The regulation of polyclonal immunoglobulin synthesis by FcR+ and FcR− monocyte subsets

CLlNlCAL

IMMUNOLOGY

AND

37, 245-252 (1985)

IMMUNOPATHOLOGY

The Regulation of Polyclonal by FcR+ and FcR-

PRYJMA, ANNA PITUCH-NOWOROLSKA, IRENA RUGGIERO. AND MAREK ZEMBALA

JULIUSZ

Department

lmmunoglobulin Synthesis Monocyte Subsets

of Clinical

Immunology,

Institute Cracow,

of Paediatrics, Poland

Copernicus

Medical

School,

FcR+ and FcR- monocyte subsets were added to the pokeweed mitogen (PWM) or aureus Cowan I-stimulated cultures of peripheral blood mononuclear cells (PBMC) or to PBMC depleted of monocytes. The numbers of immunoglobulinsecreting cells (IX) and cells with intracytoplasmic immunoglobulins (PC) were evaluated 6 days later. The addition of FcR- subset increased the number of ISC in cultures of PBMC stimulatedwith PWM andreconstitutedthe response of monocyte depleted Staphylococcus

PBMC. In contrast,FcR+ monocytessuppressed PWM-inducedresponse and, when addedin highdose,alsothat inducedby S. aureus. The FcR+ monocytes suppressed the response by inhibition of immunoglobulin secretion but not the development of PC. This suggests that FcR+ monocytes may modulate humoral response by preferential inhibition of the final differentiation of B lymphocytes into ISC. o 1985 Academic press, I~C.

INTRODUCTION Lymphocyte responsiveness in vitro to antigens and mitogens requires the presence of monocyte-macrophage cell lineage which provides antigen presenting or accessory activity [l-4]. However, monocytes also suppress the T- and B-cell responses [5-71. The recent data strongly suggest that this cell population is heterogeneous, and several functional subsets have been characterized in the mouse [S, 91. Human peripheral blood monocytes (MP)) are also heterogeneous and differ in the expression of surface markers, receptors, size, and morphology ilO, 111. We have recently reported that human MP) can be separated according to the presence or absence of Fc receptor (FcR) into the subsets which differ in their ability to support and modulate T-cell proliferation induced by mitogens or antigens and cytostatic activity [ 121. The present studies were designed to evaluate the influence of FcRf and FcRMP) subsets on polyclonal immunoglobulin production in vitro. Two different methods of polyclonal B cell activation, namely stimulation with pokeweed mitogen (PWM) and Staphylococcus aureus Cowan I, were chosen, as they represent different mechanisms involved in triggering the B-cell response. PWM-induced immunoglobulin production in vitro is T-cell dependent [13, 141, requires monocytes as accessory cells [15, 161, and is also subject of monocyte-mediated suppression [17]. Therefore, polyclonal B-cell activation induced by this mitogen 245 0090-1229185 $1.50 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.

246

PKYJMA

ET AL.

can be considered as a suitable model for studying diverse effects attributed to monocytes. In contrast, S. nureus Cowan I can induce B-cell proliferation without T-cell help [18], but T-cell factors are necessary for their maturation into immunoglobulin-secreting cells [ 191.

