Lymphocytes Binding C-Reactive Proteinduring Acute Rheumatic Fever

RALPHC. WILLIAMS, JR., and K. A. KILPATRICK, Department of Medicine,University of NewMexico School of Medicine, Bernalillo County Medical Center,Albuquerque, New Mexico 87131

M. KASSABYand Z. H. ABDIN, Department of Pediatrics, Free Rheumatic Heart Center,Cairo, Egypt

A B S T RA C T Lymphocytes binding C-reactive pro-tein (CRP) were studied in 31 patients with acuterheumatic fever and 30 controls who were chil-dren. Marked elevations in both proportions and ab-solute numbers of CRP-binding lymphocytes were re-corded in rheumatic fever (P < 0.001). No clear corre-lation was noted between plasma CRPas quantitatedby radioimmunoassay and proportions or numbers ofCRP-binding cells. Double-labeling experiments in-dicated that 60-80% of CRP-binding lymphocytes alsoshowed Fc receptors reacting with fluorescein-conju-gated IgG aggregates. Passage of lymphocytes overIg-anti-IgG columns, removed cells bearing surfaceIg but not CRP-binding lymphocytes. Studies of T-cellsubpopulations indicated no overlap between Tu- andCRP-binding cells; however about half of Ty-cellsshowed concurrent CRP binding. "Active" T-cell ro-setting cells did not bind CRP. A 12-15-h incubationof lymphocytes at 37°C in 5% C02-air showed persis-tence of CRPbinding in substantial proportions of cellsparticularly in acute rheumatic fever. CRP-bindinglymphocytes may represent a marker for immunologi-cally committed cells in acute rheumatic fever.

INTRODUCTION

C-reactive protein (CRP)l constitutes a materialwhich exists in low concentrations in normal serumbut which is often elevated during the course of acuteinflammatory reactions such as infection, trauma, oracute rheumatic fever (1-6). Recent comparative anal-ysis of amino acid sequences as well as electron mi-croscopic structure suggest that CRPmay share evolu-tionary sources with Clt related to the Clq componentof complement (7). Previous reports by Mortensen et

Received for publication 21 July 1977 and in revised form13 January 1978.

'Abbreviations used in this paper: CRP, C-reactive pro-tein; EA, erythrocyte antibody.

al. (8-10) have indicated that CRPmay bind to lym-phocytes and in particular, may actually adhere to Tcells activated by antigens but not by mitogens suchas phytohemagglutinin. Our previous studies have in-dicated a marked increase in proportions of "active"rosette-forming T cells among children with acuterheumatic fever (11). It was not clear whether active(or rapid) rosette-forming cells constituted a popula-tion of immunologically activated T cells that some-how increased during the acute rheumatic process. Re-cent reports have related increments in proportionsof active T-cell rosettes related to delayed type hyper-sensitivity reactions after in vivo skin testing (12). Pre-vious studies had appeared to indicate that proportionsor numbers of active rosettes might correlate with im-mune competence (13, 14). The purpose of the presentstudy was to examine lymphocyte subpopulations inacute rheumatic fever patients with particular at-tention to what distinct populations of lymphocytesshowed CRPbinding. It was felt that such cells mightrepresent immunologically committed or possibly anti-gen-activated cells called forth in the host responseto the disease process.

METHODSPatients studied. 31 patients with acute rheumatic fever

were studied during the first 3 wk of April 1977. All of thepatients were hospitalized at the Free Rheumatic Heart Cen-ter in Cairo, Egypt (11, 15), and were between the ages of3 and 14 with active acute rheumatic fever. The diagnosiswas established using the modified Jones Criteria as sug-gested by the American Heart Association (16). The majorityof patients studied were subjects in the relatively acutestages of rheumatic pancarditis, 1-8 wk after the onset ofsymptoms. Of these patients, 11 were experiencing their firstattack, whereas the other 20 children were studied in an ex-acerbation having had two to seven previous episodes of car-ditis or active chorea. 10 patients with acute chorea and as-sociated carditis, or evidence on physical examination of ac-tive mitral valve involvement were included in the study;the remainder were studied during the progress of active car-ditis.

J. Clin. Invest. © The American Society for Clinical Investigation, Inc., 0021-9738/78/0501-1384 $1.001384

9 of the 21 patients with active carditis had received corti-costeroids as therapy for their flagrant carditis. Concurrentdosage of prednisone ranged from 5 to 30 mg/day. Nine pa-tients with active carditis were also studied who were re-ceiving salicylates alone, and three were studied before theadministration of any therapy. The 10 patients with choreawere receiving salicylates as well as reserpine preparationsand phenobarbital for the control of their movement disorder.

The average age of patients with carditis was 9.1 years (13females and 8 males). The chorea patients averaged 10.3 yearsof age (8 females and 2 males).

Six normal control subjects were studied from control Egyp-tian children hospitalized at Cairo University Hospital on thegeneral pediatric service for correction of congenital de-formities or treatment of noninflammatory conditions. In ad-dition, a group of'24 normal children, ages 5-15 and matchedfor age and sex, were studied in Egypt and AlbuquerqIue.

