Streptococcal Antigens

Transcription

1 Cellular Reativity Studies to Streptooal Antigens MIGRATION INHIBITION STUDIES IN PATIENTS WITH STREPTOCOCCAL INFECTIONS AND RHEUMATIC FEVER STANLEY E. READ, VINCENT A. FismrI, VIRGINIA UTERMOHLEN, RuDoLF E. FALK, and JOHN B. ZABRISKE From The Rokefeller University, New York 121, and the Department of Surgery, University of Toronto, Toronto, Ontario 181, Canada A B S T R A C T The question of whether hypersensitivity to streptooal antigens plays a role in the pathogenesis of the nonsuppurative sequelae of streptooal infetions remains at present unlear. As a first step in the approah to this question, the degree of ellular reativity of peripheral blood leuoytes to streptooal antigens was investigated in a number of rheumati fever patients, patients with unompliated streptooal infetions, as well as normal healthy subjets. Using the in vitro tehnique for the inhibition of apillary migration of peripheral blood leuoytes as an index of the degree of sensitivity to streptooal antigens, the results indiate that patients with aute rheumati fever exhibit an exaggerated ellular reativity to these antigens and in partiular to streptooal ell membrane antigens. This abnormal response to streptooal membrane antigens appears to persist in rheumati subjets for at least 5 yr after the initial 'attak of rheumati fever. Only Group A streptooal membrane antigens eliited this unusual response in rheumati subjets, sine the ellular reativity to Group C and D streptooal membranes was the same in all groups. Patients with evidene of valvular disease exhibited the same degree of ellular reativity to these antigens as did patients without linial evidene of rheumati heart disease. The nature of the antigens responsible for the observed ellular response remains unknown. Enzymati treatment of streptooal ell walls and membranes designed to remove type-speifi M proteins did not alter the observed ellular reativity to the streptooal antigens. The finding that an abnormal ellular response to ertain streptooal antigens is present only in rheu- Reeived for publiation 17 July 1972 and in revised form 14 January mati patients suggests that ell-mediated fators may play an important role in the disease proess. INTRODUCTICN The question of whether hypersensitivity to hemolyti streptooi and their produts might play a role in the pathogenesis of the nonsuppurative sequelae of streptooal infetions has been the subjet of investigation for many years. Starting with the early work of Swift and Derik and Derik, Hithok, and Swift in animals (1-3), it was apparent that delayed hypersensitivity to streptooi behaved in a manner similar to that observed for tuberulin sensitivity (4). Initiation of sensitivity by prolonged foal ontat between the intat bateria and tissues of the host, inability to passively transfer streptooal-delayed hypersensitivity with serum alone, and the lak of orrelation between irulating antibodies to streptooal produts and delayed allergy were all harateristis of the streptooal hypersensitive state (5). Apropos of these studies, several intriguing observations onerning the streptooal hypersensitive state were made. First, repeated small inoulations of heat-killed streptooi were more effetive in induing the hypersensitive state than a single inoulation of living organisms (3). Seondly, extrats of streptooal ellular strutures were more effetive in eliiting the hypersensitive reation than streptooal extraellular produts. Thirdly, MWen learly desribed speifi ytotoxi and ellular inhibition effets by streptooal antigens on explanted host ells from rheumati patients in tissue ulture, as opposed to ell ultures from normal ontrols. It is interesting that these observations were made at least 2 yr before the emergene of modern in vitro tehniques of ellular immunology and ytotoxiity (6). The Journal of Clinial Investigation Volume 54 August 1974*

2 Experiments similar to those desribed in animals were also arried out in man by several investigators (7-9). In using skin tests as an index of delayed sensitivity to streptooal produts, the general onsensus of the workers was that hypersensitivity to streptooi and their produts was a ommon ourrene in man and inreased in intensity, depending on the age of the individual tested. In general, these reations were more intense in rheumati subjets than in nonrheumati ontrols (7, 8), the reativity was partiularly intense with extrats of hemolyti streptooi as ompared to nonhemolyti streptooal strains (8), and the greatest number of positive reations were obtained with autogenous streptooi, suggesting some type speifiity to the reation (1). In this onnetion, Beahey, Alberti, and Stollerman (11) and Pahman and Fox (12) have reently observed that immunization of guinea pigs with partially purified preparations of different M proteins resulted in speifi delayed hypersensitivity (skin tests and marophage inhibition tests) to the immunizing antigen. However, ross-reations to other type-speifi M proteins