Regulatory role of circulating monocytes in the differentiative and proliferative responses of human B lymphocytes

CLINICAL

IMMUNOLOGY

AND

Regulatory Differentiative G. MONTAZERI,’ The Roc~c~fellrr

16, l-10 (1980)

IMMUNOPATHOLOGY

Role of Circulating Monocytes in the and Proliferative Responses of Human B Lymphocytes’ N. CHIORAZZI, Uuit~ersiry.

1230

3 S. M. Fu,” AND H. G. KUNKEL York A~‘enrru.

NPM’ YorX.

New,

York

10021

Received June 18, 1979 Depletion of monocytes from peripheral blood mononuclear cells had a profound influence on mitogen- and antigen-induced B-cell proliferation and differentiation to antibody-secreting cells. Such depletion enhanced the generation of plasma cells identitiable by immunofluorescence and specific plaque-forming cells (PFCJ against sheep erythrocytes in cultures containing pokeweed mitogen, staphylococcal protein A (SPA), concanavalin A (Con A). and phytohemagglutinin (PHA). This enhancement was especially marked in the cases of SPA and Con A. Without monocyte depletion only 0.1 to 0.4% of the cultured cells were shown to be plasma cells and as much as a loo-fold increase was seen with monocyte removal. Similar results were also obtained in the PFC assay. These studies suggest that Con A, SPA, and PHA may be considered as inducers of B-cell differentiation to plasma cells under appropriate conditions. In two B-cell differentiation systems initiated by antigen involving allogeneic helper cells and autologous helper factors, the monocyte-dependent inhibition was also demonstrated. In addition, monocyte depletion enhanced proliferation of B cells in the presence of irradiated autologous T cells and pokeweed mitogen. This enhancement was also seen when B cells were stimulated to divide by purified anti-p antibodies. The addition of adherent cells to monocyte-depleted cultures confirmed the suppressive effect of excessive monocytes but also demonstrated that the presence of a certain number of monocytes was necessary for optimal responses in at least some of the systems studied. The striking effect of monocytes in these different systems emphasizes the importance of their consideration in B-cell stimulation studies. especially those involving human peripheral blood.

INTRODUCTION

Cells of the macrophage-monocyte lineage play an important role in both initiation and regulation of immune responses. Studies mainly in animal systems have demonstrated both enhancing and suppressing effects for macrophages on lymphocyte mitogenesis (l-3), antibody production (4), mixed lymphocyte reactivity (5), and cytotoxicity (6). In addition, a few studies in man have demonstrated an inhibitory role for monocytes in several in vitro assays (7-9). In the present investigation experiments were carried out to define the regulatory roles of circulating monocytes from normal individuals on both B-cell differentiation to plasma cells and B-cell blastogenesis. These studies demonstrated both enhancing and suppressive effects of monocytes on the production of ’ This study was supported by U.S. Public Health Service Grants RR 102, Al 10811. AM 04761. and CA 24338. ” Present address: Department of Medicine. Medical School, Shiraz University, Shiraz, Iran. ‘j N. Chiorazzi is a recipient of a fellowship from the Arthritis Foundation. All correspondence should be addressed to him. ’ S. M. Fu is a scholar of the Leukemia Society of America, Inc. 1 0090-1229/80/050001-10$01.00/O Copynght c 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

MONTAZERI

ET

Al..

antibody-forming cells in several systems. In addition, was shown on B-cell blastogenesis. MATERIALS