Special studies. CRP was determined in serum samplesusing radial diffusion and the techni(lue of Kindmark (17),and all results expressed as nanograms per deciliter of' serum.Serum samples which could not be used f'or CRPdetermina-tion because of the limits of sensitivity of' the radial diffu-sion method were assayed using the radioimmunoassav modi-fied slightly from the original techni(lue described by Clauset al. (6). In this assay, highly purified CRPwas labeled with1311 using the Bolton-Hunter reagent. Labeled CRP was re-purified after labeling by elution from C-polysaccharide col-umns to insure specificity and biologic reactivity. The rangeof values for normal serum (50 samples) was similar to thatpreviously recorded (60-8,000 ng/dl) (6). Serum samples as-sayed in parallel by the modified radial dif'fusion assay andradioimmunoassay, generally showed good agreement withnot more than a 15% diff'erence when the two methods wererun in parallel on the same samples.

Lymphocytes were isolated from 12-ml samples of' hepa-rinized blood using Ficoll-Hypa(que and gradient separation(18). After harvesting the lymphocytes f'rom the gradient,mononuclear cells were depleted of adherent cells and mono-cytes using adherence to glass petri dishes at 37°C for 30min (19). This 37°C incubation also served to allow sheddingof adherent immunoglobulin, thus depleting cell surfhaces ofthe latter as noted by Horwitz et al. (20). Nonadherent cellswere then utilized f'or assay of CRPbinding. In preliminaryassays, whole mononuclear cell preparations in which ad-herent cells had not been depleted and adherent cells, re-covered from petri dishes by gentle scraping wvith rubberpoliceman were studied, to ascertain whether such cells con-stituted a major fraction showing CRPbinding. These experi-ments did not indicate any appreciable contribution by mono-cytes or other adherent cells to CRPbinding because <1%of such cell populations showed surf;ace CRP.

CRP binding to lymphocytes utilized two parallel tech-ni(lues. CRP antisera made in rabbits were obtainecl f'romN. L. Cappel Laboratories Inc., Cochranville, Pa., as wvell asthrough generous gifts from Dr. Osmand and Dr. Gewurz,Department of Immunology, Rush Medical College, Chicago,Ill., and later from Dr. E. Gotschlich, Rockefeller Univer-sity, NewYork. In view of a recent report indicating that anti-CRP reagents might show inadvertent specificity for P-component as described by Pepys et al. (22), anti-CRP anti-serum was tested for possible anti-P specificity tusing P coimi-ponent affixed to Sepharose 4B beads (Pharmacia Fine Cheml-icals Inc., Uppsala, Sweden) in the presence of'calciumil ionls(23). Sepharose 4B beads coated with P componient werestrongly positive with specific anti-P-component antiserumilbut negative with our anti-CRP antisera. These experimentsruled out inadvertent specificity for P componient in the anti-sera used. All anti-CRP antisera used in the cell-suirf'ace

marker studies showed complete absorption of activity inthe systems studied using CRPisolated from capsular (Cs) poly-saccharide columns (8) and f'reed of contaminants by subse-quent gel filtration. IgG fractions from rabbit anti-CRP anti-serum were digested by pepsin as previously described (21)to obtain F(ab')2 fragments and were then labeled with eitherfluorescein or rhodamine. Such labeled F(ab')2 prepara-tions showed F/P ratios ranging between 1.5 and 2.0. Com-pleteness of pepsin digestioni was assayed using inhibitionof precipitation between rabbit Fc and specific goat anti-rabbit Fc antiserum in agar gel and by acrylamide gel elec-trophoresis. In several instances, pepsin-digested materialwas also absorbed with whole Cowan I protein A-containingStaphyloccus aureus to insure removal of whole IgG or ma-terial containing Fc determinants. Mononuclear cell prepara-tions were incubated with fluoreseein-labeled F(ab')2 of anti-CRP and, after washing and resuspension, examined underthe Zeiss fluorescence microscope using epifluoreseence, amercury HBO200 Wlamp with BG12 primary filter, 53 Bar-rier filter, and the fluorescein isothiocynate filter (Carl Zeiss,Inc., New York). Double labeling for CRPand other cell sur-face markers employed fluoreseein-labeled aggregates of' IgGto detect Fe receptors (11, 24) in con-junction wvith rhodamine-labeled F(ab')2 fragments of' anti-CRP. In addition, erythro-cyte antibody (EA) rosettes prepared from bovine cells coatedwith optimal sensitizing dilution of isolated rabbit anti-bovineIgG were also used for the detection of Fe receptor-bearingcells (25). IgG, coating bovine erythrocytes, was titered toprovide a plateau of optimnal dilution for producing repro-ducible Fc receptor rosettes. Preliminary studies wvere con-ducted to determine whether cells binding fluoreseein-labeled aggregates wvere idenitical to those bearing Fc re-ceptors as detected by the IgG sensitized ox-cell techni(lue.Comparative data illustrating these findings are presenited inTable I. It can be seen that virtually all the cells showingFc receptor-binding fluorescent aggregates were eliminatedafter removal by the rosetting of' cells binding to ox cells sen-sitized with rabbit IgG. Since these experiments utilizedlymphocyte populations already depleted of moniocytes andpreviously incubated at 37°C to allow for sheddinig of' cyto-philic Ig on L cells, Fe receptor-bearing cells dletected bythese techni(lues constituted T cells bearing Fe receptors,B cells, and possibly L cells w%ith Fc receptors. Double-labeling experiments wvere also condutcted on cells showingFe receptors either by EA or fltuoreseenit aggregate-bindingtechni(lue. This was performed uisinig rhodami ine-conjugatedgoat anti-human IgG. In all experiments given in Table I,cells showing EA or positive fluorescenit aggregate bindingshowed no more than 1-2% concordaniee Nvith cells bearingsurface Ig as detected by rhodaminie labeled goat anlti-huimanIgG. In many instances, simultanieous double labeling of'cellsutilized incubation of' fluoresceini or rhodlamiiine F(ab')2 anti-CRPand formation of' EA rosettes.

