A Tissue Culture Study of the Chick Fibroblast in Relation to Streptococcal Filtrates and Rheumatic Heart Disease By ROBERT J. BOUCEK, M.D., GLADYS CAMERON, M.A., VINCENT R. SAURINO, PH.D., AND ROBERT CHAMBERS, PH.D. Marked degeneration and disintegration of the chick embryo fibroblast was observed in tissue cultures after their exposure to sera of patients with active or inactive rheumatic heart disease or to the filtrates of beta hemolytic streptococci. Some tissue specificity was demonstrated; sera of the patients or the filtrates of the streptococci failed to affect contractility of the myocardial cells or secretion of the mesonephros. Sera of normal control humans or of patients with other so-called collagen diseases did not seriously affect the fibroblasts. Filtrates of nine other bacteria failed to alter the morphology of the fibroblasts unless severe toxicity was present and the tissue culture died. T HIS is a report on the response of chick embryo fibroblasts in tissue culture to filtrates of the beta hemolytic strepto- cocci and to sera of patients with a rheumatic process. It was recognized that any bacterial filtrate contains many toxic antigenic sub- stances and likewise that sera of patients with a rheumatic diathesis contain factors which might alter cells in tissue culture. Although previous investigation has directly or in- directly related rheumatic fever to the beta hemolytic streptococci, no causative role for the production of experimental or clinical rheumatic lesions by these organisms or any of their noncellular products has been clearly demonstrated. Tissue culture affords an excellent technic for the study of living cells and their reactions to exogenous substances. Nephrotoxic sub- stances in the sera of patients with glomeru- lonephritis have been demonstrated by an impairment of secretion of the embryo chick mesonephros. 1 Immunologic testing for anti- tissue antigens 2 and antibodies' has been re- ported. The fibroblastic outgrowth of the chick embryo heart was employed in this From the Research Department of Miami Heart Institute and the Medical Research Foundation of Dade County, Miami, Fla. This investigation supported in part by a research grant from the Miami Heart Association. Received for publication Sept. 24, 1953. study as test material for the observation of specific cellular derangement. Changes were observed in the fibroblasts when they were exposed to two apparently different substances, bacterial filtrates and sera of patients with active or inactive rheumatic heart disease. METHODS AND MATERIAL Bacteriology. (1) Six strains of streptococci obtained through the courtesy of Dr. Murray Streitfeld of the Bacteriology Department of the National Children's Cardiac Hospital were used. Two of the strains were classified by the USPH as streptococcus group A, type 37, USPH strain SS 53 and group A (no reaction with many typing sera); the remaining three strains isolated from children with rheumatic fever were group A, type 6, group B and an ungroupecl and untyped strepto- coccus. All were beta hemolytic. The sixth was a nonhemolytic streptococcus isolated from a stool culture. (2) Filtrates from eight unrelated bacteria prepared in the same fashion were also studied. These included filtrates of Aerobacter aerogenes, Escherichia coli, Pseudomonas aervginosa, Sal- monella lyphosa, an unknown species of bacillus isolated from a blood plate as contaminant, Slaphylo- coccus aureus beta hemolytic and Proteus mirabilis. (3) In preparing the filtrates, the inoculum was made into a standard medium consisting of: 1.5 ml. 0.2 phosphate buffer pH, 1.5 ml. 0.85 per cent saline, 1.0 ml. 1 per cent glutathione, 1.0 ml. 1 per cent sodium pyruvate, 5.0 ml. normal human serum. A trypticase, yeast extract type of filtrate, was used in preparation of the cholera filtrate. 3 (4) Filtrates were prepared after 10 ml. of the bacterial medium with its inoculum had been Circulation Research, Volume II, January 1954 by guest on July 15, 2018 http://circres.ahajournals.org/ Downloaded from
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A Tissue Culture Study of the ChickFibroblast in Relation to StreptococcalFiltrates and Rheumatic Heart Disease
By ROBERT J. BOUCEK, M.D., GLADYS CAMERON, M.A., VINCENT R. SAURINO, P H . D . , AND
ROBERT CHAMBERS, P H . D .