were seen, although the reations were always onsiderably less than those observed with the type-speifi protein used for immunization. Finally, the ellular nature of this streptooal sensitivity reation was learly delineated by Lawrene (5) when he demonstrated that delayed hypersensitivity in man ould be transferred to a streptooal skin testnegative individual via extrats of peripheral blood white ells obtained from a streptooal skin test-positive individual. While these studies were primarily onerned with the kinetis of the indution of the delayed hypersensitivity state to streptooi and their produts, other observations lent support to the onept that streptooal hypersensitivity played a role in the nonsuppurative sequelae of streptooal infetions. In studies on experimental models of rheumati fever in animals, a number of investigators (13, 14) have emphasized the need for repeated losely spaed streptooal inoulations with a marked utaneous reativity to streptooal materials as being a prime prerequisite in induing the pathologial lesions whih simulated rheumati lesions found in man. Rantz and others (15-18) have also suggested that, in man, repeated streptooal infetions are important for the disease proess, and they ite the rarity of rheumati fever before 3-4 yr of age as evidene for the neessity of an aquired hypersensitivity to streptooal materials before rheumati fever ours. Taken together, these studies strongly suggest that hypersensitivity to streptooal antigens may play a role in the initiation of events leading to the disease rheumati fever. The suess in deteting delayed hypersensitivity to a variety of partiulate antigens by using the tehnique 44 Read, Fishetti, Utermohlen, Falk, and Zabriskie of in vitro ellular migration of peripheral white blood ells (19, 2) prompted an investigation into the reativity of rheumati and nonrheumati individuals to various streptooal antigens. The following report indiates that patients with aute rheumati fever are highly reative to streptooal ellular strutures, in partiular to the streptooal membrane, and they maintain this hyper-reative state for at least 5 yr after the initial attak. METHODS Patients. The majority of the rheumati patients ame from the Rheumati Fever Servie of The Rokefeller University Hospital. Some of the aute rheumatis were from other New York City hospitals. Normal ontrols, as well as patients with unompliated streptooal infetions, ame from this same urban population and were mathed for age and sex. Streptooal strains. All strains used in this study were obtained from The Rokefeller University olletion and were kindly provided by Dr. Rebea C. Lanefield. Isolation of ellular strutures. Lyophilized ultures of Group A streptooal strains S43/197 (Type 6) and A964 (Type 5) were transferred to Todd-Hewitt broth ontaining 5% rabbit blood. After 18 h of inubation at 37C,.5 ml of the supernate of the broth ulture was transferred to 35 ml of dialysate medium prepared as previously desribed (21) and inubated another 18 h at 37C. 1 ml of this ulture was transferred to 2 liters of dialysate medium, and after 18 h of inubation, the ontents of the 2-liter flask were inoulated into 2-liter bathes of dialysate medium and inubated 18 h. Using high speed Sharples entrifugation of the 2-liter ulture (Sharples-Stokes Div., Penwalt Corp., Warminster, Pa.), the ells were isolated from the broth and washed twie in isotoni saline, ph 6.. These ells were then divided into two portions. One portion was resuspended in distilled water, the ells disrupted in a Braun disintegrator (Bio, In., Burbank, Calif.), and the ell walls isolated by methods previously desribed (22). A small aliquot of this ell wall preparation was washed in distilled water, lyophilized, and the ontents weighed to determine the dry weight of isolated ell walls in the preparation. Knowing the dry weight of the ell walls, the remaining ell wall pellet was diluted in.5 M phosphate-buffered saline, ph 7.5, to a onentration of 1 mg/ml and stored in 1-ml aliquots at -7C until use. In this manner, thawing and refreezing of the ell wall preparation ourred only one, and only one-thawed preparations of ell walls were used for eah experiment. The remaining portion of streptooal ells was resuspended in.5 M phosphate buffer, ph 6.1, ontaining 4% NaCl and.5 M EDTA with ativated phage-assoiated lysin (13). The preparation of streptooal protoplasts and isolation of the streptooal membranes from this mixture was as previously desribed (21). The proedure for the storage of aliquots of these membranes was the same as that desribed above for the isolated streptooal ell wall material. Isolation of ell walls and membranes of Group C streptooal strain C74 was ahieved by the methods desribed above. Sine the phage-assoiated lysin does not attak Group D streptooal strains, ell walls and membranes of strains from this serogroup (Group D) were isolated by differential entrifugation after mehanial disruption of the streptooal ells in a Braun disintegrator (22).