The second parallel technii(quie used for dletecting lympho-cytes bearing CRPutilized Coxvan I strain of '. aturcu.s Conl-taining surface protein A andl stuspenisionis of lightly forma-linized bacteria. Such suspenlsionls co'ul(d be stored at 4°C for2-3-wk periods withl retention of eqjuiivalent activity as in-dicator particles in the assay (26).

Suspensions of' bacteria standlardize(d at tiniform opticaldensity (560) in the Coleman spectroplhotomiieter (ColemllanlSystems, Irvine, Calif:) w%ere uitilized. 50 Al of aanti-CRP wasadded to 5 x 106 lymphocvtes in 0.25 ml of minimal essentialmedium, pH 7.4 plus 10% fetal calf'serumiii. After befing w\ashedtwice in the medium, 50 Al of' standard l)acterial stispenisionwas added, the volume was brought to 2.5 ml, and( the mixtuirewas then centrif'uged at 1,000 g to remove nonia(dlherenit bac-teria. Lymphocyte stispenisionis were genttly distributed on

C-Reactive Proteini-Bitndinig Lymphocytes 1385

TABLE IRelative Percentages of Whole Unfractionated Lymphocytes Bearing Fc Receptors

for IgG Using Parallel and Simultaneous Assays

Fe Receptors Cells with Fc Fe Receptors Cells forming Cells showing fluorescencePatient by fluorescent receptors also positive by EA ox cell EA also positive with labeled aggregatesstudied aggregates for surface Ig rosettes for surface Ig after EA ox cell depletion

1 8 0 12 1 02 9 1 14 1 03 10 2 12 2 04 8 1 14 1 15 11 2 13 1 0

glass slides, fixed with absolute methyl alcohol, and stainedwith Wright's-Giemsa stain. Lymphocytes binding CRPwerethen identified by counting 200 cells. Controls included cellscoated with normal rabbit serum as well as cells and bac-teria alone.

Before use in the respective assays, anti-CRP antiserumas well as normal rabbit sera were absorbed with lympho-cytes using 10 x 106 cells per ml of serum. Background con-trols with normal rabbit serum or bacteria and cells alonenever showed >1%apparent binding.

Specificity for both the indirect immunofluorescence aswell as S. aureus protein A methods was tested by absorptionof anti-CRP antisera with highly purified preparations of hu-man CRPprepared from pneumococcal C-polysaccharide im-munoabsorbent columns and CRP-rich pleural or ascitic fluidsas previously described by Mortensen et al. (8).

Cell subpopulation separation. Cells depleted of mono-cytes or adherent cells were passed over Ig-anti-IgGDegalan columns to further deplete cells of those bearingsurface Ig (19, 21). Cells were studied for CRPbinding be-fore and after such column passage.

In addition, the technique of Moretta et al. (27, 28) wasutilized in an attempt specifically to prepare T cells bearingFc receptors for IgM as well as those with receptors for Fcof IgG. This technique utilized enrichment for T-cell ro-setting cells with neuraminidase-treated sheep erythrocytesfollowed by overnight incubation in 5% CO2 air at 37°C inHanks' balanced salt solution. Cells undergoing this selectiveT-cell enrichment and overnight shedding were then exam-ined for T cells bearing IgM (T,u) and IgG receptors (Ty)as well as for presence of CRPusing direct immunofluores-cence and F(ab)2 anti-CRP in conjunction with bovine cellrosettes coated with rabbit IgM or IgG. These experimentsalso served to provide some evidence as to stability of sur-face binding to CRP.

Attempts were made to determine if active T-cell rosettingcells contained a population capable of binding CRP. In theseexperiments, active rosettes were prepared as previously de-scribed (11), and after rosetting at 1 h, actively rosetting cellswere separated on Ficoll-Hypaque and adherent erythrocyteswere lysed using 0.83% ammonium chloride for 10 min at37°C. Cells sedimenting to the bottom of the gradient and,therefore, lymphocytes making active or rapid rosettes, werestudied immediately or, in some instances, incubated over-night at 37°C in 5%CO2air in 20% fetal calf serum -Eagle'sminimum essential medium, and then stained by direct im-munofluorescence using F(ab')2 fragments of anti-CRP. Inparallel, the lymphocytes not forming active rosettes wereprocessed and subsequently examined for immunofluorescentCRPbinding.