a suppressive effect also

AND METHODS

Sourer crud .sepwwtion c:f’ tmmotzucle(tr culls ( MNCI. i Tonsillar tissue from routine tonsillectomy specimens was processed as described previously ( IO). Mononuclear cells from the tonsillar cell suspension and heparinized blood from normal donors were obtained on Ficoll-Hypaque gradients, washed three times, and resuspended in RPM1 1640 (Microbiological Associates, Walkersville, Md.) supplemented with either 10% fetal calf serum (FCS) or 5% human AB serum. Isolation of rosetfe~fkmimg culls (RFC). Spontaneous, nonimmune rosette formation between human lymphocytes and sheep red blood cells was performed with neuraminidase-digested SRBC as previously described (11). RFC-enriched and RFC-depleted populations of the whole MNC suspensions were isolated by centrifugation on Ficoll-Hypaque gradients. The RFC-enriched pellets were purified further by a second density gradient centrifugation. The RFC-enriched populations (T-cell fraction) routinely contained 97% or more RFC: the RFCdepleted populations (B-cell fraction) generally were comprised of 95% non-RFC. RFC were obtained by the lysis of sheep red blood cells with Tris-buffered NH,&1 solution. Depletion oj’adheretu cells. Adherent cells were removed from peripheral blood MNC suspensions by adherence to plastic. Four x 10” MNCiml in RPM1 1640-10% FCS were incubated in 24-well Linbro tissue culture plates (Linbro. Hamden, Corm.) at 37°C in 5% CO, for 90 min. The cells were then resuspended vigorously and the nonadherent cells transferred to a sterile tube. Cells to be used in wh-o subsequently were washed two additional times with RPM1 1640. In certain experiments varying numbers of peripheral blood MNC were incubated under these conditions to prepare monolayers of adherent cells. Renzo~~ul of phagocyric cells. Peripheral blood MNC were rotated in a plastic tube with a suspension of carbonyl iron (20 mg/lOi cells) in RPM1 1640- 10% FCS for 1 hr at 37°C. Cells which had ingested the metal were immobilized by a magnet and the remaining cell suspension was removed by careful pipetting. The phagocyte-depleted cells were washed twice in RPM1 1640 and resuspended in culture medium. Idenr$icariort of’tnonocytes by ttottsprct’tk pc~rmidusr srclinitlg. Monocytic cells containing peroxidase granules were identified by a modification of the technique of Kaplow (12) utilizing 3,3’-dimethoxy benzidine (Sigma Chemical Co., St. Louis, MO.). Cultrrrr conditions. All cell cultures were seeded on 24-well Linbro tissue culture plates in a total volume of 2.0 ml of RPM1 1640 medium supplemented with 100 units of penicillin/ml. 100 pug streptomycin/ml, and either loci;, FCS or 5% human serum. ’ Abbreviations used: Con A. concanavalin A: FCS. fetal calf serum: Ig. immunoglobulin: MLR, cells: PB. peripheral blood: PBS, phosphate-buffered forming SRBC.

cell: sheep

PHA. phytohemagglutinin: red blood cells; SPA.

PWM. staphylococcal

E,. neliraminida~e-clige~te~l mIxed lymphocyte saline without pokeweed protein

mitogen: A (solubleI.

reaction: Ca’. or RFC.

bheep red blood cell> MNC. mononuclear Mg”: PFC. plaquerosette-forming

cells.

REGULATION

OF

B CELL

ACTIVATION

BY

MONOCYTES

3

Cell Culture (a) Mitogen-stimulated cultures. Individual cultures contained either 2.5 x 10’ unseparated MNC or 2.0 x 10” monocyte-depleted MNC from PB or tonsillar tissue. Pokeweed mitogen (Gibco, Grand Island, N.Y.), Protein A (Pharmacia Fine Chemicals, Inc., N.J.), and concanavalin A (Sigma Chemical Co., St. Louis, MO.) were added as polyclonal activators at final dilutions of 1: 100, 10 &ml, and 10 &ml, respectively. In certain cultures 10% fetal calf serum selected for the ability to support PFC generation (Flow Laboratories, Inc., Rockville, Md.) was used in place of AB serum. (b) Antigen-stimulated, allogeneic cocultures. One to 1.5 x lo6 cells from the B-cell fractions of either tonsillar or PB MNC were cultured with or without an equal number of allogeneic PB T cells. One million washed SRBC served as stimulatory antigens. (c) Generation of autologous mi.ued lymphocyte reaction (MLR) supernatants. Four x 10” unseparated peripheral blood MNC were cocultured with 4 x 10” y-irradiated (3000 R) non-rosette-forming autologous cells (B + macrophage) in RPM1 1640-5% AB serum. After 48 hr, supernatants were collected, filtered through a 0.45pm Millipore filter, and either used immediately or frozen at -70°C.