Marked degeneration and disintegration of the chick embryo fibroblast was observed in tissuecultures after their exposure to sera of patients with active or inactive rheumatic heart disease orto the filtrates of beta hemolytic streptococci. Some tissue specificity was demonstrated; sera ofthe patients or the filtrates of the streptococci failed to affect contractility of the myocardial cellsor secretion of the mesonephros. Sera of normal control humans or of patients with other so-calledcollagen diseases did not seriously affect the fibroblasts. Filtrates of nine other bacteria failed toalter the morphology of the fibroblasts unless severe toxicity was present and the tissue culturedied.
THIS is a report on the response of chickembryo fibroblasts in tissue culture tofiltrates of the beta hemolytic strepto-
cocci and to sera of patients with a rheumaticprocess. It was recognized that any bacterialfiltrate contains many toxic antigenic sub-stances and likewise that sera of patients witha rheumatic diathesis contain factors whichmight alter cells in tissue culture. Althoughprevious investigation has directly or in-directly related rheumatic fever to the betahemolytic streptococci, no causative role forthe production of experimental or clinicalrheumatic lesions by these organisms or anyof their noncellular products has been clearlydemonstrated.
Tissue culture affords an excellent technicfor the study of living cells and their reactionsto exogenous substances. Nephrotoxic sub-stances in the sera of patients with glomeru-lonephritis have been demonstrated by animpairment of secretion of the embryo chickmesonephros.1 Immunologic testing for anti-tissue antigens2 and antibodies' has been re-ported. The fibroblastic outgrowth of thechick embryo heart was employed in this
From the Research Department of Miami HeartInstitute and the Medical Research Foundation ofDade County, Miami, Fla.
This investigation supported in part by a researchgrant from the Miami Heart Association.
Received for publication Sept. 24, 1953.
study as test material for the observation ofspecific cellular derangement. Changes wereobserved in the fibroblasts when they wereexposed to two apparently different substances,bacterial filtrates and sera of patients withactive or inactive rheumatic heart disease.
METHODS AND MATERIAL
Bacteriology. (1) Six strains of streptococciobtained through the courtesy of Dr. MurrayStreitfeld of the Bacteriology Department of theNational Children's Cardiac Hospital were used.Two of the strains were classified by the USPHas streptococcus group A, type 37, USPH strainSS 53 and group A (no reaction with many typingsera); the remaining three strains isolated fromchildren with rheumatic fever were group A, type6, group B and an ungroupecl and untyped strepto-coccus. All were beta hemolytic. The sixth was anonhemolytic streptococcus isolated from a stoolculture.
(2) Filtrates from eight unrelated bacteriaprepared in the same fashion were also studied.These included filtrates of Aerobacter aerogenes,Escherichia coli, Pseudomonas aervginosa, Sal-monella lyphosa, an unknown species of bacillusisolated from a blood plate as contaminant, Slaphylo-coccus aureus beta hemolytic and Proteus mirabilis.
(3) In preparing the filtrates, the inoculum wasmade into a standard medium consisting of: 1.5ml. 0.2 phosphate buffer pH, 1.5 ml. 0.85 per centsaline, 1.0 ml. 1 per cent glutathione, 1.0 ml. 1per cent sodium pyruvate, 5.0 ml. normal humanserum. A trypticase, yeast extract type of filtrate,was used in preparation of the cholera filtrate.3
(4) Filtrates were prepared after 10 ml. of thebacterial medium with its inoculum had been
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TARI,E 1.—Effect of Bacterial Filtrates on Chick Heart Explants in Tissue Culture (Living) [72 Hours)
Streptococcus B hemolytic(1) Streptococcus Group A—Type 37.. .(2) Streptococcus Group A(3) Streptococcus Group B(4) Streptococcus Group A—Type 6 . . . .(5) Streptococcus
In this and the other tables the conventions used are as follows: -f-response; ± = questionable response.
degree of positive response; —
±
= negative
incubated at 37 C. for 24 hours, centrifuged for15 minutes at 2,000 rpm and the supernatantsfiltered first through "F" grade fritted Corningglass filters and then through sterile "UP" gradeghiss filters. The cholera filtrate was filtered throughtwo Ertell pads and concentrated so that it repre-sented 10 times that of the original filtrate. Allfiltrates were maintained at 5 C. and kept at aneutral pH.
