Chemotactic Factor Inactivator in Normal Human Serum

JEFFREY L. BERENBERG and PETER A. WARD

From the Walter Reed Army Institute of Research, Washington, D. C. 20012and the Department of Pathology, The University of Connecticut HealthCenter, Farmington, Connecticut 06032

A B S T R A C T Normal human serum contains an inacti-vator of chemotactic factors for neutrophilic leukocytes.The chemotactic factor inactivator (CF-I) remains sol-uble when serum is fractionated with ammonium sulfate(at 45% saturation), directly and irreversibly inacti-vates chemotactic factors, and it has a broad spectrumof activity as indicated by its inactivation of the chemo-tactic fragments of human C3 and C5 (third and fifthcomponents of complement), C567, and the bacterialchemotactic factor derived from Escherichia coli. CF-Iappears as a biphasic activity according to preparativetechniques of sucrose density ultracentrifugation, elec-trophoresis, and gel filtration. Studies on the interactionof CF-I with the radiotagged C5 chemotactic fragmentfail to reveal evidence for irreversible binding as thebasis for inactivation. CF-I varies from the anaphyla-toxin inactivator in several physical-chemical respects,but evidence does not permit a conclusive statementabout the relationship of the two inactivators. CF-I mayfunction as a regulator of inflammatory responses.

INTRODUCTIONThe acute inflammatory response is mediated by a vari-ety of factors which either increase vascular permeabil-ity or lead to the leukotactic accumulation of neutro-phils. The mediators themselves are chemically diverse,ranging from peptides (the kinins, anaphylatoxins,and chemotactic factors) to substances of very low(< 1,000) molecular weight (e.g. the vasoactive amines).As typified by the complement-derived leukotactic fac-tors (from the third, C3, and fifth, C5 components ofcomplement), a diversity exists not only in structurebut also in the generation of these inflammatory media-tors. For example, C3 and C5 can be cleaved productiveof anaphylatoxin and leukotactic fragments not only byenzymes intrinsic to the complement system, but also by

Received for publication 22 August 1972 and in revisedform 26 October 1972.

enzymes extrinsic to complement, such as plasmin, tryp-sin, thrombin, and tissue derived neutral proteases (re-viewed in 1).

In spite of the remarkable diversity of mediators, theinflammatory response is kept in balance by naturallyoccurring inhibitors. For instance, the kinins and theanaphylatoxins are destroyed almost immediately upongeneration in serum. This phenomenon of inactivationhas been related to the existence in serum of a carboxy-peptidase B enzyme that inactivates the kinins and theanaphylatoxins by removal of the C-terminal arginineresidue in the peptides (2). In this report we describethe presence in normal human serum of a chemotacticfactor inactivator. It seems likely that the function ofthis inactivator is to provide regulatory control overthe leukotactic mediators generated in serum.

METHODSChemotactic assays. Chemotaxis was performed by the

micropore filter technique in modified Boyden chambers.This technique has been previously described in detail (3, 4).Filters of 0.65 gm pore size (Millipore Corp., Bedford,Mass.) were used and chemotactic activity quantitated bycounting the numbers of cells which had migrated throughchannels of filters in five high power fields under lightmicroscopy. Rabbit neutrophils from glycogen-induced peri-toneal exudates were the indicator cells. The upper com-partment of each chemotaxis chamber contained a total of2.5 X 106 neutrophils in 0.1% bovine serum albumin (BSA).The bottom compartment of each chamber contained thefluids to be tested for chemotactic activity. Hank's medium(Microbiological Associates, Inc., Bethesda, Md.) was usedfor dilution of both cells and fluids. For convenience, rabbitneutrophils were the usual indicator cell. However, theresults tested in Tables I, II, and IV were reproduced usinghuman neutrophils as the indicator cells. Only the data ob-tained with rabbit neutrophils are listed in this report.

