that the. rheumatoid lymphocytes are cells sensitized against

or causing destruction of synovial tissue. Also, it would be indicated '

' - V:-: ' ; ;37 • v ■

. tfaat in rheumatoid a rth ritis , the blood ca rrie s lymphocytes with such,

destructive potential. The foundation for this explanation lies in th ree

points of analogy to the homograft reaction. F irs t, rheumatoid a rth ritis

is thought to be a possible immunologic disorder; the homograft reaction

is known to be an immunologic mechanism. Second, the histologic p ic­

tu re seen in rheumatoid synovialiS is sim ilar to that observed during

a firs t-S et homograft rejection. Third, if rheumatoid lymphocytes are

sensitized lymphocytes,; the observed destruction could have been p re ­

dicted since hom ograft-sensitized lymphocytes will destroy their geueti-

cally:-specific ta rget tissue within diffusion chambers (33). Therefore,

it is quite plausible that within both diffusion chambers and the human

body, rheum atoid lymphocytes destroy synovial tissue by some type of

immunologic mechanism; thus, rheumatoid a rth ritis can be concluded to

be an autoimmune disease. .

However, it is plausible on the basis of this experimental study

that rheumatoid lymphocytes are m ore easily lysed than nonrheumatoid

by toxic synovial tissue pr oducts... if so , the r esult of th is type of destruc

tion of rheumatoid lymphocytes might be a complete poisoning of the '

fluid of the chamber by term inal re lease of toxic substances:. These

toxins would in turn produce nonspecific destruction before the fluid

exchange between the host and the chamber would be capable of removing

them; Two observations, if present, would support this second explana­

tion: (1) if rheumatoid lymphocytes in control cham bers showed m ore

autolysis than sim ilar nonrheumatoid controls; and (2) if tissue necrosis

seemed independent of d irect contact with lymphocytes.

Lysis of rheumatbid lymphocytes averaged th ree tim es that of ;■

nonrheumatoid lymphocyte controls (Table 7). However, in RI 7, the

rheumatoid lymphocytes had been incubated for a longer tim e before

Use than the nonrheumatoid. By elimination of both RI 7 lymphocyte

controls from consideration of Table 7, the average control lympho­

cyte necrosis becomes 1.2 for rheumatoid, 0.8 for nonrheumatoid. The

subjective plus system is not sufficiently accurate to base a qualitative

difference on these figures. The conclusion must be made that rheum a­

toid lymphocytes in them selves are only very slightly less stable than

nonrheumatoid. Other investigators using diffusion chamber technic

have not reported any difference in stability, growth, or differentiation

characteristics of rheumatoid lymphocytes as compared to other types :

(50, 51). While these two facts do not invalidate the hypothesis that

rheumatoid lymphocytes are more easily lysed than nonrheumatoid by

synovial tissue products, they certainly detract from any argument

based on a difference in stability. -

The-second hypothetical observation which would support the

possibility of nonspecific destruction is: if tissue necrosis seemed

independent of d irect contact with lymphocytes. It was impossible to

determine the mechanism of the destruction by examination of the slides

prepared from the cultured synovialis fragm ents. There was no

indication that ce ll-to -cell contact was required to produce destruction

because no great degree of invasion of cells by lymphocytes occurred.

For this point an explanation might lie in the work with the

homograft reaction (33), Where destruction within the chambers was

described.' It;was stated that contact between lymphocytes and the

target cell was necessary for initial destruction; A consequence of

contact destruction of the target cell was subsequent death of the adjoin­

ing lymphocytes. Destruction of both cell types f irs t occurred at the

surface of the tissue fragment. The longer the cham bers remained

implanted the m ore degeneration occurred in the target tissue beyond

the ,rzone of contact’5 with homOgraft-sensitized lymphocytes. Weaver

e t al. (33) explained this type of destruction on the .basis of the possible

re lease of a diffusible toxin following the initial immunologic surface

necrosiso Connective tissue fibrocytes have cytoplasmic boundaries :

Which a re very hard to ;descry^:;-Therefore, :it is possible that m icro­

scopic examination might have^failetf to • re v ea l, the' ■mechanism of initial /

destruction of synovialis. - Thus, the obvious consequence of challenge

might have been necrosis resulting from the term inal re lease of diffus­

ible toxin. The toxins might have caused tissue cell necrosis and in

addition caused the fragmentation of adjoining lymphocytes.

. Also, it is plausible on the basis of this study that the tissue

destruction Without invasion by lymphocytes is the re su lt of an artefact.

