Suppression of mitogen-induced human immunoglobulin (Ig)-A synthesis by heterologous antibody to IgA

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

19, 372-382

(1981)

Suppression of Mitogen-Induced Human lmmunoglobulin (Ig)-A Synthesis by Heterologous Antibody to IgA RICHARD Departments

J. WARRINGTON,'PATRICIA of Medicine

J. SAUDER,

AND W. JOHNRUTHERFORD

and Immunology. Faculty of Medicine. Winnipeg, Manitoba R3E OZ3, Canada

University

of Manitoba,

Received December 3, 1980 The effects of sheep anti-human IgA (SaHIgA) upon pokeweed mitogen (PWM)induced IgA synthesis and secretion have been examined in vitro, with concomitant assessment of mitogen-induced IgG synthesis. It has been demonstrated that the SaHIgA suppresses IgA synthesis and secretion while not affecting the production of IgG. The suppressive effect is not Fc dependent and is exerted upon B lymphocytes. Suppression produced by the SaHIgA is reversible by washing the cells up to about 48 hr and can be induced by sequential addition of the antibody up to -72 hr after initiation of the cultures. IgA-specific suppression was also produced by sera from IgA-deficient patients containing anti-IgA antibody. It is hoped that this in vitro model can be used in an assessment of the effects of homologous anti-human IgA. derived from IgA-deficient subjects, on PWM-induced IgA synthesis.

INTRODUCTION

There have been described a number of experimental systems in which Igspecific antisera produce class or allotype-specific suppression of normal Ig synthesis both in vivo (l-3) and in vitro (4-7). Certain of these models also demonstrate an effect by transplacental passage of allotype-specific antibodies (8). These phenomena may possibly be of relevance to selective human IgA deficiency, because of the frequency with which cw-chain-specific antibodies occur in this condition (9). Indeed, a recent local survey has demonstrated the more frequent occurrence of familial IgA deficiency in mother and child than in father and child, suggesting the possibility that a maternal influence, such as antibody to IgA, might play a role in the induction of IgA deficiency in such children (10). In order to eventually further test this hypothesis, we have developed an in vitro system for the study of the effects of such class-specific antibody in man, by assessing the activity of heterologous sheep anti-human IgA antibody upon pokeweed mitogen-induced IgA synthesis by normal peripheral blood lymphocytes, using a reverse hemolytic plaque technique (11). It has been demonstrated that the sheep anti-IgA antibody suppresses PWM-induced IgA synthesis and secretion while not affecting IgG synthesis. The kinetics, complement, and Fc dependence of the activity have been examined and it has been shown that the effect is exerted upon the bone marrow-derived (B) cell population. METHODS Cell separation. Human peripheral blood lymphocytes (PBLs) were isolated from normal donors on Ficoll-diatrizoate (LSM, Litton Bionetics, Kensington, r Reprint requests to: Dr. R. J. Warrington, Section of Clinical Immunology, General Centre, 700 William Avenue, Winnipeg, Manitoba R3E 023, Canada. 372 0090-1229/81/060372-11$01.00/O Copyright All rights

@ 1981 by Academic Press. Inc. of reproduction in any form reserved.