Direct plaque-forming cells (PFC) and immunojluorescent staining for intracytoplasmic lg. Antibody-forming cells with specificity for SRBC antigens were detected by a modification (13) of the Jerne-Nordin PFC assay. The procedure for the detection of intracellular immunoglobulin by immunofluorescence has been reported previously (14). RESULTS

Enhanced PFC Responses to Mitogens by Monocyte-Deficient Peripheral Blood MNC A comparison was made between the ability of peripheral blood (PB) MNC containing monocytes and those depleted of monocytes to differentiate to antibody-secreting cells in response to mitogens. This differentiative response was assayed by enumerating the number of anti-SRBC PFC and the number of cells containing large amounts of lg (plasma cells) generated in companion cultures. Table 1 enumerates the results of two representative experiments using pokeweed mitogen (PWM), soluble staphylococcal protein A (SPA), and concanavalin A (Con A) as polyclonal activators. In these experiments, monocytes have been defined as phagocytic and/or adherent cells with positive staining for peroxidase granules. The control cultures contained 21 and 26% monocytes, respectively, whereas the monocyte-depleted cultures contained 1.O% cells staining for peroxidase granules. Monocyte-depleted cultures generated substantially more anti-SRBC PFC than control cultures containing unseparated MNC. This enhanced antibody production occurred with PWM, SPA, and Con A as stimulants. The degree of enhancement varied from experiment to experiment and from mitogen to mitogen, with a range of 3- to loo-fold increase in response. Background PFC levels of unstimulated cultures were not appreciably elevated. In these experiments specific PFC to SRBC antigens served as a representative measure of nonspecific, polyclonal activation.

4

MONTAZERI

ET

AL.

This enhancement of antibody synthesis by PB MNC after monocyte depletion was further documented by immunofluorescent staining for cells containing large amounts of immunoglobulin. There was a direct correlation between the number of plasma cells of all antibody classes and specific direct PFC (Table 1). This enhanced generation of antibody-forming cells was seen in monocyte-depleted cultures stimulated with all three mitogens (PWM, SPA, and Con A). Five- to eight-fold increases in plasma cells were seen in PWM-stimulated cultures, 30- to lOO-fold increases after SPA exposure, and lo- lOO-fold enhancement with Con A. The plasma cells in SPA-stimulated cultures morphologically were more akin to plasmacytoid lymphocytes than plasmablasts or typical plasma cells, Supplementation of the culture medium with either AB serum or a selected lot of FCS was irrelevant to the observed responses. Three additional experiments showed similar results. In a series of similar experiments with three individuals using phytohemagglutinin (PHA) the number of plasma cells generated rose from 2 to 14% when optimal numbers of adherent cells were present. Effect oj’ Adherent Cell Rep&cement on the Grnrrcltiorl (!I’ /)ircct Anti-SRI% PFC rend Plasma Cells In order to clarify further the role of monocytes in the regulation of B-cell differentiation, monocyte-depleted MNC were cultured with PWM in the presence or absence of different numbers of autologous adherent cells. Adherent cells were obtained by incubating varying numbers of unseparated PB MNC (0.3--5.0 x 106) in plastic culture wells for 90 min at 37°C. Wells were washed vigorously three times to eliminate nonadherent cells. There was correlation between the

Percentage No.