A g reater metabolic requirem ent in rheumatoid lymphocytes than

nonrheumatoid may exist for some nutrient, also required for tissue

survival but p resen t only in low concentrations in the diffusion chamber

fluid. This would resu lt in starvation of the immobile solid piece of

the synovialis by the mobile lymphocytes. This tissue destruction with­

out lymphocyte invasion would in tu rn cause partial lymphocyte destruc­

tion due to the term inal release of toxins. With the data presented in

th is study, this possibility cannot be eliminated. Despite the lack of

reported differences in stability, growth, or differentiation characte r­

istics between rheumatoid and other types of lymphocytes (50, 51), tbe

yreasobihgism ot'm ^ ' -

The tissue destruction within chambers does not appear to be

produced by the contact immunologic mechanism reported by Weaver

e t a h ;(S3). It is reasonable to postulate an immunologic mechanism

sim ilar to homograft reactivity in which destruction is present without

cell-to^celi contact. The work of Amos and Wakefield (61 > 62, 63), ;

Gabourel (64), and Algire (65), Najarian- and Feldman (54), and a theory

Of delayed hypersensitivity published by Karush and Eisen (66) suggest

the destruction within diffusion cham bers may be the resu lt of the p ro ­

duction of a high-affinity humoral antibody in low concentrations. ;

In order to proceed, an im portant question requires answering.

. Are peripheral blood lymphocytes, used for challenge in this study,

capable of prbducing some humoral type of antibody response? Humoral

antibody is usually considered to be formed only by plasm a cells.

Fishman (68) initiated an antibody response in tissue cultures of ra t

lymph node cells as early as five days without the formation of plasm a

,cells . He detected low 'Concentrations of a diffusible type of antibody ;

by a very sensitive m ethod--neutralization of infectiyity of bacterio­

phage T2 in E. coli. With rabbit thoracic duct lymphocytes within dif­

fusion cham bers, a prim ary antibody response to a protein antigen was

obtained (55): Lymph nodes may contribute lymphocytes to the p eri- . :

pheral blood by way of the thoracic duct. Lymphocytes from the c ircu ­

lating blood make up a large portion of the hematogenous mononuclear

cells responsible for homograft rejection (13).. Finally, in man, tra n s ­

fer of peripheral blood leukocytes can readily transfer tuberculin sensi­

tivity (67), Therefore, the peripheral blood lymphocytes obtained in

buffy coat are possibly immunologically competent c e lls , capable of - ..

some type .of antibody response. Sueh antibody production might be y

effective in a diffusion chamber but extrem ely hard to detect in vitro

since the diffusion chamber has been shown to provide better nutrition

for antibody producing cells than Eagle’s , Puck’s, P a rk e r’s 199, or

T row eirs tissue culture media (68). , :

An analysis of the data presented in Tables 8 and 9 may help to

• determine whetiier the destruction is tissue specific or lymphocyte

' specific. That is , by analyzing Tables 8 and 9 together, one might be

able to determ m e which cell was the f irs t type to die within the challenge

cham bers. The assumption would be that the cells which died f irs t would

show the greatest difference in necrosis over the: controls. Thus, if

in challenge chambers tissue necrosis was three plus over the control

tissu e’s one plus and the rheumatoid lymphocyte, necrosis, was only one

plus g rea ter than the lymphocyte Control’s one plus-, then it can be

said that the destructive mechanism was tissue specific. In Table 11,

the data indicate that for challenges with rheumatoid lymphocytes, the

necrosis is in fact g rea ter for the tissue than for the lymphocytes

except in HI 11. The data show that for challenges with nonrheumatoid

lymphocytes, the necrosis is not g rea ter for the lymphocytes than for

the tissue to an extent approaching s ta tis tica l significance (p@ 0.2 -

0.1)o Not enough data exist to draw any definite conclusion about the

challenge of rheumatoid tissues. Here, the situation is complicated by

■ the presence of large numbers of autologous rheumatoid lymphocytes

in the tissue itse lf. To take exception to R I 11 would not be unreason­

able since the control rheumatoid lymphocytes were agglutinated but

definitely present. Furtherm ore, a six tim es lower inocula of lympho­

cytes was used compared to RI 9 and 10. The need for m ore experi­

mental work is indicated.

Certainly the argument for some type of immunologic mechanism

is strengthened by the following facts: (1) in challenges with nonrheu­

matoid lymphocytes, the necrosis is definitely not tissue specific; and

(2) in challenges with rheumatoid lymphocytes, 75% of the. experim ental

data indicate that the necrosis is tissue specific. Competition for some

necessary nutrient might also produce tissue specific destruction. This

type of destruction would not be produced by lysis of lyraphocytes.'