In all cases where studies of the chemotactic factor were

carried out, the chemotactic factor inactivator was mixedwith 50 dAI Escherichia coli bacterial factor (see below) or

other chemotactic factors, as indicated. After incubation atroom temperature for 30 min, this mixture was adjustedto 1.0 ml with Hank's medium and the chemotactic assaycarried out. Per cent inhibition was determined by calculat-

1200 The Journal of Clinical Investigation Volume 52 May 1973 .1200-1206

ing the ratio of chemotactic activity in the presence ofthe inactivator and in its absence, times 100 and subtractingfrom 100.

P'reparation of clhemotactic bacteria: bacterial factor wasohtained from culture supernatants (in 1-ank's medium) ofE. coli sterilized by filtration through a Micropore filter(5). 50 ul of this factor were used as the standard sourceof chemotactic factor unless otherwise indicated.

Preparation of complement cilemnotactic factors. HumanC3 and C5 were purified according to the method of Nilssonand Mfiller-Eberhard (6), which included a combination ofisoelectric precipitation, ion exchange chromatography, hy-droxyl apatite chromatography, and preparative electro-phoresis. Chemotactic fragments of C3 were produced bytreatment with trypsin (Mann Research Labs., Inc., NewYork) according to the procedure of Bokisch and Muller-Eberhard (2). Chemotactic fragments of C5 were obtainedin a similar manner (7). Except where indicated, thesesolutions were not fractionated further. In one experiment1 mg unlabeled C5, mixed with 0.1 mg radiotagged C5 (bythe iodine monochloride method, 12), was treated with 100,ug trypsin (in a reaction volume of 0.5 ml) at 320C X 20min. The reaction was stopped by addition of 200 /Ag soy-bean trypsin inhibitor (Mann Research Labs., Inc.) con-tained in a volume of 50 A1 in phosphate-buffered saline.The trypsinized (and chemotactically active) C5 materialwas then fractionated by gel filtration, as described below.A zone of radioactivity near the void volume of the elutedcolumn was found, as well as one near the cytochromemarker. The radioactivity in the latter zone was pooled andused as the source of chemotactically active CS fragment(7). Human C567, activated by trypsin treatment of C567(8), was kindly provided by Doctors C. Arroyave and H. J.Mfiller-Eberhard.Preparative electrophorcsis. Human serum was dialyzed

for 12 h against barbital buffer, pH 8.6, ionic strength 0.05.Pevikon block electrophoresis was then carried out in thesame buffer for 18 h (9). The resulting fractions weredialyzed for 12 h against phosphate buffer (pH 7.3, ionic

Presence of ChemotacticTABLE I

Factor Inactivator in Serum Fractions

Volume ofinactivator Chemotactic§ Inhibition

Source of inactivator* used+ activity

J. D., whole serum 100 210 9J. D., supernate 150 15 97J. D., precipitate 300 200 9K. J., supernate 300 20 91K. J., precipitate 300 195 10NonelI 230

* 50 1.l chemotactic factors from E. coli used. Initials refer toserum donors. Fractions of serum obtained by ammoniumsulfate at 45(%c saturation were employed. See text.t None of these preparations were chemotactically active bythemselves.§ Number of migrated cells in five high power fields, as inreference 3.11 Background chemotactic activity (in absence of bacterialchemotactic factor) count of 20.

TABLE 1I1Direct Effect of Inactimator on Bacterial Chenmotactic Factor

Inactivator* added to

cells cliemotactic Cheniotactic(upper) factor value Inhibition

Al o/0

10 165 1620 - 180 050O 190 0- 10) 25 100

20 20 100- 50 20 100- - positive control: 190

negative controlt: 30

* Soluble ammonium sulfate fraction (45%) of normalhuman serum, dialized and concentrated to 3 times theoriginal volume of serum. The inactivator was incubated for30 min at room temperature with either cells or bacterialchemotactic factor before the chemotactic assay.T Hank's medium.

strength 0.05) before being tested for their ability to in-hibit chemotactic activity.