A final control involved studies of five patients with chronicmyelogenous leukemia and eight patients with acute lympho-cytic leukemia. Small proportions (1-5%) of CRP-bindingcells in peripheral blood samples from some of these sub-jects were always associated with T lymphocytes as monitoredby concomitant E rosetting. No binding of CRPwas detectedwith immature granulocytes or blast cells from patients withvarious forms of leukemia.

RESULTS'

A marked increment in both proportions and total num-bers of CRP-binding cells was noted in peripheralblood lymphocytes obtained from patients with acuterheumatic fever when compared with values obtainedusing normal child controls. These data are presentedin Table II. Values for proportions of cells bearingCRPwere slightly higher using direct immunofluores-

TABLE IILymphocytes Binding CRPin Normal Controls and Children

with Acute Rheumatic Fever

CRP-bindinglymphocytes Total no. of CRP

% lymphocytes/mm3

Acute rheumatic fever (30) 16.3+8.3 1p < 0 821.8±509.6 1Normal children controls (30) 3.3+2.9 P<0001 137.6+61.5

1386 R. C. Williams, Jr., K. A. Kilpatrick, M. Kassaby, and Z. H. Abdin

cence and F(ab'), fragments of anti-CRP than thoseobtained in parallel using the Cowan I binding method.However, in general, excellent agreement in valuesfor CRP-binding cells was noted using the two methodsin parallel (Table III). Specificity of anti-CRP anti-serum used in immunofluorescence and Cowan I S.aureus binding was confirmed by absorption usinghighly purified CRP obtained from C-polysaccharideaffinity columns. After such absorption, no positivecells were detected using either method.

Because the high proportions of CRP-binding cellsoccurred in blood samples obtained from actutely illsubjects many with severe rheumatic pancarditis, itseemed possible that lymphocytes binding CRPmightdirectly reflect the amounts of CRPconcurrently pres-ent in plasma since CRPis known to be markedly ele-vated during the acute rheumatic process. No directrelationship was apparent between proportions or totalnumbers of CRP-binding cells and quantitative estima-tions of CRPin plasmas collected concurrently in sub-jects with rheumatic fever or normal children controls.Representative data illustrating this point are givenin Table IV. It can be seen that the highest three con-centrations of plasma CRP in carditis patients 2(550,000 ng/dl), 7 (440,000 ng/dl), and 5 (305,000 ng/dl), were associated with 795, 993, and 624 CRPcells/mm3, respectively, whereas in two chorea patients, 12and 14, much lower plasma concentrations of CRP(640 ng/dl and 3,700 ngtdl) were associated with 1,247and 853 CRPcells/mm3. In addition, a series of over-night incubations were performed and cells were re-examined after a 12-15-h inctubation at 37°C in 5%

TABLE IIIRepresentative Determinations Using Immunofluorescence

and Cowan I S. Aureus Methods for Determinationof CRP-Binding Lymphocytes

Patient studied CRP-binding cells

Patient nio. Condition Immunofltuoreseence S. aureus binding

85 Carditis 9 570 Carditis 19 1263 Carditis 35 2873 Carditis 28 2464 Carditis 14 1030 Carditis 8 1049 Carditis 13 10

329 Carditis 5 693 Carditis 10 1192 Chorea 18 1083 Chorea 23 2475 Chorea 32 24

154 Chorea 11 1169 Chorea 13 10

TABLE IVRelationshlipv between Ni in bers anid Proportions of CRP-

Binding Lymphocytes anid QuanititativeEstimations of Plasma CRP

CRP-binding Absoltute nio. ofPatient no. cells CRP-binding cells Plasma CRP

% t,g/dlRheumatic

carditis1 9 314 2,0502 19 795 550,(0()3 32 1,442 3,1004 35 2,041 125,0005 28 624 305,0006 27 2,547 26.5,0007 17 993 440,0008 14 464 3,6509 8 509 115,000

10 8 494 2,050

Chorea11 18 753 240,00012 28 1,247 64013 23 1,269 3,85014 32 853 3,70015 13 593 2,050

Normalcontrols

1 2 156 56,0002 2 147 4,0003 3 140 1,1704 4 206 6405 2 74 1,0506 1 48 3,9007 3 85 7508 4 150 3,2009 2 52 3,350

10 2 100 1,050

CO2 air incubator. Of great interest xvas the apparenitpersistence and, in some instances, slight increase inproportions of CRP-binding cells after suich incubation.Examples of such representative experiments usingacute rheumatic fever subjects' cells as well as normalcontrols are shown in Fig. 1. Cell viability as meas-ured by trypan blue exclusion remained between 95and 98% after such incubations. It was clear that lym-phocytes binding CRP did not uindergo extensiveelution uinder the eiretmstances of incubation, al-though in some instances a slight decrease or increasein proportions of CRP-binding cells was apparent. Inthe case of experiments where slight inerements inproportions of CRP-binding cells were note(d after over-night incubation, imnizunoassav of the stipernates didnot provide any clear evidenice for syntlhesis of CRPby culttured lymphoid cells.