With homograft reac tiv ity / four explanations are possible for

the absence of cytotoxicity in vitro (33, 35, 36, 37) but presence in

diffusion cham bers (33, 61) i F irs t, the degree of cellu lar contact has

been indicated as important in vitro (38, 39) but relatively unimportant ,

for challenges within diffusion chambers (33) „, Secondly, the fact that

tissue culture provides Subpptimal nutrition to antibody-producing cells

has been considered a deciding factor by some w orkers (33, 68), Third,

a large vblume of fluid medium is required to maintain cells, in vitro as

compared to the diffusion chamber. This has been suggested as being

deleterious to a challenge effect because of dilution of any diffusible

toxin or antibody produced by the lymphoid cells. However, in diffusion

cham bers, the low cellular growth ra te requires so sm all a volume of

nutrient fluid that a diffusible toxin or antibody might reach sufficient V,

concentrations to produce a clearly observable effect (33), Fourth,

complement, a complex mixture of plasm a globulins known to enter

into some immunologic reactions, has been implicated as the principal

difference between in v itro and diffusion chamber challenges (33),

Complement form s 0;5% of the total serum proteins and its

participation in immunologic reactions Is not antigen specific. When

complement is removed from the human body, its activity rapidly d is- ,

appears within 24 hours at room tem perature. It is required for lysis ..

of bacteria or erythrocytes by antibodies (69). Complement is necessary

for lysis by tuberculoprotein of leukocytes from tuberculin-sensitive

individuals or norm al leukocytes if in the presence of an undefined glob*-

ulin factor from tuberculous plasm a (70). Complement is required for

th e ly s is of .mammalian cells by heteroantibody, i.e ., an tisera from

animals of a different; species injected with such cells (71). In the papers

reporting cytotoxic activity of lymphocytes in tissue culture, one reported

, that complement increased the effect (38), the other stated complement

was without effect (39). The form er also reported that complement was

required, for the lysis of cells by heteroantibddy but isoantibody (from ■ '

homologous serum) was without cytolytic effect Oven with the addition of

complement. ' .

Pertinent to any discussion of the influenCe of complement in

tissue culture cytolysis are the comments of Pulvertaft et al.(72)„ The

principal te s t for m easuring the effects of complement is the loss of

cytolysis when the challenging fluid has previously been heated to 56° C.

for 20 minutes and its Subsequent return when guinea-pig complement

is added (69, 72). Pulvertaft cited the finding bf heat-labile antibodies

a lso destroyed a t 56°; C.> namely: . reagins, autoantibody in a case of

hepatic c irrh o sis , and autoantibody in HasMmoto se ra . He also stated:

that his thyroid cell cultures were not Susceptible to damage by Hashi-

moto se ra if the cultures were four to six days old (72). These additional

facts show the complexity of evaluation necessary in any tissue culture

report of failure in demonstrating cytotoxicity.

Complement has been shown to be an important factor for the

prpduction of cytptoxie effects by isoantibody on cells within diffusion

chambers (61 > .62, 63} and on cells in vitro (73)„ The significance of

these reports for the present investigation is that complement may be

the factor necessary to show the cytolytic action of antibody, if such is

produced against synovialis by rheumatoid lymphocytes. This possibil­

ity is strongly supported by the work of Amos (61). His system involved

diffusion cham bers within the peritoneal cavity of m ice. Substances

were added to the peritoneal fluid; subsequently the host serum , the

peritoneal fluid, and f%e:'diffusion cham bers were - sampled as a function

of time... It was'found that the M illipore filte rs of the chamber acted as

simple b a rr ie rs tb diffusion. The ra te of entry and the equilibrium con­

centration of substances in the chamber were dependent upon the com­

position of the peritoneal fluid. This was shown not only for oxygen

tension changes but also for entry of antibody and com plem ent.: As an

example, when antibody was injected into the peritoneal fluid, equili­

brium with the blood was established within thirty minutes;' but in this

time only sm all quantities had penetrated into the chamber. < Approxi­

mately one hour was required for the chamber to attain equilibrium with

the peritoneal fluid. Antibody passed very rapidly from the peritoneal

cavity into the blood, but very slowly in the reverse direction. It was

then determined that the .complement concentration within the chamber

was approximately one-tenth that found in the serum . A fter determ in­

ation of the norm al growth ra te of mouse ascites tumor cells within

diffusion cham bers, isoantibody was injected into the peritoneal cavity,

resulting in a depression of the growth ra te . Further depression was

produced only by the addition of complement to the peritoneal fluid.