~~

No mitogen

~~

Experiment

of direct

cells contaimng plasmic Ig’

PFC/culture

intracylir~

NO PWM

SPA

Con

.4

mitogen

PWM

SPA

Con

<5

145

c.5

.5

0. I

Monocyte-depleted

.55

980

II0

125

0.6

4.x

0.4

26.0

II

II.1 I

u.4

2”

Unseparated


1’5

‘C 5

I

I.6

2.1

0 I

Monocyte-depleted

,:5

360

95

1x0

-7.1

I6 7

12 I

” Cultures consisted of 2.5 x IO” addition of PWM (l:lOO), SPA (IO carbonyl iron technique. ” The percentage of cells present Ig by Rhodamine-conjugated rabbit ’ Percentage monocytes human AB serum. ” Percentage serum.

.A

I”

Unseparated

Experiment

calf


monocytes

present: present:

unseparated &ml). or

or monocyte-depleted Con A (IO &ml).

at the termination antisera to human unseparated: unseparated:

2l”r: 26’;

PB Monocytes

MNC

il.4 Ii

with or without were removed by

of culture which were stained rntracellularlv K and A light chain determinants. depleted: : depleted:

1.0%.

Culture

I .O’ ;. Culture

medium medium

I the the 1~

contatned

contalned

fetal

REGULATION

OF

B CELL

ACTIVATION

BY MONOCYTES

5

number of adherent cells and the number of cells plated. Approximately 90% of those cells found adherent to the plastic contained peroxidase-positive granules. The results of such a monocyte reconstitution experiment are illustrated in Fig. 1. After exhaustive depletion of monocytes (less than 0.1% of the cultured cells were peroxidase positive), less direct anti-SRBC PFC were detected than in the control. However, by replacing graded numbers of adherent cells, the PFC response initially increased, and then fell below control levels again. The best PFC response was obtained in cultures reconstituted with the number of adherent cells found in 1.25 x lo6 unseparated PB MNC. Marked suppression was seen when the number of adherent cells exceeded those contained in 2.5 x 10” MNC. The number of plasma cells generated in these same cultures was assayed by intracytoplasmic staining for K and A chain determinants (Fig. 1). The depletion of monocytes to the 0.1% level enhanced plasma cell generation, resulting in a 70% higher level than the unseparated MNC. The addition of the number of adherent cells contained in 0.6 x lo6 MNC to the monocyte-depleted culture gave an optimal response of plasma cell generation, i.e., 233% of the culture containing unseparated cells. When excessive numbers of adherent cells were used, the response was only 60% of the control. The Effect of Monocyte-Depletion on the Generation of Antigen-Initiated PFC In the previous sections the regulatory role of monocytes on antibody production was investigated in systems of polyclonal activation using various mitogens. The experiments presented in the next two sections were performed to determine if similar monocyte effects occurred in antigen-initiated antibody responses. Indeed, the inhibitory effects of monocytes on antibody synthesis in rlitro were also

FIG. 1. Effect of adherent cell replacement on the PWM-induced generation of antibody-forming cells. Three x IO” monocyte-depleted PB MNC were cultured in the presence or absence of the number of adherent cells present in 0.3-5.0 x IOh PB MNC. All cultures were stimulated by PWM (I: 100) for 5 days. Monocyte-depleted mononuclear cells contained 0.1% peroxidase positive cells. while the adherent cells were 90% positive. Unseparated MNC which served as controls and were positive cells. cocultured in the same manner with PWM had ?7_S peroxidase

6

MONTAZERI

ET AL.