3 Enzymati treatment of isolated streptooal ellular strutures. Trypsin experiments. Streptooal ell walls or ell membranes at a onentration of 1 mg/ml were suspended in 1 ml of.1 M Tris buffer., ph 8., to whih rystalline trypsin (Worthington Biohemials Corp., Freehold, N. J.) was added in a final onentration of 1 jug/ml. This mixture was plaed in Visking no. 24 dialysis tubing (Visking Corp., Chiago, Ill.) and the ontents dialyzed against 5 ml of.1 M Tris buffer, ph 8., for 21 h at 37C with gentle agitation. The dialyzing buffer had been prewarmed to 37C before insertion of the dialysis sa Ȧfter this enzymati digestion, the ontents of the dialysis sa were entrifuged at 2 g for 2 min, washed twie in.5 M phosphate-buffered saline, ph 7.5, then washed twie in distilled water. An aliquot was removed, lyophilized, and weighed to determine the dry weight of the streptooal ell wall and membrane preparations. The suspensions were reentrifuged and appropriately diluted to ontain 1 mg/ml in eah tube. All tubes were refrozen at -7'C until use. Preparation of tissue ulture medium. Stok solutions of tissue ulture medium (Flow Laboratories, In., Rokville, Md.) were prepared as follows: under sterile onditions, 5 ml of Earle's balaned salt solution (atalogue no. 312, 1 X onn), 1 ml of minimal essential medium amino aids (atalogue no , 5 X onn), and 5 ml of minimal essential medium vitamins (atalogue no , 1 X onn) were mixed together, plaed in sterile srew ap glass bottles, labeled "solution A", and kept at 4 C until use. For daily onsumption, 13 ml of solution A was mixed with 3 ml of sterile 8% NaHCO3, 1 ml of L-glutamine (atalogue no , 1 X onn), and suffiient sterile distilled water to make 1 liter. To this mixture, labeled "solution B", 1, U of peniillin, (Eli Lilly and Co., Indianapolis, Ind.), 1, jug of streptomyin (Eli Lilly and Co.), and 2, U of myostatin (E. R. Squibb and Sons, Prineton, N. J.) were added. This solution was stored at 4VC until use. Aliquots of solution B were mixed with either 2% fetal alf serum obtained from Flow Laboratories (Flow Laboratories, Rokville, Md., atalogue no. 455) or 1% normal AB human plasma. To prevent repeated freezing and thawing of stok serum solutions, these sera were prepared as follows: 5 ml of frozen fetal alf serum was thawed, heat inativated for 3 min at 56C, distributed in 2-ml aliquots, and refrozen at -2 C until use. Normal human AB plasma was separated from the blood ells by entrifugation, inativated at 56C for 3 min, and stored at -2'C in similar aliquots. On the day of use, aliquots of serum or plasma were removed, thawed, and mixed with medium to make the appropriate solutions. An additional 1 U/ml of peniillin were added to the omplete medium just before use. While solution A was kept for long periods at 4C, it was felt advisable not to store stok solutions of B more than 1 days at 4C. Isolation of peripheral white blood ells. 5 ml of venous blood was drawn from the median ephali vein of the antiubital fossa of eah patient into an evauated rubberstoppered bottle ontaining approximately 1-15 glass beads. The bottle was gently shaken for 1 min and the defibrinated blood was then poured into a graduated glass ylinder. An equal volume of a 2% Knox gelatin solution in Ringer's latate was added to the volume of blood and the mixture allowed to sediment for 2 min at 37C. The supernate, ontaining primarily leuoytes, was removed, plaed in either plasti (Falon Plastis, Los Angeles, Calif.) or glass tubes, and spun at 1,5 rpm for 1 min. The elltree supernate was removed and the ells from eah tube pooled and resuspended in 1 ml of.83%o ammonium hloride for approximately 5 min to lyse the ontaminating red ells. The ells were again spun at 1,5 rpm, the fluid removed, and the ells washed twie in Ringer's latate solution. After the final wash, the ells were resuspended in 1 ml of omplete medium (see setion on preparation of tissue ulture medium). The ells were ounted in a hemoytometer (Hausser and Son, Ae Sientifi Supply Co., In., Linden, N. J.). The ell suspension was again spun at 1,5 rpm and the ell pellet adjusted to 2 million lymphoytes/ml. Nonheparinized apillary tubes (Arthur H. Thomas Co., Philadelphia, Pa.) were filled with aliquots from this ell suspension and sealed with Seal-Ease lay (Clay Adams, Div., Beton, Dikinson and Co., Parsippany, N. J.). The apillary tubes were plaed in small plasti tubes and spun at 1 g for 6-8 min. The apillaries were removed and ut at the ell-fluid interfae. The portion of the apillary ontaining the ell pellet was then plaed in a small irular 2 X 2-mm planhette (Univers Mekaniska Verkstad AB Herrhagsv, 98 Endkede, Sweden) whih was then filled with.5 ml of the tissue ulture medium and sealed with a over slip. Antigens in varying onentrations were mixed in the ulture medium. Tripliate sets of planhettes were used for eah antigen onentration and the appropriate ontrols. Migration of ells from the apillary tubes was allowed to proeed for 18 h at 37C in an inubator ontaining 5% C2. A fan of migrating ells usually appeared within 2-4 h after inubation and was omplete within h. The migrating fan of ells in eah planhette was then projeted on drawing paper in one of two different ways. In the first method, a Petri dish ontaining the planhettes was plaed in a standard photographi enlarger speially adapted to ontain the Petri dish. The light soure of the enlarger was dereased suffiiently to permit easy projetion of the fan on drawing paper. The seond method made use of a Baush and Lomb mirosope (Baush and Lomb In., Rohester, N. Y.) with