Thus, it was determined that complement was a: controlling factor in .

the cytotoxicity of isoantibody to a homograft.

Some emphasis is now placed on the role of antibody in at least

the second-set homograft rejection, the reaction which is possibly

analogous to rheumatoid arth ritic inflanamation . In the work of Algire

(65) ,< Amos and Wakefield (61, 62, 63), and Gabourel (64) 5 antibody has

been implicated as being functional in Some types of homograft rej ec - :

tion. These investigators enclosed within diffusion cham bers the same

type of homologous cells which had previously been injected into the

host up to four tim es, once weekly, to ' 'hyperimmunize'? the animats

Antibody to the chamber contents was found to penetrdte the chamber

in sufficient amounts to cause either lysis or growth inhibition of the

cells. Cellular contact of host with chamber contents was not necessary.

Two facto rs should be mentioned. The chamber contents were always

cells without a supporting strom a. Gabourel (64) sta tes that no Signif­

icant growth inhibition of mouse L-fibroblasts, occurred within the

cham bers, while reactions analogous to f irs t- and second-set homograft

rejections were pcctirring outside in the host. However, this does not

rule out the possibility that the diffusion of any antibody from the '

serum , to the peritoneal fluid, and then into the chamber was insuf­

ficient to be beyond the adaptability of the cells. :

In addition, the work of Naj arian and Feldman (54) indicates

that a diffusible factor can be responsible for accelerated rejection as

early as the second-set homograft reaction. Their sensitized lympho­

cytes were within the diffusion chamber! constantly releasing into the

blood of nohimmuhized hosts some necessary diffusible factor. It

accelerated the rejection of skin grafts placed outside the cham bers.

T his; experimental resu lt certainly supports the other information con- :

cerning the role of a diffusible substance in homograft rejection.

Also, the slight but s till six tim es g rea ter necrosis of nonrheu-

matoid tissue challenged with nonrheumatoid lymphocytes when com ­

pared to honrheumatoid tissue controls, should be explainable in te rm s

of this system . This necrosis in a homologous but rionsensitized sy s­

tem is also reported by; Weaver e ta l.! (33). For homograft challenge

system s within cham bers, it was Stated that, compared to sensitized

lymphocytes, the homologous but nonsensitized lymphocytes caused

slower and leSs destruction of the target ce lls . It is possible that here

the lymphocytes slowly became sensitized in response to homologous

tissue antigens. They might have produced some diffusible-antibody !-

which was responsible for tissue destruction without ce ll-to -cell contact

- . - ' ■ V ; ■ 49

From examination of the sm all amount of data in Table 11, however>

the damage to synovialis by nonrheumatoid lymphocytes does not

appear to be tissue specific. :

A number of minor facets concerning the experim ental p ro ­

cedure employed in this study- rem ain to be considered.

.. Control and treatm ent diffusion chambers were often placed in

the sam e rat%, thus reducing the effect of biological variation of nu tri­

tional status among m em bers of the inbred ra t strain .

-Since the cham bers were picked at random for implantation,

interaction between the two chambers would have caused a random

necrotic resu lt in Tables 8 and 9. That interaction was not detected

(Table 10) is reasonable since Weaver et al. (33) noted no differences

in challenge resu lts due to the immunization status of the homologous' " ' ■ > : : ' -

chamber hosts. - v.. - -

The staining procedure with hematoxylin and eosin always p ro ­

duced over staining of undamaged nuclei, thus eliminating the critic ism

that hypochromic nuclei were not necrotic but phly under stained. P o ss i­

bilities of relative staining variations due to tim e and composition of - ;

stain were eliminated within one experim ental run by staihing all slides

and a l l filte rs simultaneously. Variations between experim ental runs

were cut down by rigidly fixing all staining tim es and using fresh solu­

tions .

; - : . 5 0

.Other sources of e rro r in this study mainly, involved the paraffih

sections. They were heterogenous, since necrosis existed in various

form s. Also, some of the fragm ents were sectioned closer to the

i center than others, etc. Reporting resu lts in term s of a plus system

prevented prejudicing of resu lts .

Quantitation of the degree of necrosis Of the chamber contents

is extrem ely vital to further work. Detection and quantitation Of some

property produced by necrosis, ra ther than quantitation, of the loss of

some healthy function, would allow experimental, variation to; produce

resu lts beyond the capability of visual estim ation.

5 1

Table 11. Challenge Necrosis - - Lymphocytes and T issue Compared.