apparent in an antigen-specific, alloantigen-driven PFC system in which potent helper activities were generated during mixed lymphocyte reactions (MLR) (10. 15). One x IO6 B or monocyte-depleted B cells and equal numbers of allogeneic T cells were cocultured in the presence of 1 x IO” SRBC for 5 days. Few specific PFC were found in cultures of PB B-cell fractions which contained a high proportion of monocytes (65% average). However, in the monocyte-depleted cultures (4% average), from 250 to 2750 direct anti-SRBC PFC were detected in four experiments with different donors. Monocyte depletion by both carbonyl iron ingestion and adherence was effective, although the carbonyl iron ingestion method was more efficient in allowing the PB B cells to produce antibody. In order to allow a more direct comparison of the potential abilities of B cells from the tonsil and peripheral blood to generate antibody-secreting cells and the effect of monocytes on this process, a similar coculture experiment was performed using PB and tonsillar cells from the same donor (Table 21. In this experiment PWM-induced responses were used for comparison. The unseparated MNC obtained from both sources gave a similar pattern of antibody responses when exposed to SRBC or PWM, i.e., few PFC were seen with specific antigenic stimulation and a moderate number of plasma cells were detectable after mitogenic exposure. However, a striking difference between the B-cell fractions from peripheral blood and tonsil of this donor was seen when the cultures were supplemented with allogeneic PB T cells and antigen. Whereas the tonsillar B lymphocytes produced large numbers of specific PFC in allogeneic coculture. the corresponding PB B-cell fraction did not yield PFC. After monocyte depletion. however, this same PB B fraction produced a level of anti-SRBC antibodyproducing cells comparable to the autologous tonsillar counterpart. An additional experiment with a different donor gave similar results. A comparison of the number of nonspecific, peroxidase-staining cells in the B-cell fractions derived TABLE COMPAKISOS

OF

I tit.

PFC

RESPONSES

BLOOD

VERSUS

7

GENER~I-ED

R\

‘THE TONSIL

FROM

MONO~I

c it AK C‘L.I t \ ot

Direct Percentage monocyteh

I t+i

Ptfutwt

~$1

THE .S.-r,wt DO,VOR”

anti-SRBC

PFC’tculture

SRBC

PWM

x70

700

PB Unseparated PB B cells PB B + allogeneic T cells Monocyte depleted PB B + allogeneic T cells Tonsil Unseparated Tonsillar B cells Tonsillar B + Allogeneic T cells

20 65 65 5

3 x

x

” 2.5 Y 10” unseparated cells, I * 10” B cells, from tonsil or PB. or 1 Y IO” B cells of either source. and 1 Y 10” allogeneic T cells were cultured in the presence of I x IO” SRBC or PWM for 5 days.

REGULATION

OF

B CELL

ACTIVATION

7

BY MONOCYTES

from tonsillar versus PB MNC demonstrated that tonsillar B-cell fractions contained an average 8% (3-20) peroxidase-staining cells whereas the PB B-cell fractions contained 65% (52-78) peroxidase-positive monocyte cells. Inhibitory Effect of Monocytes on the Antigen-Specific Stimulation of PB MNC by Autologous MLR Supernatants Supernatants obtained from 48-hr autologous and allogeneic MLR provide helper activity for antibody synthesis by monocyte-depleted PB MNC (16, 17). In the three experiments presented (Table 3), SRBC alone did not stimulate PB MNC to make PFC either in the presence or absence of monocytes. However, in the presence of autologous MLR supernatants, SRBC-stimulated cultures of monocyte-depleted PB MNC did produce antigen-specific PFC. This PFC production did not occur without inversion depletion. The responses to PWM are presented as comparisons for the degree of activation. The antigen-initiated response obtained in these monocyte-deficient cultures was 60-220% of the PWM response. In Experiment 2 the unseparated MNC showed a better response to PWM than monocyte-depleted cells. This may relate to a difference in the efficiency of depletion between Experiments 1 and 2 since a certain number of monocytes are necessary for a PWM-initiated response (Fig. 1). Studies with allogeneic MLR supernatants corroborated the findings that monocyte depletion was important for the generation of an optimal antibody response using peripheral blood MNC. THE EFFECT