a projeting attahment by whih the migrating fan was magnified 1 times and projeted on drawing paper. Irrespetive of the method used for projetion of the image, these fans were then traed on paper, the traings were ut out and were weighed on a balane sale. The index of migration was expressed as the following formula: weights of areas of migration with antigen weights of areas of migration without antigen X 1 = migration index. The degree of inhibition was alulated by subtrating the migration index from 1. Statistial analysis. Statistial analysis of the data was alulated by using the Student's t test as desribed in the Handbook of Chemistry and Physis. The degrees of freedom were alulated from the same book (24). Analytial methods. Rhamnose was determined by the method of Dishe and Shettles (25). Quantitative gluosamine determinations were done by a modifiation of the Rondle and Morgan proedure (26). Quantitative gluose analyses were done by a modified method employing gluose oxidase (Gluostat) available from the Worthington Biohemial Corporation. Ribonulei aid was determined by the orinol reation. Total nulei aid ontent and protein onentrations were determined by the absorption of solutions at 26 and 28 nm, respetively, in the Bekman UV Cellular Reativity to Streptooal Antigens in Rheumati Subjets 441

4 E 3 E ~ - was greater in rheumati patients as ompared with nonrheumati individuals, the P value of.5 for inhibition with 1 ig of ell walls is borderline. The P value of.2 for 1 *g of ell walls is aeptable and denotes a signifiant differene in the degree of reativity between the two groups. While not shown, the introdution of.1 ug of ell walls resulted in no differene between the two groups. This suggests that the antigen responsible for the inhibition is present in lower onentration in the ell wall. In ontrast, reativity to streptooal ell membranes at either 1. Ag or.1 Ag was signifiantly higher in rheumati patients when ompared to nonrheumati patients (P <.1). Leuoyte reativity in patients with aute rheumati fever. The observations that patients reently reovered from a rheumati attak had the highest leuoyte inhibition values prompted an investigation of the ellular response to streptooal antigens in a small number of patients with aute rheumati fever (within the first 2 wk of their disease). A similar group of patients with diagnosed streptooal pharyngitis were inluded as ontrols. These patients were mathes for age and sex and were from a similar urban population. All of them had elevated ASO titers, with an average of 6. Clinial signs of pharyngitis, an elevated ASO titer, and the presene of Group A streptooi isolated from throat swabs were the riteria for an unompliated streptooal infetion. The linial and laboratory diagnosis for rheumati fever was based on the aepted riteria (29) for the disease proess. The rheumati fever pa- '~ L Cell membranes 1./ig O. IH /Ig S ; - -. At 89 Add * o ; 8 i ~~~~~ * ~~~~ No.of subjets %/ Meon inhibition o/i 19 t. se Cell walls l.htg SE mean ±41 ± ±4. ±2.2 ±3.7 ±2.9 o Normals *- Rheumatis FIGURE 1 Leuoyte migration inhibition values obtained with leuoytes from rheumati and nonrheumati individuals after exposure to Group A streptooal ell walls and membranes. The differene in inhibition to streptooal membranes in rheumati vs. normal subjets was highly signifiant: P = <.1. spetrophotometer (Bekman Instruments, In., Fullerton, Calif.). Amino aid analysis of the membrane preparation revealed that there was approximately 3-5% N-aetyl gluosamine and 3-5% murami aid present. The determinations of these hexosamines was based on a modifiation of a previously desribed method (27). Serologial identifiation. The serologial identifiation of the strains used in these experiments was onfirmed with the apillary tube preipitin test using streptooal group and type-speifi rabbit antisera (28). RESULTS Cellular reativity to streptooal antigens in rheumati patients ompared to nonrheumati individuals To examine the possibility that rheumati individuals have an inreased reativity to streptooal antigens, a large group of well-doumented inative rheumati fever patients were ompared to nonrheumati individuals mathed for age and sex. The mean antistreptolysin o (ASO)1 titers were essentially the same for eah group. Migrations were done with at least two onentrations of streptooal ellular antigens in an effort to determine whether antigen onentration was a signifiant fator in the ellular reativity. Fig. 1 demonstrates that, using streptooal ell wall material as antigen, both rheumati individuals and normal ontrols were inhibited to some degree by either 1 or 1 lg onentrations of the ell wall preparation. While the degree of inhibition at both onentrations of ell wall antigens 'Abbreviation used in this paper: ASO, antistreptolysin O. 442 Read, Fishetti, Utermohlen, Falk, and Zabriskie

5 Cell membranes l.o~lg. 1,~Lg 8d Cell walls IOIL l.oelg -- SOr 1% 'CJ ~~~~~~~~ ~~~ %Mean inhibition SE mean ±5.6 ±4.1 ±3.5 ±3.6 ±6.9 ±4.9 ±3.7 ±3.7 * = Aute rheumatis = Unompliated streptooal infetions FIGURE 2 Cellular reativity studies with leuoytes obtained from aute rheumati fever patients and unompliated streptooal infetions. There is marked ellular reativity of rheumati leuoytes to streptooal ell walls and partiularly ell membranes. U). Sed. rote ASO titers CRP E H.W. 7 yr Chinese g +4 admitted 7/15/ x r 1 * 6. -& Disharge 9/17/7 x x1 fg ell walls * /ig membranes -C o FI o O[ Prednisone (mg) Aspirin (g) Undiluted :5 1*+ -1: S 6' i ' 9 Days )/ - 1 C - 2 -D oo 3 -C S Patients PIX 2.I..- Heartreative ontibody titers x L. L 4 56 Mo FIGuRE 3 A omparison of the serial migration inhibition studies, heart-reative antibody determinations and therapy in patient H. W. with aute rheumati fever and rheumati arditis. Note the hange in ellular reativity both during and after prednisone and aspirin therapy. 14 Cellular Reativity to Streptooal Antigens in Rheumati Subjets 443