MATERIALS

AND

METHODS

Lymphocytes. Peripheral blood mononuclear cells (PBMC) were isolated from EDTA-treated venous blood of healthy donors by standard Lymphoprep (Nyegaard, Norway) gradient. After washing the cells were resuspended in RPM1 1640 (GIBCO) medium, supplemented with 10% of foetal bovine serum (FBS, GIBCO), 2 mM L-glutamine (GIBCO) and antibiotics (complete medium), and adjusted to the concentration of 1 x 106/ml. Monocyte depleted PBMC. Ten milliliters of PBMC (2 x 106/ml) in complete medium were incubated (37”C, 5% CO,) for 1 hr on glass petri dish (100 mm). The non-adherent cells (NAC) were carefully collected, washed, and adjusted in the complete medium to the concentration 0.5 x 106/ml. Alternatively, PBMC were depleted of monocytes by passing through Sephadex G-10 (Pharmacia) column as described by Alonso et al. [21] and adjusted to the concentration 0.5 x lO?ml. These would also be called subsequently NAC. M@ content was monitored by euchrysine fluorescence microscopy [4]. Only NAC preparations contaminated with less than 1% of monocytes were used. Isolation of monocytes and their subsets. PBMC depleted of T cells [20] were allowed to form rosettes with human “0,” Rh+ (D) erythrocytes coated with human IgG anti-D antibody (Biotest, West Germany) and then overlayered on Lymphoprep. After centrifugation for 30 min at 400g cells from pellet (rosetting) and interphase (non-rosetting) were recovered. Both populations were then allowed to adhere onto FBS-precoated plastic petri dishes (Sterilin) for 45 min, and adherent cells were released with 10 mM EDTA. FcR+ M@ (adherent cells for the pellet) and FcR- M@ (adherent cells for interphase) were 87-95% pure MO as determined by euchrysine staining. Whole population of MQl (AC) was obtained by adherence from T-cell depleted PBMC. (For details see [12].) Mitogens. Pokeweed mitogen (Gibco) was used in cultures at a final dilution of 1: 100. Suspension of killed S. aureus Cowan I [18] was added to the culture in a final concentration of 1:2500 (v/v). Culture conditions. PBMC were cultured (37°C. 100% humidity, 5% CO,) for 5-6 days in the presence of PWM or S. aureus in round-bottom Falcon 2057 tubes in l-ml aliquots. To some tubes various numbers of AC, FcR-, or FcRf M@ were added at the beginning of the culture. NAC (0.2 ml) were cultured for 6 days in Falcon 3040 microplates in quadruplicate alone or together with various numbers of M@ subsets and in the presence of PWM or S. aureus. Evaluation of response. The number of cells actively secreting immunoglobulins (ISC) and the percentage of cells with cytoplasmic immunoglobulin (PC) were evaluated accordingly by the reverse plaque technique and a direct immunofluorescence as previously described [22, 231.

MONOCYTE

REGULATED

Ig SYNTHESIS

25

. FcR.----;.=z=.::

20

I

,'

10

2'5

x

247

IN VITRO

20

o FcR+ fi AC 10

5

25

10

20

Monocytes 1 FIG. 1. PBMC depleted of monocytes by passing through Sephadex G-10 column (experiment I and experiment 2 ---) or by adherence (experiment 3 -.-) were cultured with FcR+ and FcR- monocyte subsets or adherent cells (experiment I) added. The response was evaluated by the reverse plaque assay. (x 103

RESULTS Both FcR- and FcR’ Non-adherent Cells

M@ Subsets Can Reconstitute

Response

of

Monocyte-depleted PBMC (NAC), when cultured in the presence of PWM at low cell density, cannot be triggered into immunoglobulin production unless adherent cells are added [IS]. This cell culture system was chosen to test the ability of MIZ) subsets to act as accessory cells. NAC (lO’/well) were cultured in the presence of PWM with various numbers of AC, FcR-, and FcR+ M@ subsets. The results obtained in three representative experiments are shown on Fig. 1. PWM could not trigger substantial response in culture of NAC, although some ISC were detected in the majority of experiments (200-4000 ISC/106). Addition of 2.5 x lo3 of FcR+ or FcR- M@ (2.5%) increased the response irrespective of the subset. The addition of higher number of FcR- subset further increased the response, while FcR+ M@ suppressed the response almost to the level seen in NAC cultured alone. Unseparated AC, when added to NAC, in general followed the pattern of FcR’ subset. S. aureus was usually able to induce substantial immunoglobulin production in cultures of NAC. However, in some experiments, when response was low, it was increased by adding back monocytes irrespective of the subset, although FcR- subset was more effective (unshown). Thus, the response to S. aureus showed different dependency on M@ subsets. Modulation of Irnmunoglobulin Production by FcR- and FcR’ MQ Subsets in Cultures of PBMC Stimulated with PWM or S. aureus Cowan I The observation that FcR+ M@ can suppress, while FcR- rather enhance the number of ISC in PWM-stimulated cultures of PBMC [12] was confirmed in this