Preparation of Sera. Human sera were obtain eunder sterile conditions from normal male andfemale individuals, from random sampling ofpatients with rheumatic heart disease, active orinactive, and from patients with other forms ofso-called collagen diseases. No anticoagulants wereadded to the blood and, upon retraction of the clot,the expressed sera were collected. All the filtratesand the patients' sera were kept and used reepatedlyin the experiments.
Preparation of Tissue Cultures. The doublecoverslip method as described by Cameron6 wasuniformly used. Hearts of 7 to 9 day chick embryoswere cut into 12 to 14 fragments and two fragmentsplaced on each coverslip in 2 drops of medium.The experiments were made in duplicate so thatat least four explants were used per experiment.The control medium consisted of 2 drops of fowlplasma, 1 drop of Tyrode solution, 1 drop of 10per cent chick embryo extract and L drop of normalhuman serum. In the experimental preparationsthe drop of normal serum was replaced by a dropof bacterial filtrate or of the patient's serum. Totest the tissue specificity, cultures of chick embryomesonephros containing phenol red were made.
The cultures were examined at intervals during72 hours for the quality of contraction of the
myocardial cells, the condition of the hbroblustsgrowing from the explant and the secretory activityof the mesonephros. Original nitrates and patients'sera were kept and the experiments repeated.
Representative cultures were fixed in 5 percent formalin solution and stained with hema-toxylin and eosin for further study.
RESULTS
With the exception of two bacterial filtratesthe various filtrates and sera did not seriouslyaffect the viability of the cultures.
(A) Bacterial Filtrates in the Tissue Cultures.Filtrates of the hemolytic streptococci isolatedfrom the throats of patients with rheumaticfever or typed as group A hemolytic all gave auniform response. The fibroblasts becamevacuolated, sometimes cytolysed, and the out-growth was not luxuriant. The secretoryactivity of the mesonephros and the contractionof the myocardial cells did not appear to beaffected (table 1). In the control cultures,the growth of the fibroblasts (fig. 1.4) wasorderly and normal, and mitosis was observed.No chromatin debris was seen. In experimentalcultures with the filtrates of the beta hemolyticstreptococci, sparse outgrowth and a markeddisorganization of the fibroblasts were noted(fig. 1JS). Most of the mitotic figures becameabnormal; round, monocytic types of cellswith eccentric hyperchromatic nuclei appeared
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• *FIG. 1. (A) Fibroblasts grown in a normal medium; fixed, H. and 1']. stained tissue culture. Note
mitotic figure in upper and lower portion of picture and orderliness of the cytoplasm. (X133) (6)Fibroblasts treated with filtrate of beta hemolytic streptococci; fixed and stained. Note vacuolation,chromatic dust, in the lower portion of picture and the bizarre mitotic figure in the center of the figure.(X133)
FIG. 2. Appearance of fibroblasts after exposure tofiltrates of beta hemolytic streptococci or sera ofrheumatic patients. Note small hyperchromic nucleiwith eccentric nuclcoli, disorderly character of thecytoplasm and naked nuclei of fibroblasts. (X133)
FIG. 3. Effect; of filtrate of beta hemolytic strepto-cocci or sera of rheumatic patients on the cytoplasmof the fibroblasts: Note chromatic dust. (X133)
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TABLE 2.—Effect of Filtrates and Sera on the Cytologyof Fibroblasls in Tissue Culture, Fixed and Stained
H. and E.
Bacterial Filtrates(1) Filtrate with-
out inoculum. ...(2) Group A—
Type 6(3) Nonhemolytic.
strep
Sera—Human(1) Normal(2) Active rheu-
matic(3) Inactive rheu-
matic(4) Hyperergy. . . .