C319 Health Sciences

ANTIBODY-INDUCED

SUPPRESSION

OF

IgA SYNTHESIS

373

Md.). When isolation of thymus-derived (T) lymphocytes and B lymphocytes was undertaken, adherent cells were first reduced by incubation of the PBL at 37°C for 45 min in plastic flasks or petri dishes. PBLs were then rosetted with aminoethylthiouronium bromide (AET, Aldrich Chemical Co., St. Louis, MO.)-treated sheep red blood cells (SRBC) and the rosetted T cells were separated from nonrosetting B cells on a second LSM density gradient (12). B cells were further purified by a second rosetting step with AET-SRBC. T cells were isolated following rosetting by ammonium chloride lysis of the SRBC (13). Surface Igpositive cells were identified and enumerated by staining with fluoresceinconjugated rabbit anti-human IgG, IgA, and IgM (Behringwerke AG, Marburg, West Germany), centrifuged at 20,m to remove aggregates. Monocytes were identified by peroxidase staining (14). Cell cultures. Lymphocytes were cultured in triplicate in disposable tissue culture tubes, 10 x 60 mm (Falcon; Becton, Dickinson Co. Canada, Mississauga. Ontario, Canada) or in round-bottomed microwell tissue culture plates (Linbro; Flow Laboratories, Rockville, Md.) at 37°C in 5% CO* in air, in RPM1 1640 (Grand Island Biological Co., GIBCO, Grand Island, N.Y.) with 2 mM L-glutamine and 50 &ml gentamicin (Schering Corporation, Kenilworth, N.J.) supplemented with 10% heat-inactivated serum from an &&deficient donor (IgA < 2 rig/ml) previously screened for a-chain-specific antibodies (by the National Reference Laboratory, Canadian Red Cross) and shown to support normal PWM-induced IgG and IgA synthesis and secretion. PWM (Gibco) was added at 1: IO- 1: 1000 dilution to appropriate tube or microwell cultures and cells were incubated for a period of 6-7 days, being fed on alternate days with a cocktail containing RPM1 1640, essential and nonessential amino acids, L-glutamine, glucose, and serum. Cultures were harvested and cells counted using hemocytometer chambers, cell viability being assessed by trypan blue exclusion. Proliferation in microwell plates was measured by incorporation of [3H]thymidine (sp. act. 2 Ci/mmol, Amersham Co., Oakville, Ontario, Canada), 1 &i in 0.01 ml being added to each milliliter of culture and cells were incubated for a further 5 hr and then harvested using a Skatron automatic cell harvester (Flow Laboratories). Radioactivity was assessed by liquid scintillation counting in a Beckman LS 335 counter (Beckman Instruments. Fullerton, Calif.). Preparation of antiserum. Sheep anti-human IgA was prepared by immunization with 0.7 mg of DEAE-cellulose (Pharmacia; Uppsala, Sweden) purified human colostral IgA in complete Freund’s adjuvant at approximately 15-day intervals for four doses. The antiserum obtained was then immunosorbent purified by adsorption with an IgA-deficient serum (IgA < 2 &ml) coupled to Sepharose 4B (Pharmacia) by cyanogen bromide (J. T. Baker Chemical Co., Phillipsburg, N.J.) (15). This antiserum was a-chain-specific by double diffusion in gel and gave a single line against IgA on immunoelectrophoresis against normal human serum. It showed no reactivity with purified IgG or IgM by enzyme-linked immunoassay using alkaline phosphatase (Sigma, St. Louis, MO.) linked to rabbit anti-sheep IgG, while binding to polyclonal IgA (16). Affinity purification of antibody. IgA was semipurified from a 40% saturated ammonium sulfate precipitate of pooled normal serum, by gel filtration on AcA 34 (Ultragel, LKB Industrie Biologique Francaise), followed by purification of the