248

PRYJMA ET AL. TABLE

THE

IMMUNOREGULATORY PKODUCTWN

Cells added to

EFFECT OF FrR’ AND IN CULTURES STIMULATED

Exp I

___-

0

I

FcRMUNWYTE WITH PWM

Sussf?rs

ON IMMUNWI.OBI!I.I~V XN l

OK S. ~U,~L’II.S Cow __ -. ISC/IO” Exp

-.-

~~~

Exp 3

2

__--

I 1,200

4400

8,200

7680

I I .700

4280

AC

5 x IO4 I x IO5 3 x 105

ND ND 7,000

ND ND 5600

ND ND ND

ND ND ND

12,500 9.500 6.250

ND ND ND

AC FcR-

5 x I04 I x IO” 3 x 105

36,000 36,500 14,000

ND 8200 7800

27,000 22,000 8,000

4800 4850 9000

12,800 19.900 10,680

3430 3060 4000

AC FcR+

5 x 104 1 x 105 3 x I05

8.000 4,400 2,500

5200 7100 3600

ND 3.600 3,300

ND 5140 IO50

IO.940 4,500 4,764

3500 5500 2058

study (Table 1). The FcR- subset greatly increased the PWM induced response of PBMC in low doses, while higher doses (approx. 30% MO) were usually not effective. The response of PBMC stimulated with S. aureus was not consistently effected, and the results observed were variable. The FcR+ subset added to PBMC suppressed PWM induced response in a dose-dependent manner. The cultures stimulated with S. aureus were suppressed only by high doses of FcR+ subset. The Differential Effect of Monocyte and Occurrence of Plasma Cells

Subsets on the Immunoglobulin

Secretion

The FcR+ MO, when added to the PWM-stimulated cultures of PBMC, caused significant suppression of ISC. However, when the number of PC was assessed in the culture, the suppressory effect of FcRf M@ was not so evident (Fig. 2). To analyze this further, PWM-stimulated cultures of NAC were reconstituted with graded doses of both subsets. Figure 3 shows that, as found earlier, cultures

z 15 Y

0 SC l PC

b 10 (D 0 b ‘; :: 0‘-

5

lklh

0 2.5 (~10~ FcR’ Monocytes

5 )

2. PBMC were cultured in the presence of PWM and FcR+ monocyte subset. The response was evaluated by the reverse plaque assay (IX) and cytoplasmic immunoglobulin presence (percentage of PC). FIG.

MONOCYTE

REGULATED

Ig SYNTHESIS

IN VITRO .

249

FcR-

FIG. 3. The influence of FcR+ and FcR- monocyte subsets added to NAC on production and secretion of immunoglobulins assayed as ISC or PC.

reconstituted with FcR- M@ gave excellent response, while FcR+ M@ were unable to provide accessory function. In contrast, both subsets were comparable in increasing the appearance of PC in the cultures. In another experiment NAC were reconstituted with FcR- M@, which caused significant enhancement of the response when assessed both by number of ISC and PC percentage (Fig. 4). Additional admixture of FcR+ M@ led to the suppression of ISC, but the number of PC was not decreased. It was concluded that FcR+ MP) suppressed the appearance of ISC but not PC, which suggested that modulatory effect of this subset on B celf response is exerted at the final differentiation step into ISC. DISCUSSION

Monocytes are necessary as accessory cells for PWM-induced polyclonal immunoglobulin production in cultures of human PBMC [15, 16, 241. They also inhibit both proliferative response [5] and immunoglobulin production induced by PWM [17, 251. This paper shows that these two opposite functions can be easily dissected within a population of peripheral blood monocytes by their separation into FcR- and FcR+ subsets. The FcR- monocytes were shown to be superior as accessory cells and, in contrast to suppressory activity of FcR+ subset, could enhance the response induced by PWM. Depressed response in PWM stimulated cultures of human PBMC rigorously depleted of monocytes, adherence to glass 10 z x 6 "OS Z ? 0 x =: 0 0

5 0

10 0

10 25

10 5

10x103FcR+

FIG. 4. Graded number of FcR+ monocytes were added to NAC supplemented with FcR- subset. The response of NAC cultured without monocytes or with low number of FcR- is also shown. Number of ISC 0 and percentage of PC I are shown.

250

PRYJMA

ET AI..

or plastic, preincubation with silica or L-leucine methyl ester, can be restored by adding back viable monocytes [15, 16, 261. In the present studies PBMC, depleted of monocytes by adherence to glass or passage through Sephadex G-10, were cultured at low cell density in flat-bottom microwells as suggested by Rosenberg and Lipsky [lS]. This cell population did not respond or responded poorly when stimulated with PWM. FcR.