6 en U) L- ' o) -1 Sed. rate ASO titers CRP r 4 2- CL 2+ 2 I+. B.S. 9 yr White Y xilg ell walls admitted 4/2/7 * 1.g membranes Disharged 5/11/7 x _- _- x -- -* F1O!8 ~2 6 None x mo Days Mo FIGURE 4 Serial migration inhibition results and heartreative antibody titers in patient B. S. with aute rheumati fever who had no evidene of arditis and who was treated with bed rest alone. Migration inhibition values rapidly returned to normal in this patient in spite of persistene of heart-reative antibody titers. tients had an average ASO titer of 8. Blood samples were drawn from these patients either at the time of the onset of rheumati fever and before the institution of anti-inflammatory therapy, or during the onvalesent phase (1 days to 2 wk) after the diagnosis of linial pharyngitis. While the number of patients with aute rheumati fever is admittedly small, Fig. 2 demonstrates that patients with aute rheumati fever were highly sensitive to streptooal membranes even at.1 ig of this preparation when ompared to patients with unompliated streptooal infetions. The differene in ellular reativity was highly signifiant (P <.1). In ontrast, there was no differene in the ellular response of both groups when streptooal ell walls were used as the antigen whih suggests that the leuoytes of all individuals reat equally to omponents of the streptooal 444 Read, Fishetti, Utermohlen, Falk, and Zabriskie ell walls. Although the degree of inhibition with streptooal ell membranes (4%) in rheumati subjets appears to be signifiantly higher than the inhibition values obtained with streptooal ell walls (28%), statistial analysis of the data failed to reveal any signifiant differene between the two antigens (P =.1). Repeated determinations of the degree of leuoyte reativity to streptooal antigens were arried out in two patients during and after aute illness in order to (a) ompare the level of heart-reative antibodies (3) with the migration inhibition values, and (b) observe the effet of the treatment shedule on these two parameters of host response to the streptoous. Fig. 3 summarizes the results obtained with patient H. W. On admission, the patient had mitral insuffiieny with an elevated sedimentation rate of 85 mm/h, C-reative protein 4+, and ASO titer of 2,. The heart-reative antibody titers were elevated. In addition, the migration inhibition values were between 5 and 6%, whih shows a high degree of ellular sensitivity to streptooal antigens. After 3 days of prednisone therapy, the usual laboratory parameters of an inflammatory proess had returned to normal as had the migration inhibition values. During a follow-up period of 14 mo, in the absene of any treatment, the migration inhibition values gradually showed more ellular streptooal reativity but never to the degree observed during the aute attak. By 14 mo, the heart-reative antibody values and other laboratory tests were apparently within normal limits, but ellular reativity to streptooal ell walls and membranes still persisted. The seond ase, B. S. (Fig. 4), illustrates a milder ase of aute rheumati fever, with arthritis and fever as presenting symptoms, in whih the only treatment was bed rest. On admission, the sedimentation rate was 6 mm/h, C-reative protein was 2+, and ASO titer was 3,, but there was no evidene of arditis either by ausultation or repeated eletroardiogram. Although the migration inhibition test initially showed inreased ellular reativity to streptooal ell walls and membranes, these values rapidly returned to normal within 6 mo of the aute attak. This ourred at a time when elevated heart-reative antibody titers were still present in the serum of this patient. Persistene of ellular reativity to streptooal antigens in rheumati fever patients Sine an analysis of leuoyte inhibition values in aute rheumati fever patients indiated a signifiant degree of reativity to streptooal membranes, leuoytes obtained from patients who had their attaks 1-1 yr previously were tested with streptooal antigens in an effort to determine how long this sensitivity to membrane antigens persisted. For the sake of onveniene,

7 Rheumati attak < 5 yr Type 6 streptooal antigens Rheumati attak > 5 yr Type 6 streptooal antigens - 3 E :93 ~ 4- d5- Patients % Mean inhibition Cell membranes ~ Cell walls Il/Ig S., 8 s I Cell membranes SE mean ± 5.6 ±3.8 ±6. ± 2.9 ±6.8 ±3.8 ± 8.5 ±t 2.9 o:= Normals * = Rheumnatis FIGURE 5 A omparison of the ellular reativity studies to Group A streptooal ell wall and membrane antigens in rheumati fever patients who had their attak either less than 5 yr ago or more than 5 yr ago. Sen-sitivity to streptooal membrane antigens is maintained in rheumati subjets up to 5 yr after an initial attak. * S Cell walls I II 2:2 these patients were divided into two groups; those patients who had their attak within the past 5 yr and those patients whose attak ourred more than 5 yr ago. An examination of the leuoyte inhibition values in Fig. 5 demonstrates that patients who had their attak less than 5 yr ago ontinue to exhibit a heightened reativity to streptooal membranes even at a onentration of 1 iag of membranes. While there appears to be an inreased reativity to ell wall antigens in