374

WARRINGTON,

SAUDER,

AND

RUTHERFORD

IgA by passage through a column of Sepharose 4B (Pharmacia) coupled to sheep anti-human IgA F(ab’), (see below) by cyanogen bromide. The IgA after elution using 0.25 M acetate buffer, pH 4.0, followed by 0.25 M acetate and 4 M guanidine was shown to be pure by immunoelectrophoresis, immunodiffusion, and by hemagglutination (17), using rabbit anti-human IgA (Cappel Laboratories, Cochraneville, Pa.) and anti-IgG and IgM (Dako, Cedarlane Laboratories, London, Ontario, Canada). This IgA was subsequently coupled to Sepharose 4B and the sheep anti-human IgA prepared from a 40% saturated ammonium sulfate precipitate was passed through this column or a control column of Sepharose 4B coupled to bovine serum albumin (BSA) (15). Preparation of F(ab’), fragments of sheep anti-human IgA. The IgG2 fraction of the 40%-saturated ammonium sulfate precipitate of the sheep anti-human IgA was isolated by protein A-Sepharose (Pharmacia) followed by elution of the bound fraction with 0.58% acetic acid (18). Both bound and unbound (containing IgGl) fractions of the antisera were shown to have similar suppressive activity (see below). F(ab’), fragments were prepared by pepsin digestion (3%, 2500 units/ml, Worthington Biochemical Corporation, Freehold, N.J.) of the sheep IgG, previously dialyzed against 0.1 M acetate buffer, pH 4.0. Digests were brought up to pH 7.0 with 0.1 M NaOH. The F(ab’), fragments were isolated by gel filtration on AcA 34 and were shown to contain antibody activity against human IgA by immunodiffusion. Detection of cytoplasmic immunoglobulin by immunofluorescence. Cytocentrifuge preparations of the cultured cells were fixed in 5% acetic acid and 25% ethanol at - 12°C rehydrated in PBS, and stained at room temperature for 20 min with rabbit anti-human IgG or anti-IgA antibodies labeled with fluorescein isothiocyonate (molar F/P ratio, 2.3-3.1). Slides were examined using a Leitz Ortholux II microscope (E. Leitz, Wetzlar, West Germany). The percentage of cells with detectable amounts of cytoplasmic immunoglobulin was determined and the number of immunoglobulin-containing cells per culture was calculated. Assay of immunoglobulin secretion by reverse hemolytic plaque technique. IgG- and IgA-secreting cells were quantitated by a reverse hemolytic plaque assay using protein A-coated red cells (I), using a slight modification of the technique described by Rector et al. (19). Rabbit anti-human IgG and anti-IgA (DAKO, Cedar-lane, and Cappel Laboratories, respectively), and shown to be y-chain- and a-chain-specific by hemagglutination were utilized at dilutions of 1:40 and 1:20, respectively (shown to be optimal in this system) and four times washed preparations of PWM-stimulated PBLs and control cells, at a cell density of 2-5 x lo4 viable cells per plate. Following incubation of the plates for 4 hr at 37°C in 5% CO, and air, a lo- to 1Zfold dilution of normal guinea pig complement was added (Hemlo, Cedarlane Laboratories, London, Ontario, Canada) and after a further 30-min incubation, plaques were scored using 4x magnification. Results are expressed as PFC/106 viable cells. RESULTS

Several recent studies have demonstrated the sensitivity and reproducibility of the protein A reverse hemolytic plaque technique for assessing class-specific Ig synthesis and secretion (11, 19), including its relevance to human studies (20, 21).

ANTIBODY-INDUCED

SUPPRESSION

OF

IgA

SYNTHESIS

375

In the studies described here, although there are variations between different individuals with regard to the level of response (for 10 normal subjects, IgG response was 4590 -t 3284 PFC/106 viable cells and IgA was 1560 k 1564 PFC/lO’ cells) and the concentration of PWM required to elicit maximal IgG and IgA synthesis, there was consistency in the response of the same individual from day to day with regard to the relative levels of IgG and IgA production. The optimal concentration of PWM was the same for IgG and IgA synthesis (Fig. 1) and for most individuals, optimal synthesis of both IgG and IgA occurred on Day 6. The same individual gave the following responses when cultures were carried out in the presence of 10 different normal serum samples: IgG response was 8297 + 2456 PFCl106 cells and IgA response was 2105 + 542 PFC/106 cells. Thus the system could be used to assess the suppressive effect of sera containing class-specific antibodies upon mitogen-induced Ig synthesis and secretion. Effect of Sheep Anti-Human IgA Antiserum The addition of increasing dilutions of the sheep anti-human IgA (SaHIgA) to PWM-stimulated peripheral blood lymphocyte cultures resulted in suppression of IgA synthesis at a final concentration of the antiserum of 1:50, with the reappearance of IgA production in a dose-response manner. IgG production was not affected by the presence of the antiserum (Fig. 2). The antiserum interfered with both IgA synthesis and secretion, as is seen by assessment of cytoplasmic immunofluoresence using fluorescein-conjugated rabbit anti-human IgA and anti-human IgG. Here, in two experiments, IgA-positive cells were reduced from 9.5 to 0.8% by the presence of the SaHIgA while IgG-positive cells made up 10.5% of the PWM-stimulated cultures and 6.5% of the PWM-stimulated cultures containing SaHIgA at a dilution of 1:50. These experiments indicate that the effect of