OF MONOCYTE

TABLE 3 REMOVAL ON ANTIGEN-INITIATED PFC MLR SUPERNATANTS AND SRBC” Direct

anti-SRBC

INDUCED

BY AUTOLOGOLIS

PFClculture

Nothing

SRBC

SUP

SRBC + MLC sup

Experiment 1” Unseparated Monocyte-depleted

15 <5

<5 <5

<5 <5

<5 109

<5 125

Experiment la’ Unseparated Monocyte-depleted

<5 <5

C.5 <5

<5 10

<5 185

15 196

Experiment 2” Unseparated Monocyte-depleted

<5 <5

<5 <5

<5 55

<5 72

<5

MLC

PWM

20

” 3.5 x 10” unseparated or monocyte-depleted MNC were cultured in 2 ml RPM1 1640 serum for 5 days with SRBC in the presence or absence of an optimal dilution of autologous MLR supernatant. Experiments 1 and 2 were performed using PB MNC from two different individuals; Experiments 1 and la were performed with the same donor MNC on separate days with separate autologous MLR supernatants. ” Monocytes present: unseparated-20%: depleted < lv. ’ Monocytes present: unseparated-13%; depleted 11%. ” Monocytes present: unseparated-22%: depleted < 19.

8

MONTAZERI

E.1

EjJect of Monocvtes on the Prolijkrati~~e Mitogen and Anti-p Chain Antibodies

AL.

Response

of‘ B Cells to Pokelr!crd

Since the previous experiments demonstrated a regulatory role for circulating monocytes in the differentiation of PB B lymphocytes, the effect of monocytes on the proliferation of B cells was also evaluated. This was approached by stimulating B cells by two different methods in which only B-cell division could occur: (i) PWM and irradiated T cells, and (ii) anti-p antibodies. B-cell fractions containing monocytes or depleted of monocytes were incubated with irradiated (3000 R), syngeneic T cells and PWM for 5 days (Table 4). In order to avoid the difficulties in interpretation that varying numbers of potential proliferating cells per culture might produce, two dilutions of B cells were used. In this way comparisons were made both on the basis of the total number of cells/ culture and on the number of pure B cells/culture. Monocyte removal significantly enhanced B-cell proliferation to PWM. In particular, 0.75 x lo5 cells of the monocyte-depleted B-cell fraction incorporated six to eight times more thymidine than 1.5 x lo” cells of the untreated B-cell fraction, although approximately the same number of pure B cells was present in each culture. An assay of the number of SRBC-RFC present at the initiation of culture was only 2% and about 6% at the end of the culture. Therefore, the majority of cells dividing were of the R-cell lineage. Total cell counts and blast counts at the end of the culture paralleled [“Hlthymidine incorporation results. The effects of monocyte removal on B-cell proliferation were tested in the second system, i.e., by stimulating with purified antibodies to IgM (18). This proliferative stimulus has been shown to be effective in the absence of T cells. Untreated and monocyte-depleted B-cell fractions with 38 and 5% monocytes. respectively, were cultured with anti-p antibodies for 3 days and then assayed for [“Hlthymidine incorporation. The monocyte-depleted B-cell fraction gave a count of 25,000 and the untreated B-cell fraction only 3500. In other experiments division was not observed when purified peripheral T cells were stimulated in a similar

EIUtlAi5Cf.f~

POKE WFFI)

MI

IOGLN

(‘FI)

T-ABLE 4 DNA SYN I HI.SIS

MONOO

I t Rthzo\,sr

IN01

Number ceils/culture Experiment

PtRIPtiERAl

BI

~

B

Cct

L 5

RI

of

Percentage monocytes

Medmm

PWM

I

Unseparated Monocyte-depleted Unseparated

B cells

Experiment 2 Unseparated Monocyte-depleted Unseparated Monocyte-depleted

I.5 I.5 0.75

‘\ IO’ b IO’ * lo”

B cells

0.75

* lo”

I.5 1.5 0.75 0.75

1 k x ‘<

B cells B cells B cells B cells

‘I I .5 * IO” irradiated either with or without with

B cells B cells

Monocyte-depleted

pulsed

OI

”

2 /*Ci

(3000 monocyte

of I:‘H]thymidine

R),

autologous depletion and

,

5,227 761 2,078

6.5 5

60

I@5 lo” lo” lo”

.: 6U 3

7‘ cells were in the presence harvested

92s

6s

cocultured of PWM

12 hr later.

with ( I: 100).