Vacuo-lization
+ +
±
+ +
+ +
Chro-maticDust
+ +
+ +
+ +
Con-tractedDegen-eratedCells
+ +
+ +
+ +
NuclearChanges
+ +
+ +
+ +
Ab-normalMitosis
+ +
+ +
+ +
TABLE 3.—Effect of Human Sera on Chick HeartEx-plants in Tissue Culture (Living) (72 Hours)
(!) Normal serum 11. A.:C. C.:*
(2) Rheumatoid arthritisM. G. and D. M.*
[3) GlomeiulonephiitisM. S. and G. P.*
(4) Lupus ci'ythematosisII. II.*
(5) Hyperergy response se-rum sickness, M. N.*
(6) Rlieumatic heart dis-ease
(u) Active A. F. :M. C : G. P.*
(b) Inactive E. B.:G. R.: R. P.: E. D.:M. H.: H. S.*
(c) Inactive T. R.*
Via-bility
+
+
+
+
4-
+
Myo-cardial
Con-traction
+
-|--f
±
+
-f-
+
Out-growth
ofFibro-blasts
+
±
+
±
+
±
±
+
Degen-eration
fot
Fibro-blasts
_
—
-
+ + +
+ + +
—* Initials of patients studied.
(fig. 2) and cystic nuclear areas could be seen.The cytoplasm became markedly vacuolated(fig. IB). Chromatic dust and spindly, pyknoticand, at times, naked nuclei were observed(figs. 2 and 3). The bacterial filtrates weretested in dilutions of 1:2, 1:3 and 1:4. A more
profound degenerative reaction was producedwith the greater than with the lower dilutions.
Other bacteria including the nonhemolyticstreptococci failed to demonstrate any strikingfibroblastic disorganization (table 1). Some ofthe filtrates, such as that of Pseudomonasaeroginosa, were universally toxic. The filtrateof nonhemolytic streptococci failed to produceany histocytologic changes comparable to theabove (table 2).
(B) Human Sera in the Tissue Cultures.Addition of normal sera from control subjectsfailed to produce any demonstrable effect onthe explants or on the outgrowing fibroblasts.The sera of patients with diseases other thanrheumatic fever failed to produce the fibro-blastic change (table 3). The contracting myo-cardium was not influenced by the sera of anypatients.
The cytologic changes in cultures containingthe sera of rheumatic patients (table 2) wereidentical in every way with those seen incultures with filtrates of the beta hcmolyticstreptococci. Rounded monocyte-like cells witheccentric hyperchromic nuclei were present. Inaddition, abnormal mitoses, spindle-shaped andnaked nuclei, pyknotic nuclear remnants,vacuolated cytoplasm and nuclei and chromatindust (figs. 2 and 3) were observed. The serumof one patient (T. R. table 3), with a mitralmurmur but without a history of rheumaticfever produced no granularity or vacuolationof the fibroblasts.
DISCUSSION
The demonstration of identical changes infibroblasts in tissue culture exposed to sera ofpatients with rheumatic heart disease andfiltrates of beta hemolytic streptococci confirmsan inter-relationship which previously had beenonly suspected. However, the similarity of thefibroblastic response must be interpretedcautiously. It does not necessarily mean thatthe responsible agent in both test substances isthe same.
The reaction of the fibroblasts to thefiltrates of the beta hemolytic streptococcivaried inversely with the dilution of thefiltrates. A more profound response wasobserved in a 1:4 than with a 1:2 or a 1:3
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dilution of the filtrate. Such a phenomenonmay be similar to the "paradoxic zone phe-nomenon" of Eagle5 which was so designatedbecause stronger concentrations of penicillindid not necessarily produce a reduction ofbacterial activity. But he failed to demonstratethis reaction when he used the beta hemolyticstreptococci.6 Another explanation for theparadoxic reaction of the 1:2 dilution of thebeta hemolytic streptococci filtrates in tissueculture might be the so-called zones of minimalequivalents of an antigen-antibody combina-tion. More work using tissue culture technicsin conjunction with biochemical analyses willclarify this phase of the problem.
In the bacterial nitrates, the substancereacting on the fibroblast is presumably anexotoxin, perhaps streptolysin 0. Our frag-mentary and, as yet, inconclusive data suggestthat cholesterol added to the filtrates exerted a"protective" action, resulting in a less severefibroblastic derangement. This reminds one ofthe protective action of cholesterol, noted byHewitt and Todd,6 against the lethal effect ofstreptolysin 0 in mice.