rheumatis, the wide variation in response to the ell wall preparations in both groups was refleted in the borderline statistial signifiane (P <.5, but >.2). Examination of the ellular reativity of leuoytes obtained from patients who had their attak more than 5 yr before the study (Fig. 5) indiates that abnormal ellular reativity to streptooal antigens is no longer detetable in this group of patients.' While an oasional patient reated strongly to a given onentration of either streptooal membranes or ell walls, the average inhibition values were the same for this group over two different onentrations of either ell walls or membranes. Cellular reativity to streptoooal antigens in rheumati patients with and without valvular disease Reent reports by Goldstein, Rebeyrotte, Parlebas, and Halpern (31, 32) indiate that mammalian strutural glyoproteins (obtained from mammalian heart valves) ontain antigens similar to those isolated from Group A streptooal ell walls. The shared antigeniity appears-to be due to the presene of ommon N-aetyl gluosamine terminal groups in mammalian tissue and the Group A streptooal arbohydrate. This work is further strengthened by the observations of Dudding and Ayoub (33) that the sera of patients who develop rheumati valvular disease ontain high titers of antibodies to the streptooal Group A arbohydrate (of whih the antigeni determinant is N-aetyl gluosamine) and that these antibodies may persist for years after the initial attak. In ontrast, some of the patients with non valvular rheumati fever, as well as patients with rheumati horea, also ontain Group A arbohydrate antibodies, but the titers fall off rapidly after the aute attak. In view of the known ross-reation between the strutural glyoprotein present in valvular tissue and the streptooal group-speifi arbohydrate, it has been suggested that antibodies to either the strutural protein or the streptooal arbohydrate moiety are responsible for the valvular damage. Inative rheumati fever patients with and without valvular disease were ompared (mathed for age, sex, and time from original attak) to determine if there was a seletive reativity to streptooal ell wall strutures as ompared to membrane antigens. Fig. 6 summarizes the results of these studies, and it is lear that there is no signifiant differene in the degree of leuoyte inhibition in either group. Although one would Cellular Reativity to Streptooal Antigens in Rheumati Subjets 445

8 - 3 E 1 D8 3 o 5 Patients % Mean inhibition.se mean Type 6 streptooal antigens Cell membranes 1. g.1 g 1Ig ±13. ± ± ±9.8 *= Patients with volvulor disease i av * : ±7.2 ±5.3 CeJI walls * OaQ ±8.3 ±6.8 O= Patients without volvulor disease FIGURE 6 Migration inhibition responses to streptooal antigens in rheumati subjets who had evidene of rheumati heart disease ompared to patients without evidene of valvular damage. The response to streptooal ell walls and membranes was the same for both groups. not expet to see a differene in reativity to streptooal membranes, the failure to demonstrate any differene with streptooal ell walls was surprising and may be due to the limited range of antigen onentrations used. To date, only humoral antibodies, ross-reative with heart glyoprotein and Group A streptooal arbohydrate, have been demonstrated in patients with valvular disease (33). Enzymati treatment of partiulate streptooal antigens: its effet on leuoyte reativity in rheumati subjets Hypersensitivity studies in both animals and man have stressed the fat that the grqatest degree of ellular or skin reativity to streptooal antigens was observed when the type-speifi strain was used as the test antigen. More reent studies by Beahey et al. (11) and Pahman and Fox (12) have indiated that the typespeifi M protein moiety was responsible for the inreased ellular reativity in immunized guinea pigs. These studies suggested that the heightened leuoyte inhibition response to Group A streptooal antigens might be related to the type-speifi M protein surfae antigen on streptooal ell walls. In addition, previous studies involving enzymati treatment of ell membranes (21) resulted in the release of streptooal antigens whih showed ross-reative determinants with mammalian musle tissue. Aordingly, both streptooal ell walls and membranes were treated with rystalline trypsin (see Methods) for 2 h at 37C. After this inubation, the ell walls and membranes were washed several times in saline and resuspended to the original onentration. Idential preparations of untreated ell walls and membranes were used as ontrols. Preipitin reations or agar immunodiffusion studies with typespeifi antisera failed to reveal the presene of M protein in the trypsin-treated ell walls. However; in spite of the absene of type-speifi surfae proteins on these ell walls, Fig. 7 demonstrates that the ellular reativity to both preparations was essentially equal. In addition, there was no loss of reativity of leuoytes from rheumati patients to trypsin-treated streptooal membrane antigens. Cellular reativity to streptooal antigens from other hemolyti streptooal groups In an effort to determine whether the heightened response to streptooal antigens was peuliar for Group A streptooal antigens, ell walls and membranes from Group C and D streptooi were also prepared. Leuoytes obtained from rheumati individuals and normal individuals were ompared with respet to the degree of ellular reativity to these antigens. Fig. 8 summarizes these studies, and while reativity to Group A strepto- 446 Read, Fishetti, Utermohlen, Falk, and Zabriskie