- monocytes increased the response in a dose-dependent manner with plateau effect seen at the dose of approximately 10%. This contrasted with FcR’ monocytes, which restored the response when added in low numbers, but strongly suppressed when used in higher doses. The superior accessory activity of FcR- monocytes correspond to their increased antigen presenting capacity [12]. This function is known to be related to the expression of class II MHC antigens [27-311 and is reinforced by IL-I [32]. As we have shown previously [12], both FcR+ and FcR- monocytes, when stimulated with LPS, produce comparable amounts of IL-1 and do not differ in the expression of a common DR antigen. However, we cannot exclude a possibility that they differ in the expression of some other class II determinants relevant for accessory function, e.g., HLA-DS determinants [33]. The present data show that monocyte subsets when added to cultures of PBMC differ also in the regulatory activity, as: (1) FcR- monocytes do not suppress but enhance PWM-induced immunoglobulin production, (2) FcR+ monocytes are responsible for suppression of the PWM-induced immunoglobulin production, (3) ability of differentiated B lymphocytes to secrete immunoglobulins is more suppressed by FcR+ monocytes than immunoglobulin synthesis. It could be argued that the FcR- monocyte-mediated enhancement of immunoglobulin production was due to cells contaminating this population, e.g., B lymphocytes. However, this trivial explanation seems unlikely because contamination of this monocyte subset with surface immunoglobulin-positive cells as a rule did not exceeded 5%, and the number of ISC in PWM stimulated cocultures of FcR- monocytes and T cells was negligible (not shown). Other observations, namely reduction of the response seen after adding large numbers of FcR- monocytes to PWM-stimulated cultures and especially the lack of the consistent enhancing effect on parallel cultures stimulated with 5’. uureus, also exclude contaminating B cells as candidates responsible for the observed phenomenon. PWM-induced polyclonal B cell activation, proliferation, and maturation into ISC is strictly T-cell dependent [ 13, 141. In contrast, S. UUY~US Cowan i-induced response requires T cells only for B-cell maturation into ISC [ 191, since B-cell proliferation induced by this mitogen is T-cell independent [18]. In this context the influence of FcR- monocytes on PWM- but not S. aureus-induced response strongly suggests that T-cell dependent proliferative response of B lymphocytes is a target for a modulatory mechanism mediated by this monocyte subset. In contrast to PWM-induced response the immunoglobulin synthesis after stimulation with S. aureu~ was relatively resistant to FcR+ monocyte mediated suppression, which suggests that the inhibition is not due to direct effect of monocytes on B cells. A prominent feature of FcRf monocyte-mediated suppression was their different effect on synthesis and secretion of immunoglobulins. It is known that free FcR shed in vitro can inhibit immunoglobulin secretion in cultures

MONOCYTE

REGULATED

Ig SYNTHESIS

IN

VITRO

251

stimulated with PWM and Nocardiu mitogen [34-361, and the question arises whether similar mechanism occurs in the FcR+ monocyte-mediated suppression. If so, it remains to be determined whether FcR+ monocytes mediate suppression by shedding their receptors or whether free receptor, when added to the culture, is incorporated into monocyte membrane which results in the increase of their suppressory activity. Since neither FcR+ monocyte nor soluble FcR mediated suppression was studied in a totally T-cell independent system, it is difficult to decide whether B-cell maturation into ISC can be directly affected or whether this phenomenon requires T-cell presence. The possibility of the former is suggested by the finding that loosely adherent FcR+ cells present in bone marrow are responsible for arrest of immunoglobulin secretion from plasma cells [37], and unseparated monocytes were shown to suppress EBV-induced immunoglobulin production [ 171. However, dissection between synthesis and secretion of immunoglobulins was also attributed to T cells as well [38], therefore, a possibility of “cooperation” between monocyte subsets and subsets of T lymphocytes cannot be ruled out. In summary, we conclude that FcR- and FcR+ monocytes have a differential modulatory effect on T-cell dependent PWM-induced polyclonal immunoglobulin production and that the FcR+ subset may interfere with the final B-cell maturation process. ACKNOWLEDGMENTS This work was supported by Grant 10.5 from the Polish Academy of Sciences. The skillful technical assistance of Ms. B. Tata and the excellent secretarial help of Mrs. R. Hinz are gratefully acknowledged.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

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