. 1 . . ' . ' ' ' t _ '

. ’ ' t ' .Experiment ,

: . L* + 't ,’ . Corrected Average ? • N ecrosis1._ . -t L + 't ;

• : : " V;': h ; : ■ ' L* r

TT . t .:. ■■■ :y^;

'R IB .,: ; : d. -'d" . .'"2,0 - .2.3 ' -y -I d'2.0 0.0

R I9 ' I;, 1.8 2,2 : :■ o.o 0.3

RI- IO' " / . 1.0 1,3 ' 1.0 1.0

■■ Hi i i ' - ' '■ 3.0 2.0 ; l,o 0,7 •

•Average 1 2.0 ' -2,0/ \ 1.0 0.5 ,

Note: Rheumatoid: £«* - lymphocytes, t* - synovialis; nonrheurnatoid: L lym phocytes,/t - synovialis; corrected necrosis ® challenge necrpsis - eohtrol necrosis. '% . - ' ' '

CHAPTER V

'StMMARY

Cix synovial tis su es , - two rheumatoid and four nonrheumatoid,

were challenged within diffusion cham bers by inclusion of lymphocytes

obtained from subiects with and without rheumatoid a rth ritis

In. chambers containing rheumatoid lymphocytes in contact with

either type of tissue, necrosis averaged three tim es that in controls i,

.1 The necrosis was observed to occur without extensive ce ll-to -eell con­

tact- Following are the four possible m echanisms, here arranged in

descending order . F irs t, am immunologic mechanism: peripheral blood

lymphocytes were possibly capable of producing or releasing a diffusible

, high-affinity:: antibody. Second, an artefact: a difference in the two types

of lymphocytes existed in the requirem ent f o r .some essential nutrient

present only in low concentrations in the diffusion cham ber fluid. This

would resu lt in different degrees of starvation of the immobile, cen tra l­

ized tissue fragment by the mobile lymphocytes at the periphery of the

chamber. Third, an artefact: a difference in stability of lymphocytes

existed when in the presence of tissue metabolic w astes, resulting in

: dichotomous; degrees of poisoning of the chamber' flu id , by the• te rm in a l'; •

5 2

release of toxins. Fourth? an immunologic mechanism: initial necro­

sis was contact produced. The term inal release of diffusible toxins

caused the observed necrosis and lys'ed the adjoining. lymphocytes.

More experim ental work on an exact quantitative basis is needed to

arrive at a definite conclusion concerning the cause and meaning of the

observed necrosis attendant upon challenge with rheumatoid lympho-

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Figure 1. Rheumatoid synovialis cultured alone. (Note the large number of undamaged nuclei.)

Legend:Figures 1, 2, and 3: Hematoxylin and eosin stained paraffin sections; 300X.

63

Figure 2.

Figure 3.

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Rheumatoid synovialis cultured with nonrheumatoid lymphocytes. (Note the large number of undamaged nuclei.)

Rheumatoid synovialis cultured with rheumatoid lympho­cytes. (Note the absence of undamaged nuclei.)

64

Figure 4. Nonrheumatoid synovialis cultured alone. (Note numerous undamaged nuclei.)

Legend:Figures 4, 5, and 6: Hematoxylin and eosin stainedparaffin sections; 300X.

Figure 5. Nonrheumatoid synovialis cultured with non-rheumatoid lymphocytes. (Note the undamaged nuclei.)

Figure 6. Nonrheumatoid synovialis cultured with rheumatoidlym phocytes. (Note the scarcity of undamagednuclei.)

66

Figure 7. Rheumatoid lymphocytes cultured alone. (Note the num­erous undamaged nuclei.)

Legend:Figures 7, 8, and 9: Hematoxylin and eosin stainedM illipore filter surfaces; 300X.

Figure 8. Rheumatoid lymphocytes cultured near rheumatoidsynovialis. (Note reduction in number of undamaged nuclei.)

Figure 9. Rheumatoid lymphocytes cultured near nonrheuma-toid synovialis. (Note scarcity of undamaged nuclei.)

68

Figure 10. Nonrheumatoid lymphocytes cultured alone. (Note large numbers of undamaged nuclei.)

Legend:Figures 10, 11, and 12: Hematoxylin and eosin stainedM illipore filter surfaces; 300X.

69

w e- • ' _ w S H B B M H I

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Figure 11. Nonrheumatoid lymphocytes cultured near rheum a­toid synovialis. (Note large numbers of undamaged nuclei.)

Figure 12. Nonrheumatoid lymphocytes cultured near non­rheumatoid synovialis. (Note the large numbers of undamaged nuclei.)