Salt fractiowntion. Normal human serum was allowedto clot for 2 h and then p)recil)itate(l with ammonium sulfateat 40 or 45% saturation at 50C. (No differences in yieldor behavior of the chemotactic factor inactivator were notedunder these two conditions of fractionation.) The super-nate was then dialyzed for 48 h against phosphate-bufferedsaline. Unless otherwise indicated, the dialyzed fractionswcre concentrated with PM-10 membrane (Amicon Corp.,Lexington, Mass.) to one-third the original volume of serumbefore assaying for chemotactic inactivator activity.

Ultraeentrifugation and gel chromatography . Ultracen-trifugal characteristics of various preparations were deter-mined by fractionation in a sucrose density gradient (7.5-35%c sucrose in phosphate buffer, pH 7.3, ionic strength0.05) with centrifugation at 55,000 rpm for 16 h at 4VCin an International Hematocrit Centrifuge (InternationalEquipment Co., Boston, Mass.), B-60 using a swingingbucket rotor (4). Gel chromatography was carried out withBiogel P200 (Bio-Rad Laboratories, Richmond, Calif.) inphosphate-buffered saline. The proteins human IgG, BSA,and cytochrome c were used as markers in ultracentrifugaland gel filtration techniques. The same markers have beenused previously (7).

RESULTSSalt fractionation of chemtotactic factor inactivator.

When the bacterial factor from E. coli was treated with100 al whole human serum no significant loss of chemo-tactic activity was noted (Table I). At the present timeat least 20 different whole normal human sera have beentested and found to be lacking in ability to inhibit chem-otactic activity. However, when the various ammoniumsulfate fractions from each of two sera were studied, itwas found that the soluble fraction of serum was sig-

Chemotactic Factor Inactivator in Normal Human Serum 1201

IgG BSA cytochrome

k-x

0

uJI

z0

TZIz

2 4 6 8 10 12 14 16FRACTIONS

FIGURE 1 Patterns of chemotactic factor inactivator inammonium sulfate-soluble fractions from two normal humansera, separated by density gradient ultracentrifugation. 50,al bacterial chemotactic factor was incubated with eachfraction from the gradients at 25'C X i h, the mixture thenassayed for residual chemotactic activity. In each of thepreparations two zones of inhibitor are present.

nificantly inhibitory for the chemotactic factors whenappropriate volumes were used. No inhibitory activitycould be demonstrated in the redissolved precipitate,indicating that the chemotactic factor inactivator isassociated with a fraction of serum other than theIg-rich fraction.

Direct action of inactivator on the chemotactic factor.These experiments were designed to determine if the in-activator was directly affecting the chemotactic factorsor the indicator leukocytes. Using the ammonium sul-fate soluble (concentrated) fraction from normal serum

TABLE II IIrreversible Effect of Chemotactic Factor Inactivator

ChemotacticMaterial tested* activity: Inhibition§

Blank 25Bacterial factor 240Bacterial factor boiled (15 min) 270 0Bacterial factor + inactivator,370C X 20 min, then boiled (15 min) 100 59

Bacterial factor + boiled (15 min)inactivator, 370C X 20 min 205 15

Boiled (15 min) bacterial factor + in-activator, 370C X 20 min 90 63

* Source of bacterial chemotactic factor was 50 IAI culturesupernate (sterilized by filtration) from E. coli. Chemotacticfactor inactivator was the concentrated and dialized solublefraction after precipitation of normal human serum withammonium sulfate at 457% saturation. A volume of 20 ulwas used.t Counts of migrated cells in five high power fields.§ Per cent.

described in Table I, increasing amounts ot this materialwere added either to the leukocyte suspension or to thebacterial chemotactic factor and then incubated for 20min at room temlperature. Table II shows the results ofthese experiments. When 10-50 /l inactivator was in-cubated with cells, 11o effect on chemnotactic activitywas noted. However, as little as 10 /l inhibitor addeddirectly to the bacterial factor completely eliminatedchemotactic activity. These results indicate that theinhibitor present in the 45% ammonium sulfate solublefraction of human serum acts directly on the chemotacticfactor (rather than on the cells) to render it inactive.When large amounts of inhibitor (> 50 /Al) were added tothe cell suspension, decreased chemotactic responseswere found, presumably due to an effect of the inhibitoron the diffusing chemotactic factor.