Suppressive

IOO-

l/loDo

A

l/loo

DILUTK)N

l/10 PWM

B

4 567810 DAYS IN CULTURE

FIG. 1. (A) IgG (closed symbols) and IgA (open symbols) pFC/106 viable lymphocytes present in peripheral blood lymphocyte cultures stimulated for 6 days with increasing concentration of pokeweed mitogen (PWM). (B) Kinetics of IgA production as PFCIloG viable lymphocytes in response to stimulation with PWM.

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WARRINGTON,

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RUTHERFORD

FIG. 2. Effect of increasing dilution of heterologous SaHIgA on PWM-induced response of normal peripheral blood lymphocytes as assessed by IgG (closed circles) and IgA (open circles) PFC/lOG viable lymphocytes.

the SaHIgA is not simply one of interference with the developing antiserum in the reverse plaque assay. IgA class-specific suppression was also induced by the immunosorbent-purified SaHIgA, in the presence of this antibody, IgA PFC being reduced from 8900 to 940 PFC/106 viable cells, while the equivalent amount of unpurified antiserum reduced the number of IgA PFC to 705 PFC/106 viable cells (mean of two experiments). The presence of SaHIgA in concentrations suppressive to IgA synthesis and secretion did not affect PWM-induced lymphocyte proliferation as assessed by [3H]thymidine incorporation (Table 1). Nor did the antiserum itself induce significant lymphocyte proliferation. Kinetics

of the SaHIgA-Induced

Suppression

Sequential addition of SaHIgA to PWM-stimulated lymphocyte cultures at a dilution of 1:SO resulted in suppression of the IgA response when addition was carried out from Days 1- 3 (Fig. 3) but did not suppress as completely when added at Day 4 (2 days prior to harvesting the cultures). The latter fact argues against the effect of the antisera being simply exerted upon the reverse plaque assay, by interfering with the effects of the developing antiserum. The IgG PFC response was not significantly affected by the addition of the SaHIgA as demonstrated previously. Similarly, sequential removal of the anti-IgA antibody at 4, 24, 96, and 144 hr showed that increasing suppression of the IgA response resulted with increasing duration of exposure of the PWM-stimulated cells to the SaHIgA (Fig. 4). Again, the IgG PFC response was not significantly affected by this maneuver (shown here because washing the cells during culture could affect immunoglobulin synthesis in general).

ANTIBODY-INDUCED

SUPPRESSION

TABLE EFFECTS

OF SHEEP ANTI-HUMAN INTO

OF

IgA

1

IgA UPON PWM-INDUCED [3H]T~~~~~~~~ PERIPHERAL BLOOD LYMPHOCYTES

Incorporation PWM, 1:lOO SaHIgA PWM + SaHIgA

377

SYNTHESIS

INCORPORATION

of [3H]thymidine (S - C)”

10,085* 265 9,610

12,476 57 10,736

” S - C = cpm (stimulated - control). ’ Mean of six cultures.