X.YI)S 3 I.392 24,xxo X,09

3,760 9.506

14.636 40.540

2,400 4,343

47,833 X6.756

I

I .5--0.75 y IO9 PB B cells After 5 days cultures wet-e

REGULATION

OF

B CELL

ACTIVATION

BY MONOCYTES

9

fashion with the same antibody preparation. Similar results were obtained in additional experiments using monocyte-depleted peripheral blood B cells from several separate donors. Quantitation of the proliferative response using [“Hlleucine label or blast cell enumeration gave comparable results. DISCUSSION

The present studies demonstrate that monocytes exert a very significant regulatory role in the differentiation of B cells to plasma cells in vitro. Removal of monocytes enhanced the generation of both plaque-forming cells and plasma cells by peripheral blood mononuclear cells in the presence of mitogen. In the case of pokeweed mitogen, a three-fold increase was seen. The other two mitogens, namely staphylococcal protein A and concanavalin A, have not been generally recognized as inducing B-cell differentiation and, indeed, they were shown not to result in significant B-cell maturation in unseparated mononuclear cell cultures. However, after monocyte depletion as much as loo-fold increase of plasma cell generation was detected. In addition, evidence was also obtained showing that PHA induced a 7-fold increase in plasma cell generation of peripheral blood mononuclear cells in the presence of optimal numbers of adherent cells. Thus, under these conditions staphylococcal protein A, concanavalin A, and phytohemagglutinin act as B-cell mitogens. Their action on B cells is probably similar to that of PWM, i.e., via T-cell factors. This inhibitory effect of monocytes was also evident in two systems in which antigen-initiated B-cell differentiation was evaluated, i.e., the allogeneic helper cell and MLR supernatant systems. These systems have been shown to result in both antigen-initiated and polyclonal responses (15 - 17) and thus provided important corroborative data to those generated with the various mitogens. Monocyte-dependent inhibition was most dramatically illustrated in the experiments involving tonsillar B layers and peripheral blood B layers from the same individuals. Reconstitution experiments provided additional evidence for this inhibitory effect. It also was apparent that a small number of monocytes were necessary for the generation of antibody responses. This latter finding is in agreement with that previously reported (19). In the present investigation the potential inhibitory effects of monocytes on human PB B-cell proliferation were also apparent. The two blastogenesis assays employed require further comment. In the case of PWM stimulation, T cells are known to be needed to generate mitogenic factors which affect B cells (20). In the present report T cells were irradiated and analyses at the initiation and the termination of the culture indicated that insignificant proliferation of T cells occurred. Thus, the DNA synthesis represented B-cell proliferation under these conditions. In the case of stimulation by anti-p antibodies, extensive evidence has been obtained to document that Ig+ B cells are the target for these antibodies and B-cell proliferation is independent of T cells (18a). Thus, monocytes exert suppressive effects on B-cell proliferation in two apparently unrelated systems. The exact mechanism responsible for the monocyte-dependent suppression in these different systems remains to be clarified. Reconstitution experiments utilizing cell populations comprised of greater than 90% peroxidase-staining cells indicated that monocytes play a central role in the inhibition of B-cell responses. In the case of mitogen stimulation, some T-cell factors probably act on monocytes

10

MONTAZERI

ET Al.

which in turn suppress B-cell function either directly or indirectly. This is indeed the case in the Con A-induced suppressor cell system (21, 22). Whether this is also the mechanism for monocyte action in the two antigen-dependent systems is not apparent. In the case of anti-p antibody stimulation, T cells are not involved and this mechanism cannot be the explanation. Recently prostaglandins have been incriminated as playing a major role in various macrophage suppression systems (23) and indomethacin has been shown to reverse some of these suppressive effects. Indeed, our experience has shown that indomethacin also reversed partially the inhibitory effect of monocytes. However, since this reversal was not complete, it suggests additional factors are involved. Profound suppressive effects of monocytes on antibody responses have been reported in certain disease states such as multiple myeloma (71, Hodgkin’s disease (81, and sarcoidosis (9). In the present studies these suppressive effects have been demonstrated in normal individuals in a variety of different systems. Although monocytes by themselves may not be responsible for the varying degrees of responsiveness in all cases, it is evident from our data that these cells require consideration in any in llitro study on antibody synthesis especially in human peripheral blood where the monocyte concentration is so high. ACKNOWLEDGMENTS The authors assistance and

thank Linda

Mary Light

Margaret Zansitis, Alice for secretarial assistance

Mayer. and Ruth in the preparation

Brook? of this

for skilled manuscript.

techntcai

REFERENCES I. 2.