Irreversible nature of the chemotactic factor inhibitor.The inactivator (described above) was mixed with thebacterial chemotactic factor under a variety of condi-tions. Bacterial chemotactic factor boiled for 15 minshowed no loss of activity (Table III). When the in-activator was added to the boiled bacterial chemotacticfactor, 63% loss of chemotactic activity resulted. Theprior boiling of the inactivator abolished most of theinhibitory effect on chemotactic activity (Table III, 15%loss), whereas boiling the mixture of inactivator andchemotactic factor (after they had been incubated to-gether) failed to restore the chemotactic activity (59%loss). These results indicate that the chemotactic factorinactivator acts in an irreversible manner on the bac-terial chemotactic factor to render it inactive.Broad spectrum of activity of inactivator on chemo-

tactic factors. The previous experiments indicated thatthe inactivator present in the soluble (and concentrated)fraction of ammonium sulfate-treated human serum couldinactivate the bacterial chemotactic factor. The data inTable IV demonstrate that the inactivator has a rather

100

4

O IgG BSA Cytochrome0Uj~~~~~

. 50 STARTING MATERIAL:O SOLUBLE FRACTION OFE

SERUM fKJ) IN 40%.Z AMMONIUM SULFATE EU

0~ ~ ~ ~~6 4

CUMULATIVE VOLUME

FIGURE 2 Elution from Biogel 200 of the soluble fractionof human serum obtained with ammonium sulfate at 45%osaturation. From each sample 100 gI volumes were incubatedwith 50 ,tl bacterial chemotactic factor at 250C X a h, thenassayed for residual chemotactic activity. Two zones ofinhibitor are present. Protein was measured in the Folinreaction.

1202 J. L. Berenberg and P. A. Ward

broad range of activity on chemotactic factors. Thechemotactic activity of human serum, treated with zy-mosan (4), the complement-derived chemotactic factorC567 and the chemotactic fragments of C3 and CS, andthe E. coli bacterial factor were all inhibited. Thus, thecheemotactic inactivator obtained from human serum isable to inactivate both complement-derived and comnple-ment-independent chemotactic factors. Reasons for thedifferent degrees of inactivation (59-96%,, Table IV) ofvarious chemotactic factors are not known at the presenttime.

Ultracetarifuigal analysis of chesnotactic factor blacti-vator. Sucrose density gradient ultracentrifugation wasperformed to determine physical-chemical characteristicsof the chemotactic factor inactivator. Two inhibitor-richpreparations, threefold concentrates of ammonium sul-fate-soluble fractions, as described above, from twodifferent human sera were studied. The results areshown in Fig. 1. For these experiments 50 ,/l bacterialchemotactic factor was added to each of the 16 fractionsfrom the density gradients incubated for 2 h at roomtemperature, then diluted to 1 ml in Hank's medium andtested for residual chemotactic activity. Both frames inFig. 1 indicate that inactivator activity is biphasic, ap-pearing at or below the lgG marker, and near the albu-min (BSA) marker. These results suggest that thechemotactic factor inactivator in serum is heterogenousand can be resolved into at least two different fractions.