The presence of the SaHIgA did not simply delay the appearance of IgA PFC. Thus at Day 6, the presence of a 1:50 dilution of the antiserum suppressed IgA PFC from 2500 per lo6 cells to 0, while at Day 8 the response was reduced from 1410 per lo6 cells to 5 and at Day 10 from 1130 per lo6 cells to 0. Over this time, IgG PFC fell from 4400 per 106 cells to 1950. The SaHIgA did not affect the IgG PFC response. Fc Dependence of the Suppressive Effect of SaHIgA It has been previously demonstrated that of the two sheep IgG subclasses, IgG2 but not IgG1 binds to protein A (IS). In addition, there is evidence that the IgG2 subclass does not efficiently bind guinea pig complement, when compared to the IgG1 subclass (22). Confirmation of this was afforded by the demonstration that protein A purified sheep anti-human IgA did not produce plaques in the reverse hemolytic plaque assay. However, the protein A-purified fraction was able to inhibit PWM-induced IgA synthesis and secretion as efficiently as the intact antiserum, indicating that the ability to fK complement was not a prerequisite for suppression of the IgA response by the antiserum. In addition, F(ab’), of the Sa HIgA suppressed specifically IgA synthesis and secretion as efficiently as the intact antibody, similarly indicating the lack of Fc requirement for the suppressive effect (Table 2).

DAY OF ADDITION

OF SaHlgA

3. Effect of sequential addition of a suppressive (1150) dilution of SaHIgA on PWM-induced IgA PFC, expressed as percentage suppression compared to PWM cultures to which the SaHIgA was not added. FIG.

378

WARRINGTON,

SAUDER,

AND RUTHERFORD

1I0244872%

144

TIME OF WASHING

tin

OF CELLS

FIG. 4. Effect of sequential removal by washing of a suppressive (1150) dilution of SaHIgA on PWM-induced IgG (closed circles) and IgA (open circles) PFC/lOG viable lymphocytes.

B-Cell Dependence of the SaHIgA Effect

To assess the B-cell dependence of the SaHIgA effect, T and B lymphocytes partially depleted of monocytes by adherence were stimulated in vitro by PWM for 2 days, following which, T cells were removed by AET rosetting. The B cells were then returned to their original supernatant and half the cultures were exposed to a suppressive concentration of the SaHIgA and the cells were incubated for a further 4 days, at which time IgG and IgA PFC were quantitated by reverse hemolytic plaque technique. As can be seen from Table 2 while the SaHIgA did not affect IgG PFC, there occurred significant suppression of IgA PFC by 8%. TABLE Fc INDEPENDENCE IgA

2

AND B-CELL DEPENDENCE OF THE EFFECT OF SHEEP ANTI-HUMAN ON PWM-INDUCED IgA SYNTHESIS AND SECRETION

IgG PFC/lOB cells PWM, 1:lOO + SaHIgA” + SaHIgA F(ab’), B cellsb B cells + SaHIgA B cells + PWM B cells + PWM + SaHIgA

IgA PEC/ lo6 cells

13,400 12,450 11,300

4030 220 560

380 49

210 30

17,900 16,006

9600 1040

n SaHIgA and F(ab’), used at dilution of 1: 100. b B cells in medium from PBL cultures incubated with or without PWM for 2 days, then T cell depleted.