If, as originally suggested by Mortensen et al. (8- 10),

C-Reactive Proteini-Bitndintg Lymphocytes 1387

BEFORE AFTERINCUB. INCUB.

*

*RFo NORMAL

0-------

O---

A further attempt to define lymphocyte subpopula-tions bearing CRPwas performed using Ig-anti-IgGDegalan column passage to deplete surface Ig con-taining cells. The results obtained after such immuno-absorbent column depletion are given in Table Vb.The increase in proportions of CRP-binding cells afterIg-anti-Ig column passage along with virtually conm-plete elimination of' cells bearing surface Ig, indicatedthat B cells with surface immunoglobulin did not con-stitute a major population showing CRPbinding, andthat the residual population-presumably mainly T orL cells without surface Ig but possessing Fc receptors(29, 30), were largely responsible for surface CRP. Toexamine these findings further, Fc receptor-bearingcells were specifically enriched for using EA rosetteformation with ox cells coated with rabbit IgG anti-body and isolation of such rosette-forming cells byFicoll gradients. Proportions of CRP-binding cells innon-EA rosette-forming lymphocyte cell preparationswere compared to those enriched by rosetting for EAFc receptor bearing cells. Again, as in the case of dou-ble-labeling studies described above, it was clear from

FIGURE 1 Results of changes in proportions of CRP-bearingcells as identified using fluorescein-labeled F(ab'), fragmentsof anti-CRP before and after 12-15-h incubations at 37°C in5% CO,-air incubator. RF, rheumatic fever.

CRP-binding cells represent antigen-activated T cells,their concomitant cell-surface markers might providean indication as to what lymphocyte subpopulationswere actually involved in such a process during therheumatic fever episode. Accordingly, a variety of dou-ble-labeling experiments focused on this particularpoint. Among unfractionated lymphocytes depleted ofmonocytes and adherent cells, about one-half of all thecells reacting with fluorescein-conjugated aggregatesshowed concurrent staining for rhodamine-conjugatedF(ab')2 fragments of anti-CRP. Representative experi-ments illustrating these findings are provided in TableVa. Cells showing staining for both CRPand humanaggregated IgG showed distinct and separate immuno-fluorescence. Thus, the staining for rhodamine-labeledanti-CRP appeared as red fluorescent spots and patchesclearly distinct from the apple-green particulate sur-

face immunofluorescence produced by the Fc receptoradherence to isothiocyanate-labeled IgG aggregates.Observation of cells after incubation at 37°C f'or 30-60 min showed a tendency for polarity or capping ofanti-CRP-binding structures which was clearly sepa-

rate from Fc receptors identified with the fluorescentgreen IgG aggregates. However, endocytosis and dis-appearance of CRPwas not observed after observationsup to 3 h. From these experiments, it was apparentthat a considerable proportion of CRP-binding cellswere lymphocytes which showed IgG Fc receptors.

TABLE VDouble-Labelitng Experiments to Determine Overlap of Cell

Surface Markers with Lymphocytes Binding CRP

Proportion ofcells staining

Proportion of with pepsin- Proportioncells staining digested of cellswith fltiores- anti-CRP showing

(A) ceinated IgG (rhodamine- dotiblePatient studied aggregates labeled) staining

82, Carditis 14 11 75105, Chorea 14 15 9329, Carditis 28 16 13

Experiments before and After Passage of Cellsover Ig -Anti-Ig Degalan Columns

Proportion of Proportion of Proportioncells staining cells with of cellswith pepsin- complement showing

(B) digested receptor sturfacePatient studied anti-CRP EAC Ig*

106 Before Ig-anti-Igcolumn 23 30 25

After Ig-anti-Igcolumn 40 5 1

114 Before Ig-anti-Igcolumn 17 0 23

After Ig-anti-Igcolumn 24 0 0

* Cells with surface Ig identified using fluorescein-labeledF(ab')2 f'ragments of' rabbit anti-human Ig.

1388 R. C. Williams, Jr., K. A. Kilpatrick, MI. Kassaby, and Z. H. Abdin

35

30

25

20

15

F--C-)0Ia-

a-C-)

10

5

TABLE VIRelative Proportions and Total Numbers of T-Cell Subpopulations in Patients with Rheumatic Carditis

or Chorea and Normal Controls

T cells bearing receptors for Fc IgC (Ty) T cells bearing receptors for Fc IgM (T,u)

Patient group % Absolute no. % Absolute no.