Isolation of chemotactic factor iniactivator by mtolecu-lar sicvinig. The inactivator-rich fraction of serum pro-duced by ammonium sulfate treatment was chromato-graphed in Biogel-200 (Bio-Rad Laboratories) at pH7.3 (Fig. 2). Two zones of inhibitor activity weredemonstrated: the first eluted between the IgG andBSA markers, and the second near the cy}tochrome cmarker. As with the ultracentrifugal fractionation, thesedata indicate a heterogeneity in the characteristics ofthe chemotactic factor inactivator. In experiments notlisted here rechromatography of each of the two zonesof chemotactic factor inactivator has resulted in thesame chromatographic position of each inactivator asin the original separation. This finding together withthose to be presented below suggests that the hetero-geneity of the inactivator is due to two separate anddistinct substances.

Electrophoretic separation of the inactivator. 8 mlnormal human serum not previously fractionated waselectrophoresed in a Pevikon block at pH 8.6 and thefractions (100 nml each) tested for ability to inhibit thebacterial chemotactic factor was found to be present intwo zones corresponding to a- and P-globulin positions.(Fig. 3). To what extent these two zones of inactivatorrelate to the two zones found by ultracentrifugation andgel filtration (Figs. 1 and 2) is not known.

- 80-z -0

40-I _z

0'

INHIBITOR OF CHEMOTACTIC(BACTERIAL) FACTOR

.11 --1

0 4 8 12 16

FIGURE 3 Electrophoretic separation of normal humanserum in Pevikon, pH 8.6, barbital buffer, ionic strength0.05. Detection of inactivator was carried out, as describedin Fig. 2. Two zones of inhibitor are present, one in thea-position, the other in the a-position towards the anode(to right).

Aiechamisins of action/ of clheinotactic factor inactiva-tor. None of the preceding experiments defined themechanism by which the inactivator interacts withchemiotactic factors to render them inactive. To studythis question. the trypsin produced (and radio-labeled)CD fragment was isolated and then incubated with theinactivator. The source of the inactivator was the mate-rial eluting near the IgG marker front a Biogel column(Fig. 2). This inactivator was concentrated to a volumeof one-third the original serum volume. 10 Ol inacti-atorwas incubated with 50 /4 C5 fragment (equivalent toapproximately 5 tug original CS) at room temperaturefor 20 min, and then ultracentrifuged. The results areseen in Fig. 4. Neither the amounts of radioactivity inthe gradient nor the position of the radioactivity seemed

TABLE IXInactivation oJ Several Cheniotactic Factors by Inoctiacltart

Chemotactic activity

Inhibitor InhibitorFactor tested absent iresent Inhibition

Activated serum* 200 5 96C567 (5jg) 280 30 89C3 fragment: 145 35 59C5 fragment$ 280 10(0 65

Bacterial factor(50,l, from E. coli) 250 90 64

* One-tenth human serum incubated with 1 mg zymosan (4).In all experiments 20 ,ul inhibitor was used.t 50 mg C3 or C5 chemotactically activated with trypsinl.(See text).

Chemotactic Factor Inactivator in Normal Human Serum 1203

IgG BSA

Position of Inhibitor

ii

4 6 8 10FRACTIONS

12 14 it

FIGURE 4 Sucrose density gradient analysistrifugation of radio-tagged and chemotacticafragment before (solid line) and after (dottement with inhibitor isolated in Fig. 2 (the inwas the one that eluted near the IgG marlactivator does not cause a shift in the main Iactivity of the C5 fragment.

altered as a result of inactivation (Fig. 4,when compared with the pattern for tchemotactic fragment (Fig. 4, solid line).tests of the various fractions showed thehigh levels of activity in fractions 12-14whereas chemotactic activity in the same

lost as a result of pretreatment of the C5 fthe inactivator (Table V). It should be po

in the preparation of C5 fragment treainactivator a small zone of radioactivity wE

ultracentrifugal position of the inactivatortions 8 and 9). The significance of the sm

radioactivity ('- 4% of total radioactivitfragment preparation) is unknown. Whi]rule out a highly reversible interaction, thgest that however the inactivator alters theit is probably not due to substantial, irreveto the chemotactic factor.