ANTIBODY-INDUCED

SUPPRESSION

379

OF IgA SYNTHESIS

Since it could be argued that the suppression induced by the SaHIgA in these circumstances could be mediated by an activity against some class-specific helper factor in the PWM-conditioned supernatants, Qday activated B cells, after T-cell removal by rosetting, were cultured after washing in fresh medium and serum for a further 3 days in the presence or absence of SaHIgA. In these circumstances also, IgA PFC were reduced from 416 per lo6 cells to 150 per lo6 cells, without a concomitant reduction of IgG PFC. This amounts to a 64% suppression of IgA PFC. By Day 4, maximal suppression of IgA PFC is not attainable (Fig. 3). However, B cells activated for a shorter period and then transferred to unconditioned medium did not maintain an adequate response. Although the possibility cannot be excluded, it seems unlikely that the effects of the SaHIgA were exerted through an activity against helper factors in these circumstances, since this would have to be directed against bound helper factor exposed on the activated B-cell membrane. @fects of IgA-Deficient Sera on PWM-Induced ZgA Synthesis Since it was proposed that the above in vitro model would be utilizable in the assessment of the effects of class-specific human anti-IgA antibody on mitogeninduced IgA synthesis, a small number of IgA-deficient sera were examined with regard to their ability to sustain PWM-induced IgA production. Such sera did not contain IgA by immunodiffusion and had less than 2 ng IgA/ml by radioimmunoassay (National Reference Laboratory, Canadian Red Cross). Two such sera [(a) and (b)], heat inactivated and at 10% in the culture, markedly suppressed PWMinduced IgA synthesis as compared to normal control AB serum (Table 3). Both these sera contained high levels of class-specific anti-IgA antibodies by radioimmunoassay (as demonstrated by the National Reference Laboratory, Canadian Red Cross) and by hemagglutination. In contrast, IgA-deficient sera without cy-chain-specific antibodies did not suppress the IgA PFC response. DISCUSSION

The data presented in this paper indicate that antibodies directed against IgA can have striking effects upon mitogen-induced IgA synthesis and secretion in

TABLE SUPPRESSION OF PWM-INDUCED IgA CONTAINING CLASS-SPECIFIC

Serum samples

&A-specific antibodies

3 PFC BY IgA-DEFICIENT ANTI-IgA

SERA

ANTIBODY

IgG PFC/lOG cells

IgA PFC/lOG cells

Percentage suppression

Control AB serum Serum (a) Serum (b)

+ +

11,000 10,800 12,400

4300 1980 965

54 78

Control AB serum Serum (c) Serum (d)

-

5,260 6,240 ~,~

4560 4240 4100

2 10

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SAUDER,

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RUTHERFORD

vifvo, causing suppression of IgA production while leaving IgG synthesis unaffected. The effect of the antiserum is mediated by the Fab portion of the antibody and is not Fc or complement dependent. These findings are similar to but not completely consistent with studies reported by Finkelman and Lipsky (7) using rabbit anti-human 6 and F antisera, in the form of F(ab’), where it was found that the antisera blocked PWM-induced IgG secretion while under some circumstances augmenting IgM production. The latter effect was dependent upon cell density and concentration of the anti-Ig, being more apparent at lower cell densities and antibody concentration. At higher concentrations, IgM secretion was suppressed. It was suggested that the anti-Ig might direct a PWM-induced switch from IgM to IgG secretion during mitogen-induced Blymphocyte differentiation. In the studies reported here, no enhancement of IgA secretion occurred under any circumstances. It may be that the initial stages of B-cell activation are more readily stimulated than later stages. Although other explanations for the effects of the antisera are possible, suppression of the switch mechanism would be consistent with the data reported in this study, since the full suppressive activity of the antiserum was not exerted if added after Day 3 of culture and could be removed by washing up to that time. Available evidence appears to indicate that in man detectable PWM-induced IgG and IgA synthesis occurs concomitantly rather than sequentially (29, 30). These experiments show that up to at least 24 hr in culture, there is little effect exerted by the antiserum, perhaps because the suppression is reversible initially and proliferation and differentiation of B lymphocytes committed to IgA synthesis and secretion has not yet progressed significantly while T-cell activation has begun. However, the subsequent appearance of comitted IgA-bearing cells may result, in the presence of the antiserum, in the expression of suppression. Similarly the sequential addition of anti-IgA inhibits IgA secretion maximally when present from Day 1 to 3. However, addition of the antiserum on Day 4 failed to achieve as complete suppression, suggesting that some cells already committed to IgA synthesis and secretion are resistant to the effects of the antiserum at this time. This may indicate that there exists a critical period prior to secretion and at the time of the switch when the effect of the antiserum is maximal. However, the actual effects of the heterologous anti-IgA upon the expression of surface IgA by activated lymphocytes remains to be evaluated. Although the activity of the SaHIgA in this study was not Fc dependent, being exerted by the F(ab’), of the antibody, as demonstrated also by other workers for anti-p, anti-y, and anti-8 (7, 23), the activity of the Fab monomers was not examined, so the dependence for the suppressive effect of capping and endocytosis of sIgA following exposure to the SaHIgA cannot be commented on in this system. However, other studies in experimental animals have suggested that such a phenomenon is not a requirement for anti-Ig-induced suppression of B-cell activity (24, 25). In such circumstances, other explanations must be sought. One theoretical possibility is an effect increasing cyclic AMP levels, resulting in inhibition of proliferation and possibly differentiation (26, 27). Alternatively, changes may be induced in the cell membrane preventing further activation of the cell by mitogen (28).