Normal controls (30) 10.5-±4.5* p < 001 313.1+184.5 p < 001 49.5+10.4 p < 001 1X577+721 64Rheumatic fever and carditis (16) 15.9±6.6J 811.4+±742.4 J * 33.4±+10.3 J 1,654.9+1,3484Rheumatic fever and chorea (8) 10.9+3.5 392.8±+148.5 30.1+7.3 1,158.1+5604

* Numbers refer to mean-+SD.4 Difference between absolute no. of lymphocytes bearing Fc IgM receptors NS.

several experiments that Fc receptor-bearing cells andCRPbinding were occurring together. In these experi-ments, cell populations specifically enriched for lym-phocytes bearing Fc receptors in four experimentsshowed 21-25% concurrent double labeling for CRP.However, fluorescence CRP identification in the pres-ence of concurrent EA rosettes was technically judgedmore difficult.

During various physiologic states, particularly thoserelated to activation, T cells are known to show Fcreceptors (31, 32). Accordingly, a series of experimentswhich focused on specific T-cell populations were con-ducted. T cells bearing specific IgM or IgG Fc recep-tors were prepared by the techniques of Moretta etal. (27, 28). Results of T,t-and Ty-cell determinationson children with acute rheumatic fever as well as nor-mal controls are given in Table VI. It was clear thatT,u cells predominated and that Ty cells constitutedonly a small fraction of the total T cells that were spe-cifically enriched by neuraminidase sheep cell ro-setting and overnight incubation to allow shedding andmembrane stabilization. Of interest were the findingsof significant elevation of proportions and numbers ofTy cells in rheumatic children with decrease in pro-portions of T,u cells as compared to controls. After iso-lation of T cells using rosetting with neuraminidase-

treated sheep erythrocytes and overnight incubationat 37°C in IgM-free medium, concurrent labeling ofT,u and Ty cells was performed using fluorescein-labeled F(ab')2 fragments of anti-CRP. These parallel-labeling experiments showed that no T,u cells bearingreceptors for Fc of IgM showed CRPbinding, whereas25-60% (mean 46%) of Ty cells bearing IgG Fc re-ceptors bound CRP. Thus, when specific T-cell pop-ulations were examined for CRP, only Ty cells showedsurface staining. Representative data illustrating thesefindings are given in Table VII.

Experiments were also conducted on cells remainingat the Ficoll-Hypaque gradient interface after neur-aminidase-rosetting of E-binding T cells. In five experi-ments, 12-16% of cells remaining in this B-cell layershowed CRP binding-even after overnight incuba-tion at 37°C. Cell surface-marker studies in this pre-sumptive B-cell layer showed 50-65% cells with sur-face Ig and 20-26% cells with Fc receptors by bovineEA rosettes. In two experiments where enough cellswere present for accurate double-labeling studies onthese isolated B cells after overnight incubation, 40and 38% showed concurrent binding of pepsin-di-gested fluorescein-labeled anti-F(ab')2 and rhodamine-labeled F(ab')2 of anti-CRP. This clearly establishedthat there was a definite overlap between cells bearing

TABLE VIICRP-Binding T-Cell Populations in Acute Rheumatic Fever

Patient studiedUnfractionated Total isolated

Patient lymphocytes T cells positiveno. Condition binding CRP* for CRP* Ty cells Ty + CRP* TM cells TM + CRP*

73 Acute carditis 26 12 16 25 20 092 Chorea 18 20 10 40 37 056 Chorea 28 17 13 50 37 065 Carditis 24 20 23 60 47 083 Chorea 26 18 12 58 35 0

* Unfractionated cells and isolated T cells studied by indirect immunofluorescence usingF(ab')2 anti-CRP.

C-Reactive Protein-Binding Lymphocytes 1389

surface Ig and those capable of binding CRP. In addi-tion, double-labeling experiments using EA rosettesand anti-CRP F(ab')2 showed concomitant occurrenceof these surface markers in 40 and 52% of isolated B-cell fractions. T-cell contamination as monitored byE rosettes was <2%. These data are given in TableVIII.

Experiments were next designed to examine whetheractive rosette-forming cells participated in CRPbind-ing. Very little overlap in these two populations oc-curred. Separation of active or rapid rosette-formingcells and comparison with remaining whole lympho-cyte populations either immediately before or after 12-14-h incubations at 370C showed that very few (0-1%) of the active rosette-forming cells bound CRP. BYcontrast, a considerable fraction of cells which didnot form early rosettes were shown to bind CRP (Ta-ble IX). Stubsequent double-labeling experiments withthis latter population again confirmed the presence offrequent concurrent Fc receptors and CRPbinding incells not participating in active T-cell rosette forma-tion.

DISCUSSION

The current findings indicate that a marked increasein cells binding CRPoccurs during the course of' acuterheumatic fever. This was demonstrated using two,parallel independent methods of' lymphocyte-CRPbinding, which included direct immunofluorescent la-

TABLE VIIIStudies of CRPBinding in Unfractionated antd Isolated

Lymphocyte Populations in Patients tvith AcuteCarditis of Rheuimatic Fever

Double-labelingmiiethods on B-cell

fractioniUnfrac-

tionated Cells Cellslympho- CRPbinding showing showiig

Patient sttdied cytes stirf'ace EAbinding T B Ig pltus rosette

No. Condition CRP cells* cellsl CRP pluts CRP

103 Acute 14 7 8 38 40carditis

108 Acute 16 7 11 40 52carditis

* T cells isolated by neuraminidase E rosetting and allowedto shed or incubate in 5%C02-air at 37°C in medium for 12 h.I B cells refer to the cells not separated by Ficoll-HypaIqueas neuraminidase rosette-forming cells, but remaining atFicoll-Hypa(que interface. Cell-surface marker studies onthese cell fractions showed 50-65% cells with sturface Igand 20-26% cells with Fc receptors by bovine EA rosetteformation.