DISCUSSION

The data presented in this paper indicaexists in normal human serum an inactivatactic factors. This inactivator is heterog4by a variety of fractionation techniques,appears in two positions. The chemotacactivator acts directly and irreversibly o

tactic factor, and it has a broad spectruiinactivating C567, the chemotactic fragme'C5, and the bacterial chemotactic factor

E. coli. It is not possible to completely ex

sibility that some of the effect of the chemotactic factorinactivator is cell-directed, but the bulk of the evidencefrom these studies strongly suggests otherwise. It shouldbe noted that normal human serum contains this in-activator, but the amount present is usually too low todetect in the absence of any fractionation and/or con-

FRAGMENT centration procedures. These would explain why it ispossible to chemotactically activate normal human serumwith agents that trigger the complement system, in spite

c5 FAGMENT of the fact that chemotactic factor inactivator exists inINACTIVATOR the serum.

The mechanism by which the inactivator interactswith the chemotactic factor is not precisely known. Thedata in Fig. 4 and Table V do not permit an unambigu-ous conclusion regarding the mechanism by which the

6 chemotactic factor inactivator abolishes the chemotacticactivity. It can be clearly stated that this is through a

by ultracen- direct effect on the chemotactic factor rather than by anIly active C5 effect on leukocytes.ed line) treat- If the mechanism of inactivation of the chemotactic

ker). Thoe in- factors is indeed enzymatic, then the broad spectrum ofpeak of radio- action by the chemotactic factor inactivator may imply

a common functional and structural region in each of thechemotactic factors. There may be an analogy in previ-ous chemotactic studies where it was demonstrated that

brokent lie)d deactivation of leukocytes by way of complement chemo-tactic factors eliminates subsequent chemotactic respon-

Chemotactic siveness to any of the complement-derived or the bac-

p(TsblnceV)f terial (E. coli) chemoattractants (10). This could be

(Tableons V, interpreted to indicate a common receptor on the neu-

ragment with trophil for structurally different factors, or it could:ragment with also indicate structurally similar chemotactic factors allted with tht acting on the same receptor. In the latter context, the

as seen in the(Fig. 4, frac- TABLE Vall amount of Chemotactic Activity in CS Fragment*ty in the C5le we cannotLese data sug-

C5 fragmentrsible binding

Lte that there.tor of chemo-enous in that,the material

tic factor in-tn the chemo-mn of activity,nts of C3 andderived fromclude the pos-

Chemotactic activity in ultra-centrifugal fractions.

Fraction Loss oftested fragment fragment chemotactic(25 jA) untreated treated$ activity

12 110 20 8213 160 40 7514 165 65 6115 20 30 0

* Obtained by isolation of trypsin treated C5. 5 pug C5 frag-ment were fractionated in each of two gradients.$ The chemotactically active C5 fragment from 5 Ag (volumeof 50 ul) was incubated with 10 ul (approximately 25 pugprotein) of inhibitor-rich fraction of serum. After incubationat 370C X 20 min, ultracentrifugal separation, shown in

Fig. 2, was performed. Chemotactic activity in various frac-tions from each of two gradients was then assessed.

1204 J. L. Berenberg and P. A. Ward

'-" 5.0-I0x

L-

I.4o 1.0-

I-

uZ 0.5-

04U 7U,

broad activity of the chemotactic factor inactivatorwould be readily explicable.

It is not surprising that in human serum there shouldexist an inactivator of chemotactic factors, whetherthese be products of intrinsic proteins such as comple-ment, or products of extrinsic agents such as bacteria.In the cases of the kinins and anaphylatoxins, potent in-hibitors present in serum inactivate these biologicallyactive peptides in an irreversible and enzymatic manner(2, 11). Other inhibitors such as the C-1 inhibitor (12),a2-macroglobulin (13) and a,-antitrypsin (14) exert aregulatory function by stoichometric-binding with anumber of enzymes, resulting in their inactivation,thereby limiting the action of kinin-producing, fibrino-lytic, complement and coagulation pathways. ai-anti-trypsin also inactivates the neutrophil-derived neutralprotease and elastase (15), providing a control mecha-nism to prevent undue destruction of tissue proteasesby leukocytic enzymes. It would be anticipated that ac-tive substances such as the chemotactic factors wouldfall under similar control, otherwise one would predictuncontrolled mobilization of leukocytes into foci con-taining chemotactic factors.A major question arising from these studies concerns