Parkhouse. Calderon.

3. 4.

Yoshinaga, Mosier.

5. Oehler, 6. Weiss, 7. 8.

R. M. E., and .I.. and Unanue.

Dutton. E. R..

R. W.. ./. I~I!!II~~I,I/. 96, 663. 1966. ,t’
M.. Yoshinaga. A., and Waksman. D. E., .Scirrlc,e 158, 1573, 1967. J. R.. Herberman, R. B., Darrell. A., and Fitch, F. W., ./. //>tn~r/rr~,/.

B. H..

J. R., and 119. 510,

Knapp, W.. and Baumgartner. G.. ./. /r~t~vlr!t~~l. 121. Goodwin, J. S., Messner. R. P., Bankhurst, A. D.. R. C., Jr., h’. El/g/. J. Mrd. 297. 963. 1977.

9. Katz. P., and Fauci. A. S., C/i),. E.\-/I. Irrr/~lr,/~o/. 32. IO. Chiorazzi. N.. Ferrarini. M., Montazeri, G.. Hoffman. tion pp.

in Man: 159-271.

I/r Virro Synthesis Academic Press,

I I.

Hoffman, T.. (B. R. Bloom

12. 13.

Kaplow, Fauci.

14.

Fu.

and and

Winchester.

USA 71.4487.

tt/r

Djeu. 1977.

f..

Implication” 1979.

136.

J. Y.. f’e/i.

1177, Peake. 5%.

.2lt,tl.

1978. G. I‘.. lY7X. and Fu,

(A.

Saikt.

S. M..

S. Fauci

1965. .I. t.r~1.

R. J.. Feizi,

T..

tletl. Walzer.

144,

674.

956.

1972

/n~,,,/r&.

and

Kunkel. H. G.. f/t “1,~ I’iiro Method\ tn Cell Medtated J. R. David. Eds.). pp. 71-X1. Academic Press, New

L. S., RIO& 25, 215. A. S., and Pratt, K. R.,

S. M.,

and Clinical New York,

./

J. H..

29.

238.

and

1977.

Williams.

/,I “Antibody

Produc-

R. G. Ballieux, and 1.umor York. 1976.

Ed<.).

Immuntty‘

1976,

P. D., and

Kunkel,

H. G.. I’ro(.

.%(I/. il( (id

1974.

15. 16. 17.

Chiorazri, N., Fu. S. M., and Kunkel, H. G.. ./. ,%r/‘. ,Z/rll. 149, 1543, 1979, Chiorarzi. N.. Fu. S. M.. and Kunkel. H. C., /f,l,,rrfrr,,/. KVI. 4.5. 719. 1979. Yu, D. T. Y.. Chiorazzi, N., and Kunkel. H. G.. CC,//. /rtvn,~,r~~/.. 50. 305. 1980.

18. 18a. 19. 20. 21. 22. 23.

Chiorazzi. N.. Fu. S. M.. and Kunkel. H. G.. I‘(>(/. Prr>< 38. 1082, 1979. Chiorazzi. N., Fu, S. M., and Kunkel. H.. Clirl. Ivtrncc~~r~. /,rrvr/,n~,/~trrh. 15, 301, IYXO. Rosenberg. S. A.. and Lipsky, P. E.. .I. /,r~~vrr/ro/. 122, YX. 1979. Insel. R. A.. and Merler. E.. .I. I~~tnro,l<~l. 118, 2009. 1977 Rich, R. R.. and Pierce. C. W., J. Er11. Me
.s(./