TABLE IXRapid Rosette-Forming Cells and Nonrapid Rosetting

Cells Double-Labeled for CRP

Binding CRP

Patient Nonrapid rosette-fonrmingstudied Rapid rosettes cells

1 2 142 0 243 0 174 0 15

beling of' cells using F(ab')2 fragments of' anti-CRP aswell as binding of Cowan I S. aureus bacteria coatedwith anti-CRP through Fc-reactive sites on protein Aof the staphlococcus. Use of pepsin-digested F(ab')2fragments of anti-CRP avoided inadvertent adsorptionof' reagent to cell surface Fc receptors (33). It is pos-sible that other nonimmunologic changes may actuiallyaff'ect lymphocyte-binding to CRP. Availability of'eniough purified CRPat the time of' the work in Cairoprevented studies aimed at this point, utilizing lym-phocytes from rheumatic fever children and additionof exogenous CRP after appropriate shedding or in-cubationi. Wle have, however, confirmed the f'act thatCRP added to normal lymphocytes does show memil-brane binding. This does not appear to be a fiunctionof P component and was observed using CRP free ofthe latter material.

CRP-binding lymphocytes could not be directly cor-related with quantitative elevations of' plasmiia CRPsince high proportions and total numbers ocecurred bothin patients with elevated as well as much lower (uIail-titative amouints of plasma CRP. It is clear, however,from the data presented in Table IV that some linmit-ing increase in plasma CRP or threshold mayvbe in-volved in lymphocyte CRPbinding. A majority ofCRP-binding lymphocytes did not appear to be convenitionialB cells with surface Ig since depletion of' B cells UsinlgIg-anti-IgG columns resulted in relative increase andleinrichnmenit of CRP-binding lymphocytes. It mllUst berecognized, however, that such procedures could alsohave (lepleted adherent T cells and other cells besidesthose with surface Ig. When isolated B cells weire stud-ied, however, a definite proportion (38-40%) shovedconcordanice between CRP and cell sturf:ace Ig uisinigF(ab')2 reagents. Several approaches at double label-ing showed that CRP-binding cells often occtrred inassociation with cell sturface IgG Fe receptors. In ad-dition, studies of cell populations enrichecl for T cellsby neuramiiinidase-sheep cell rosetting and sulbsequentovernight incubation showed that T,u cells didc notbind CRP, but that about one-half of Ty cells wereassociated with CRPbinding. Thtus, the CRP-binding

1390 R. C. Williams, Jr., K. A. Kilpatrick, M. Kassablj, anid Z. H. Abdini

lymphocytes in rheumatic fever represent a definite pro-portion of B cells, but appear to be mainly associatedwith presence of' cell surface receptors for Fc of IgG.Our findings in children with acute rheumatic feverare somewhat different from the CRPbinding primarilyto T cells in normal peripheral blood initially reportedby Mortensen et al. (8, 9). Data shown in Table VIIIindicate some binding to B cells as well as T cellsin the subjects studied. It seenms possible that CRPmay bind to lymphocytes in diseased patients perhapson a different basis f'rom those of' normal subjects. Itis conceivable that CRP binding may, instead of re-flecting an immuinological mechanism, merely be re-lated to lymphocvte membrane damage of some kind.Some of the cells bearing Fc receptors and CRP ap-pear to be Ty cells as defined by Moretta et al. (28).From the data cutrrently available, it appears pos-sible that other lymphocytes bearing Fc receptorsperhaps similar to L, null, or K cells (30)-may alsobind CRP. Experiments are now in progress to definethis possibility, uising CRP-binding cells in an anti-body-mediated cytotoxicity system (34, 35). CRP-bind-ing cells did not overlap with T cells forming activerosettes. These findings appear to confirm recent ob-servations by West et al. (36, 37) showing that cellsactive in T-cell rosette formation at temperattures of32°C do not overlap with T cells bearing Fe receptors.

If CRP-binding cells actually represent in part anti-gen-activated T cells, then such cells could providean interesting possible source of cells activated towardsstreptococcal products or possibly cross-reacting car-diac antigens. Our cu-rrent work has been specificallydirected at this possibility. The use of' anti-CRP af-finity columns as a method to isolate CRP-bindingcells and to study their specific reactions to mitogensas well as to a variety of streptococcal or cardiac anti-gens could provide direct data on whether or not suchCRP-binding cells are indeed activated by identifiableantigens. The stability of CRPbinding in lymphocytesincubated for 12-15-h periods is of' considerable in-terest in this regard. Since data in Table VIII indicatedthat a proportion of CRP-binding cells appeared to bindto cells with Fc receptors which were not T cells asseparated by neuraminidase rosetting, it is also pos-sible that suclh cells might function as nonspecific ef-fector cells in such systems as natural killing as de-scribed by Nelson et al. (38, 39).