the relationship of the inhibitor of chemotactic factorsand the anaphylatoxin inactivator (AI) 1 described byBokisch et al. (2). AI is a pseudoglobin with an elec-trophoretic mobility of an a-globulin, a molecular weightof 325,000, and is present in normal human serum. Thisinactivator has carboxypeptidase B activity and largelyaccounts for the ability of normal human serum torapidly destroy the kinins generated by kallikrein andthe anaphylatoxin peptides C3a and C5a. To what ex-tent are AI and the inactivator of chemotactic factoridentical? On the basis of the data presented in thispaper there are some similarities between the two in-hibitors. On the other hand, there are several significantdifferences. As shown by ultracentrifugal, electropho-retic, and molecular sieving techniques, the chemotacticfactor inhibitor is heterogenous, consisting of two dif-ferent substances. Bokisch et al. (2) described a singleform of AI in serum, but later work has revealed thatAl may exist in serum as a polymer, raising the possi-bility that under different conditions AI may have vari-able physical features (16). Evidence to date indicatesthat the chemotactic factor inactivator is not impairedin the presence of phenanthroline at 10' M. Such resultswould suggest that the chemotactic factor inactivatoris not identical with AI (2). The resolution of the basicquestion about identity or nonidentity of AI and thechemotactic factor inactivator must await availability

1Abbreviation used in this paper: AI, anaphylatoxin in-activator.

of a highly purified preparation of the latter. In part,the difficulty in resolving this question relates to theunknown chemical relationship between the chemotacticpeptides of C3 and C5 and the homologous anaphyla-toxins, C3a and C5a. There is highly suggestive evidencein favor of the nonidentity of the homologous pep-tides with anaphylatoxin and chemotactic function. Al-though treatment of purified C3 and C5 with trypsinleads to the production of fragments with anaphylatoxinand chemotactic activity, continued treatment with tryp-sin results in the disappearance of anaphylatoxin,whereas there is no demonstrable loss in chemotacticactivity (17, 18). Since there is a fundamental questionregarding structure relationship between the anaphyla-toxins and the chemotactic factors, extrapolations be-tween the data from AI and those of the chemotacticfactor inactivator are difficult. Furthermore, it shouldbe pointed out that while human serum cannot be acti-vated to produce anaphylatoxin (2, 17), there is littledifficulty in activating human serum with an immunecomplex so as to generate chemotactic activity (3, 4).These findings indicate that chemotactic activity whichis generated in human serum by complement activatingsubstances is resistant to the effects of inactivatorsnaturally present in serum, either because the chemo-tactic factors are not susceptible to the inactivators, orbecause they are generated in quantities too great to behandled by the levels of inactivators. Whatever the casemay be, the ability to generate chemotactic activity inhuman serum certainly stands in sharp contrast to thatinability to generate anaphylatoxin activity in wholehuman serum. It should be stressed that, while someobvious differences exist between AI and the seruminhibitor of chemotactic factors, as revealed by fraction-ation procedures, there is no definitive answer to thequestion of identity or nonidentity of the two inacti-vators. Perhaps the most relevant point in our datais the demonstration of a naturally occurring chemo-tactic factor inactivator in human serum. Data will bepresented in a subsequent paper to show the presenceof this inactivator in super-normal amounts in patho-logic human sera.The problem of a better assay for the chemotactic

factor inactivator is obvious. When the inactivator issufficiently purified, it should be feasible to develop animmunochemical assay that would allow detection of theinactivator in whole serum. Alternatively, when thestructural definitions of the various chemotactic factorsare obtained, it is possible that this information can betranslated into chemical changes in the chemotacticfactors reflecting the action of the chemotactic factorinactivator. For the present, however, the functionalassay will remain.