ANTIBODY-INDUCED

SUPPRESSION

OF IgA SYNTHESIS

381

The present findings regarding the ability of heterologous anti-human IgA antibody to suppress mitogen-induced IgA synthesis and secretion may have little relevance to the clinical situation. However, it is intriguing that a-chain-specific antibodies occur in a significant proportion of IgA deficients (9), perhaps 40% of such subjects, depending upon the level of serum IgA (31). It therefore is possible that these IgA-specific antibodies may play a role in some individuals in (a) the maintenance of IgA deficiency and (b) the pathogenesis of IgA deficiency, particularly in familial cases (10). With regard to the latter, experimental models are somewhat supportive of this hypothesis despite the fact that the suppressive effects of anti-IgA when administered neonatally may be reversible in part, perhaps reflecting a difference in the susceptibility of more mature cells to the antiglobulin as compared to immature B lymphocytes (32, 33). This may explain the longlasting effects of antibody seen in allotype suppression in the rabbit induced by transplancental passage of allotype-specific antibody (8), because of increased susceptibility of the fetus. An alternative explanation could be the generation of suppressor T cells in this situation, as has been demonstrated in a mm-me model of neonatally induced chronic allotype suppression (3). There is some support for the existence of both types of abnormality, i.e., suppressor T cells and defects in B-cell function, in some patients with IgA deficiency (34- 36). We have been able to demonstrate that serum samples from IgA-deficient subjects known to possess cu-chain-specific antibodies (as demonstrated by the National Reference Laboratory, Canadian Red Cross) will similarly suppress PWM-induced IgA synthesis and secretion. It is hoped to now show that this suppression is antibody mediated, to purify these antibodies and compare their mechanisms of action with that of heterologous anti-human IgA antibody. In so doing, valuable information may be obtained regarding one potential mechanism of IgA deficiency in man as well as useful data on the processes of activation and differentiation of human B cells. ACKNOWLEDGMENTS We would like to acknowledge the helpful discussions and collaboration of Dr. R. E. Petty in the development of this study and to thank Dr. Petty, Dr. M. A. Schroeder, and the Canadian Red Cross Blood Transfusion Service, Winnipeg, for supplying normal and IgA-deficient serum samples. We thank the National Reference Laboratory, Canadian Red Cross Society, for supplying data on IgA levels and the presence of anti-IgA antibodies in IgA-deficient sera. This work was supported by grants from the Manitoba Medical Services Foundation, Inc., and the Medical Research Council of Canada.

REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Manning, D. D., J. fmmuno/. 109, 1152, 1972. Murgita, R. A., Mattioli, C. A., and Tomasi, T. B. Jr., J. Exp. Med. 136, 209, 1973. Herzenberg, L. A., Okumura, K., and Metzler, C. M.. Transplunr. Rev. 27, 57, 197.5. Pierce, C. W., Solliday, S. M., and Asofsky, R.. J. Exp. Med. 135, 675, 1972. Schufiler, C., and Dray, S., Cell. Immunol. 10, 267, 1974. Anderson, J., Bullock, W. W., and Melchers, R., Eur. J. Immunol. 4, 715, 1974. Finkelman, F. D., and Lipsky, P. E., J. Immunol. 120, 1465, 1978. Dray, S., Nature (London) 195, 677, 1962. Ammann, A. J., and Hong, R., Medicine 50, 223, 1971. Oen, K. G., Petty, R. E., and Schroeder, M. L., Submitted for publication. Gronowicz, E., Coutinho, A., and Melchers, F., Eur. J. immunol. 6, 588. 1976. Kapfan, M. E., and Clark, C., J. Immunol. Methods 5, 131, 1974.