CRP elevation is known to accompany many acuteor subacute inflammatory conditions (1-6, 40, 41). Theexact origin or sites of CRPsynthesis as well as local-ization within inflammatory tissue previously havebeen studied by several groups (42-46). It is wvellknown that marked CRP elevation in serum accom-panies the acute process in rheumatic fever (4). Noprecise definition of the role of CRPin such disordersas acute rheumatic fever has yet been identified. Be-

cause of' its elevation in so many inflammnatory reac-tions, CRPhas long been regarded merely as an actute-phase reactant similar perhaps to other serumii proteiiisof' this type such as ceruloplasmin, haptoglobin, orseromucoid (41, 47, 48). It is clear that CRP is presentin small amounts in normal human serumil (6) so thatits presence does not necessarily reflect synthesis of'an abinormal protein. The following hypothesis mightbe suggested by our own data as well as the previousreports of NMortensein et al. (8-10). If' CRP is indee(dcapable of' pref'erential binding to antigen-activated T,K, or nattural killer cells, it is conceivable that suchCRPbinding might serve to defuise or atten-uate self:directed cellular responses possibly harmiif'uil to thehost.

The studies involving attempts to determine pos-sible lymphocyte subpopulations binding to CRPbearsome comment. Double-labeling techniq(uies clearly in-dicated that a substantial proportion of' CRP-bindingcells were T cells. In addition, when T cells wereselectively enriched UsiIng the neuraniinidase erythro-cvte-rosetting techni(que, T cells showing IgG Fc re-ceptors (Ty) but not those bearing IgM Fc receptors(T,) were positive for CRP binding. These findingsare of coinsiderable interest since T,u cells have beensuggested as putative markers for helper cells (49, 50).In the presenit study, proportions of'T,i cells have beennoted to be decreased during the acute rheumilatic proc-ess, whereas Ty cells were significantly inierease(l.However, T cells binding CRP clearly appeared alsoto have IgG Fe receptors. WVhether such Ty cellscarry a specific functionial role such as suppressor orkiller fuinction remains to be determined. Recent datapresented by Moretta et al. (50) appear to indicate asuppressor role for Ty cells in some in vitro assay sys-tems involving B-cell activation.

Our studies of' CRP in supernates from lymphocytecultures or merely incuibation experiments have notprovided clear evidence for active synthesis of' CRPby isolated T cells. These possibilities have been re-cently pursued in view of the report of' extensive se-quence homologies between Cl and CRPand the P-amyloid component (7). It is important in this regardto point out that F(ab'), fragments obtained from anti-CRP antiserum generously provided by Dr. E. Got-schlich showed similar reactivities in the lymphocytecell surface assays to those obtained from Dr. Osmandand Dr. Gewurz. The antiserum provided by Dr.Gotschlich was prepared against crystallized CRPwhereas that obtained from Dr. Ossmand and Dr.Gewurz was prepared from affinity column-purifiedmaterial. Our testing for anti-P-component specificityin anti-CRP antisera proved negative (22, 23). If lym-phocytes were under certain circumstances capable ofCRP synthesis, they might under previous or currentevolutionary circumstances be capable of CI synthe-

C-Reactive Proteini-Bintdinig Lymlphocytes 1391

sis as well. However, the vast bulk of evidence clearlyimplicates the liver as primary site of synthesis for CRP(42, 46).

Recent extensive studies (51-57) have describedmany of the important potential biological effects ofCRPboth in vitro and in vivo. Previous work has in-dicated that mixed leukocyte reactions are diminishedwhen non-HLA-compatible lymphocytes from two pa-tients with acute rheumatic fever are studied togetherin one-way stimulation (58). Gewurz et al. (8, 53) havealso shown that mixed leukocyte reaction in normalsubjects is diminished by CRP. In the latter instance,CRP effect was attributed to modulation of antigen-activated cells in the mixed leukocyte reaction. It nowseems important to recognize that proportions of CRP-binding cells may directly influence several expres-sions of the immune response. These collective find-ings are of particular interest in the light of currentreports indicating that streptococcal antigens producean altered cell-mediated immune response in patientswith acute rheumatic fever (59).

ACKNOWLEDGMENTSWe are indebted to Dr. H. Gewurz and Dr. A. P. Osmandfor providing us with highly purified C polysaccharide andanti-CRP and helpful discussions during the course of thiswork. Wewould also like to thank Dr. E. Gotschlich for gen-erously providing anti-CRP prepared against previouslycrystallized antigen. We also thank Dr. M. B. Pepys ofHammersmith Hospital, London, for generously providinganti-P-component antisera, P-component-coated Sephadex,and for helpful suggestions. It is a pleasure to acknowledgethe excellent technical assistance of Mrs. Jean Montano,and the secretarial help of Mrs. Carol Montman, Mrs. VohndaStarr, and Ms. Carol Lombardi.

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C-Reactive Protein-Binding Lymphocytes 1393