Chemotactic Factor Inactivator in Normal Human Serum 1205

ACKNOWLEDGMENTS

This paper was supported in part by National Institutes ofHealth Grant AI 09651-02.

REFERENCES

1. Ward, P. A. 1970. Neutrophil chemotactic factors andrelated clinical disorders. Arthritis Rheum. 13: 181.

2. Bokisch, V. A., and H. J. Miller-Eberhard. 1970. Ana-phylatoxin inactivator of human plasma: its isolation andcharacterization as a carboxypeptidase. J. Clin. Invest.49: 2427.

3. Ward, P. A., C. G. Cochrane, and H. J. Mfiller-Eber-hard. 1965. The role of serum complement in chemo-taxis of leukocytes in vitro. J. Exp. Med. 122: 327.

4. Ward, P. A., C. G. Cochrane, and H. J. Mfiller-Eber-hard. 1966. Further studies on the chemotactic factor ofcomplement and its formation in vivo. Immunology. 11:141.

5. Ward, P. A., I. H. Lepow, and L. J. Newman. 1968.Bacterial factors chemotactic for polymorphonuclearleukocytes. Am. J. Pathol. 52: 725.

6. Nilsson, U. R., and H. J. Mfiller-Eberhard. 1965. Iso-lation of pif-globulin from human serum and its char-acterization as the fifth component of complement. J.Exp. Med. 122: 277.

7. Ward, P. A., and L. J. Newman. 1969. A neutrophilchemotastic factor from human C'5. J. Immunol. 102: 93.

8. Arroyave, C. M. 1972. Interaction between C5, C6 andC7. Fed. Proc. 31: 659. (Abstr.)

9. Mfiller-Eberhard, H. J., A. P. Dalmasso, and M. A.Calcott. 1966. The reaction mechanism of fBic-globulinC'3) in immune hemolysis. J. Exp. Med. 123: 33.

10. Ward, P. A. 1970. Complement derived chemotacticfactors and their interactions with neutrophilic granulo-cytes. Int. Arch. Allergy Appl. Immunol. 108.

11. Erdds, E. G. 1966. Hypotensive peptides: bradykinin,kallkdin, and eledoisin. Adv. Pharmacol. 4: 1.

12. Austin, K. F. 1971. Chemical mediators of the acuteinflammatory response. In Progress in Immunology. B.Amos, editor. Academic Press Inc. 724.

13. Ganrot, P. 0. 1967. Interaction of plasmin and trypsinwith a2-macroglobulin. Acta Chem. Scand. 21: 602.

14. Laurell, C. B., and S. Erickson. 1963. The electro-phoretic ai-globulin pattern of serum in ai-antitrypsindeficiency. Scand. J. Clin. Invest. 15: 132.

15. Janoff, A. 1972. Neutrophil proteases in inflammation.Annex. Rev. Med. 23: 177.

16. Mfiller-Eberhard, H. J., V. A. Bokisch, and D. B.Budzko. 1970. Studies of human anaphylatoxins and oftheir physiological control mechanism. In Immunopath-ology, VIth International Symposium (Grindelwald).P. A. Miescher, editor. Grune and Stratton, Inc., NewYork. 191.

17. Cochrane, C. G., and H. J. Miiller-Eberhard. 1968. Thederivation of two distinct anaphylatoxin activities fromthe third and fifth components of human complement.J. Exp. Med. 127: 371.

18. Ward, P. A., and J. H. Hill. 1970. C5 chemotactic frag-ments produced by an enzyme in lysosomal granules ofneutrophils. J. Inirnumol. 104: 535.

1206 J. L. Berenberg and P. A. Ward