9 Type 6 streptooal antigens Z 3 E 1 Cell membranes Il./Lq Cell walls 1z9 1 D 3 - S 5 F * * No. of patients 7 7 % Mean inhibition SE mean ±35 ± ±49 ±5. = Trypsin treated preparations Untreated preparations FIGURE 7' A omparison of the ellular migration response in rheumati subjets to both trypsinized and nontrypsinized Group A streptooal ell walls and membranes. Enzymati digestion of both ell walls and membranes made no differene in the ellular response of rheumati subjets to these antigens. oal antigens is heightened in rheumati individuals, ellular reativity to Group C and D streptooal antigens is the same for both groups of patients. Cellular reativity to streptooal antigens in newborn infants A number of reent reports have stressed the fat that baterial antigens (34, 35), and in partiular streptooal antigens, may have mitogeni properties. In view of these findings, the ellular response to streptooal ell walls and membranes was studied in leuoyte populations obtained from newborn infants. Fig. 9 summarizes the leuoyte migration values in 1 newborn infants. It an be seen that the degree of ellular reativity to both Group A and D membranes, as well as Group A ell walls, is within normal limits in these infants. When the degree of inhibition in newborns was ompared to values obtained in normal individuals, no statistial differene between the two groups was observed (P =.1 -.8). The lak of statistial differenes between the two groups probably reflets the relative insensitivity of the test system at low inhibition levels. Of greater importane is the fat that these streptooal antigens did not eliit a nonspeifi mitogeni response in newborn infants. DISCUSSION Many studies have now demonstrated that the inhibition of migration of human peripheral blood leuoytes in the presene of their speifi sensitizing antigen is an aurate in vitro refletion of the state of delayed-type hypersensitivity to that partiular antigen (reviewed in 36). It has been shown that only partiulate antigens give onsistent and aurate results in this diret migration inhibition system (37). Reent studies by Roklin (38) have shown that a speifi fator, leuoyte inhibitory fator, is released by sensitized lymphoytes _ 3 "' 2 2 I9 V)o o 1 2-Do: 2 s 3 Group C membranes I/pg 3T Streptooal antigen 8 8 Patients 8 1 %/Meon inhibition S E mean 4.4 ±2.8 o = Normals * = Rheumatis Group D membranes i ig ±2.5 ±2.1 FIGuRE 8 The lak of heightened ellular response to streptooal Group C and D membranes in rheumati patients and normal ontrol subjets. Cellular Reativity to Streptooal Antigens in Rheumati Subjets 447

10 g 5 I E 3 an C In v 3F 5 p No. of subjets % Mean inhibition SE Mean Group A streptooal Group D streptoool Cell membrdnes Cell membranes H9g O. jig Jig O. I tlg * I FIGURE 9 A omparison of leuoyte migration inhibition to streptooal born infants. Group A streptooal Cell walls l9g i.9g [) - us - e I a S S VI antigens in new- after stimulation by speifi antigen and that this substane inhibits the migration of human polymorphonulear leuoytes. He showed that the leuoyte inhibitory fator was distint from migration inhibitory fator both in moleular weight and in biologial ativity. He also demonstrated again the orrelation between delayedtype skin reativity and diret leuoyte migration inhibition. Capillary migration patterns of peripheral blood leuoytes obtained from patients with rheumati fever and patients with unompliated streptooal infetions were studied in the presene of various streptooal frations. Beause only partiulate antigens work in this system, no soluble streptooal antigens or partially purified wall omponents, suh as M protein, were used diretly. While the number of patients studied to date is admittedly small, the following general onlusions may be made. First, there is a heightened ellular reativity to streptooal membrane antigens in patients with aute rheumati fever, even at a onentration of.1 og/ml. This inreased ellular reativity to membrane strutures persists in rheumati patients up to 5 yr after the initial attak without any linial or laboratory evidene of an interurrent streptooal infetion or rheumati reurrene during this period. Afte 5 yr the majority of rheumati individuals lose this altered sensitivity to membrane strutures and respond in a normal fashion to these antigens. In ontrast, patients with unompliated streptooal infetions with elevated antibodies to streptooal produts, e.g. high ASO titers, reat to membrane antigens in a normal fashion. Both groups exhibit a somewhat heightened ellular reativity to streptooal ell walls. However, this reativity drops off quikly in both groups and leaves a high and persistent sensitivity to membrane strutures in patients with rheumati fever only. Seondly, the speifiity of this ellular reativity for Group A streptooal membranes was evidened by the fat that Group C and D membranes eliited normal responses in both rheumati and nonrheumati individuals. The possibility that streptooal ell surfae proteins might play a predominant role in the observed sensitivity to streptooal ell walls was exluded by the fat that trypsin-treated ell walls (extrats of whih ontained no M protein as determined by the apillary preipitin method) produed the same degree of ellular reativity as did untreated ell walls. Thirdly, antigen-antibody omplexes do not appear to play a predominant role in the heightened ellular response to streptooal antigens. The ells used in these experiments had been washed at least five times before use, and only heterologous alf serum was used in the test system. While humoral antibody