If the h'broblastic-reacting substance in thefiltrate of the beta hemolytic streptococci isstreptolysin 0 or any of the other exotoxins, itis unlikely that this would persist in the seraof patients long after cessation of the activedisease. It must be remembered that thefibroblastic degeneration occurred wheneverthe sera of patients with inactive as well asactive rheumatic heart disease were studied.Some of the patients with inactive rheumaticheart disease had had the last rheumaticrecrudescense many years before. From thevarious sera-antibody studies, gamma globulindeterminations, C-reactive protein or others,no prolonged reactions have been noted.7
The in vitro fibroblastic specificity wasfurther demonstrated by the failure of the seraof the patients with rheumatic heart diseaseto inhibit the secretory activity of the chickmesonephros in tissue culture or the contractionof the myocardium. Such a specificity of tissueantibodies for selectively affecting the cellulargrowth and function of a particular type ofcell has been previously observed.1 Theproduction of iso-antibodies and their role in
the pathogenesis of disease has been previouslyadvanced. Acute hemolytic anemias andthrombocytopenic purpuras are thought bymany to be examples of the effect of auto-tissue antibody formation with resultantdestruction of the cell.8' 9 It is possible that asimilar phenomenon occurs in patients withrheumatic heart disease.
Further critical investigation is necessarybefore a coherent theory on the pathogenesisof rheumatic fever can be safely advanced.The tissue culture technic offers a promisingapproach not only to the problem of patho-genesis but also to diagnosis and therapy ofrheumatic fever.
SUMMARY AND CONCLUSIONS
1. A selective fibroblastic derangement wasdemonstrated in tissue cultures of chickembryo heart after exposure to filtrates of betahemolytic streptococci.
2. Filtrates of nine bacteria other than thebeta hemolytic streptococci failed to produce aselective fibroblastic derangement.
3. Identical fibroblastic derangement wasnoted following exposure of similar cultures tothe sera of patients with active and inactiverheumatic heart disease.
4. Neither the filtrates of streptococci norsera from patients with rheumatic heartdisease affected the contraction of the myo-cardium or the secretory activity of themesonephros.
5. Sera of patients with rheumatoid arthritis,glomerulonephritis, lupus erythematosus and ahyperergy response with joint manifestationsfailed to produce serious fibroblastic derange-ments.
ACKNOWLEDGMENTS
It is with pleasure that the authors acknowledgethe constant encouragement and advice of Dr.George T. Lewis and Dr. E. Sterling Nichol.
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2 LANDSTEINEH, K.: The Specificity of SerologicalReuctions. Cambridge, Mass., Harvard Uni-versity Press, 1947.
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3 GHIITITTS, J. J.: T Agglutination Phenomena inMan and Animals. 2nd Annual Meeting, As-sociation of Blood Banks. Seattle, Wash. Nov.3, 1949.
4 CAMERON, G.: Tissue Culture Technique, ed. 2.
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"HEWITT, L. F., AND TODD, E. W.: The effect ofcholesterol and of sera contaminated withbacteria on the haemolysins produced by hemo-lytic streptococci. J. Path. Bact. 49: 45, 1939.
7MCCARTY, M.: The Immune Response in Rheu-matic Fever. Rheumatic Fever Symposium.
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8DAMESHEK, W., AND BLOOM, M. L.: The Eventsin the Hemolytic Crisis of Hereditary Sphero-cytosis with Particular Reference to the Reticu-locytopenia, Pancytopenia and an AbnormalSplenic Mechanism. In Dameshek, W., andTaylor, F. H. L.: George R. Minot Symposiumof Hematology, New York, Grune & Stratton,1949. Pp. 160-1S9.
a EVANS, R. S., TAKAHASI, K , DUANE, R. T.,PAYNE, R., AND LIU, CHI-KONG: Primarythrombocytopenic purpura and acquired hemo-lytic anemia. Arch. Int. Med. 87: 48, 1951.
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