382

WARRINGTON,

SAUDER,

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RUTHERFORD

13. Boyle, W., Transplantation 6, 761, 1968. 14. Kaplow, L., Blood 26, 215, 1965. 15. Cuatrecasas, P.. and Antinsen, C. G., In “Methods in Enzymology” (W. B. Jakoby, Ed.), Vol. 22, p. 351, Academic Press, New York, 1971. 16. VolIer, A., Bidwell, D., and Bartlett, A., In “Manual of Clinical Immunology” (N. R. Rose and H. Friedman, Eds.), pp. 506-512, American Society for Microbiology, Washington, D.C.. 1976. 17. Gold, E. R., and Fudenberg, H. H., J. Zmmuno/. 99, 859, 1967. 18. Lind, I., Live, I., and Mansa, B., Acta Pathol. Micro&o/. &and. (B) 78, 673, 1970. 19. Rector, E. S., Carter, B. G., Kelly, K. A., Lang, G. M., and Sehon, A. H.. Eur. J. Immunol. 9, 471, 1979. 20. Lanzavecchia, A., Sacchi, G., Maccario, R., Vitiello, A., Nespoli, L., and Ugazio, A. G., &and. .I. Immunol. 10, 297. 1979. 21. Freijd, A., and Kundri, T., Stand. J. Immunol. 11, 283, 1980. 22. Feinstein, A., and Hobart, M. J., Nature (London) 223, 950, 1969. 23. Chiorazzi, N., Fu, S. M., and Kunkel, H. G., C/in. Immunol. Immunopathol. 15, 301, 1980. 24. Schuffier, C., and Dray, S., Cell. Immunol. 11, 367, 1974. 25. Sidman, C. L., and Unanue, E. R., J. Exp. Med. 144, 882, 1976. 26. Hadden, J. W., Coffey, R. G., Ananthakrishnan, R., and Hadden. E. M., Ann. N. Y. Acad. Sci.. 241, 1979. 27. Parker, C. W., Ann. N. Y. Acad. Sci., 255, 1979. 28. Kearney, J. F., Klein, J., Bockman, D. E.. Cooper, M. D., and Lawton, A. R., J. Immunol. 120, 158, 1978. 29. McLachlan, S. M., Rees Smith, B., and Hall, R., J. Immune/. Methods 21, 211, 1978. 30. Pryjma, J., Munoz. J., Virella, G., and Fudenberg, H. H., Cc/l. Zmmunol. 50, 115, 1980. 31. Holt, P. D. J.. Tandy, N. P., and Anstee, D. J., J. Clin. Pathol. 30, 1007, 1977. 32. Sidman, C. L., and Unanue, E., Nature (London) 257, 149, 1975. 33. Raff, M. C., Owen, J. J., Cooper, M. D., Lawton, A. R., Megson, M.. and Gathings. W. E., J. Exp. Med. 142, 1052, 1975. 34. Waldmann, R. A., Broder, S., Krakauer, R., Durin, M., Meade, B.. and Goldman, C., Trans. Assoc. Amer. Physicians 89, 215, 1976. 35. Atwater, J. S., and Tomasi, T. B. Jr., Clin. Immunol. Immunopathol. 9, 379, 1978. 36. Cassidy, J. I.. Oldham, G., and Platts-Mills. T. A. E., C/in. Exp. Immunol. 35, 296, 1979.