does not appear to play a role in the inhibition of leuoyte migration, ell-bound antibody ould still be present on the surfae of these antigen-reative ells and, indeed, may play an important role in the initiation of events in the leuoyte migration inhibition system. Repeated apillary migration tests in the presene of streptooal antigens were arried out in only two aute rheumati fever patients, but the results obtained in these two ases are perhaps worthy of omment. Patient H. W.'s loss of ellular reativity during aspirin and steroid therapy is probably related to the effet of these drugs on lymphoytes (39, 4). The rapid return and persistene of ellular reativity to streptooal antigens in this patient after essation of therapy ontrasts with the relatively rapid disappearane of migration inhibition in patient B. S. This differene raises the question of whether the intensity and duration of a heightened ellular response to streptooal antigens plays a role in the severity of the attak or in the sus- 448 Read, Fishetti, Utermohlen, Falk, and Zabriskie

11 eptibility to rheumati reurrenes. It should be noted that in the two ases presented, we are omparing only overall severity of disease and not the manifestation of arthritis vs. valvulitis. We would postulate that a patient with severe arthritis or horea might show just suh a persistene of ellular reativity to streptooal membranes and that this reativity piture is not speifi for arditis only. Previous studies using streptooal antigens have left some doubt regarding the speifiity of the ellular response. Franis, Oppenheim, and Barile (41) felt the lymphoyti response to streptooal ellular antigens was speifi, sine ord blood lymphoytes did not respond to these antigens. In ontrast, Keiser, Kushner, and Kaplan (34), using essentially the same type of antigens, noted a definite response of ord blood lymphoytes to these antigens and felt the ellular reativity was "nonspeifi" in nature, akin to the response indued by phytohemagglutinin and other mitogens. These onfliting results may be due to the preparations of antigen used. For example, it has been shown by Taranta, Cuppari, and Quagliata (35) that streptolysin "S" preparations ontain a separable hemolyti omponent and a nonspeifi mitogeni omponent. Studies by Keiser et al. (34) suggest that at least part of the transforming material present in extrats of streptooal ell walls and membranes may be related to the presene of just suh streptolysin S-related materials. Our ontrol studies, inluding normals mathed with the rheumatis studied, and espeially the studies on ord blood lymphoytes, showed that we were not dealing with a nonspeifi mitogen in our antigen preparation. Our studies have shown a persistent heightened reativity of peripheral blood lymphoytes of rheumatis to ell membranes of Group A streptooi. This is of partiular interest, as this struture was previously shown to ontain antigen(s) ross-reative with sarolemma of mammalian heart and smooth musle of blood vessel walls (21, 42). The nature of the antigen (s) responsible for the observed reativity is at present unknown. Trypsin treatment of ell walls (and ontaminating membrane fragments) did not appear to alter the lymphoyti response to these streptooal antigens. In addition, the lak of M protein on these treated walls would tend to exlude this protein as an ative partiipant in the reation. Treatment of streptooal membranes with trypsin also did not materially affet the ellular response. Whether the lipoprotein portion of the ell membrane plays a role in this reation has not been asertained at present, and studies designed to delipidate the membrane are in progress. As mentioned previously, the possibility that irulating antigen-antibody omplexes play a major role in the inhibition proess appears to be exluded on the basis of the use of heterologous serum in the migration system. While these results strongly suggest that there is a heightened response to streptooal antigens in rheumati individuals, the exat role these sensitized ells play in this disease proess remains unknown. The present finding that there is an abnormal ellular response to membrane antigens, oupled with previous reports of an abnormal humoral antibody reative with streptooal membrane in rheumatis, ould argue strongly for an important role of this ell struture in the disease proess. The ross-reative properties of these antigens might oneivably result in autosensitization to tissue antigens produing ytotoxi effets in the tissues of the host. This onept is in agreement with the histologial findings of a large number of lymphoyti ells in and near the pathologial heart lesions of rheumati fever (43). The observed humoral response might either prepare the tissues for this autosensitization proess or at in onjuntion with sensitized ells to produe the damage to the tissues. Experiments to determine the ytotoxi effet of these sensitized ells for mammalian ardia ell monolayers and the effet of autologous sera on this reation are now in progress. ACKNOWLEDGMENTS This investigation was supported by a grant from the National Institutes of Health, no. HE-3919, a grant from the New York Heart Assoiation, and a grant from the Ontario Heart Foundation. REFERENCES 1. Derik, C. L., and H. F. Swift 1929, Reations of rabbits to nonhemolyti streptooi. I. General tuberulin-like hypersensitiveness, allergy or hyperergy following the seondary reation. J. Exp. Med. 49: Swift, H. F., and C. L. Derik Reations of rabbits -to nonhemolyti streptooi. 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