Cytokines produced in lymph follicles

Immunology Letters, 22 (1989) 129-134

Elsevier IMLET 01270

Cytokines produced in lymph follicles Rikiya T s u n o d a 1, N a d i n e C o r m a n n 2, E r n s t H e i n e n 2, K i k u o O n o z a k i 3, Pierre C o u l i e 4, Yukio A k i y a m a 5, K a z u y u k i Yoshizaki 6, C6cile Kinet-Den0612, L e o n J. S i m a r z a n d M i z u K o j i m a 7 Xlnstitute of Clinical Medicine, University of Tsukuba, Japan," 2Institute of Histology, University of Liege, Belgium; 3Institute of Basic Medical Science, University of Tsukuba, Japan; 4Ludwig Institute for Cancer Research, Universitd Catholique de Louvain, Brussels, Belgium," 5Central Research Laboratories, Ajinomoto Co. Ltd., Japan; 6The 3rd Internal Medicine, Osaka University Hospital, Japan," 7Department of Pathology, Mito General Hospital, Japan

(Received23 February 1989; accepted 20 April 1989) 1. Summary

2. Introduction

The events occurring inside lymph follicles during a germinal center reaction are poorly understood. Using B and T lymphoid cell populations prepared from human tonsillar lymph follicles, and enriched or not in macrophages or in follicular dendritic cells, we examined the production of cytokines by these cells in vitro. Interleukin 6 (IL-6) and tumor necrosis factor (TNF) were found in the supernatants of cultures stimulated with phytohemagglutinin or pokeweed mitogen. IL-1/3 was occasionally detected; its secretion apparently depends on the origin of the tonsils, the stimulation, and the cell populations. IFN-'y and IL-2 were not produced in significant amounts by these lymph follicle cells. IL-4 was only found in very low concentrations in the supernatant of the different cell cultures. The cell populations containing follicular dendritic cells produced more IL-6 and TNF than the others, especially than those composed of only B and T ceils.

In germinal centers (g.c.), B cells divide and differentiate in memory or in plasma cell precursors. The phenotypes of the g.c. cells have been extensively studied: non-circulating B cells at different maturation stages and T cells have been characterized [4, 9, 16, 17]. Beside the lymphoid cells, two other typical g.c. cell types have been described: the follicular dendritic cells (FDC) and the tingible body macrophages. FDC represent only 2°70 of the g.c. cells [10]. They extend long and complex cytoplasmic processes between the lymphoid cells and are able to retain immune complexes via their C3b and Fc receptors [15, 20, 21, 25] for long periods of time and without endocytosis. FDC can be isolated using mild enzymatic digestions [18], and appear as cell clusters in which they surround lymphoid cells. These FDCclusters exhibit surface antigens and receptors typical of g.c. cells [8, 26, 27]. They are very sensitive to most cell separation techniques, therefore their ratio to free lymphoid cells is low (0.5 to 2070). However, morphometrical analysis indicated that in these cell suspensions from g.c. FDC possess around 40070 of the total cell surface [14]. A lot of phenomena occur inside the g.c.: B cells are attracted into the g.c., some of them divide, others undergo an isotype switch [3] and mutations in the V hypervariable zones occur, improving the affinity of the Ig [19]. But the parameters controlling these events remain largely unknown. In order to determine them, we have isolated FDC-clusters and cultivated them in vitro. FDC-containing cell suspensions are representative for lymph follicles [27].

Key words: Cytokines; Germinal centers; Follicular dendritic

cells Abbreviations." anti-IL-2R, antibodies against IL-2 receptors;

BSA, bovine serum albumin; FceR, receptor for immunoglobulin E Fc fraction; FDC, follicular dendritic cell; g.c., germinal center(s);IFN, interferon;IL, interleukin;PHA, phytohemagglutinin; PMA, phorbol myristate acetate; PWM, pokeweedmitogen; TNF, tumor necrosis factor. Correspondence to: Dr. E. Heinen, Institute of Human Histology, 20, rue de Pitteurs, B-4020, Liege, Belgium.

0165-2478 / 89 / $ 3.50 © 1989 Elsevier SciencePublishers BY. (BiomedicalDivision)

129

Previous experiments have shown that FDC influence the B cells: they increase their survival rate [5], improve their multiplication capacity and apparently modulate their differentiation in Igsecreting cells [6]. Lymphoid cells in turn can establish contacts with FDC and transfer immune complexes to them [7]. In the present work, we examine the production of cytokines in supernatants of cultures containing FDC ( B + T + F D C ) , compared to lymphoid cells cultured alone (B +T) or in presence of macrophages (B +T+ M). 3. Materials and Methods

3.1. Source of tonsils Tonsils and adenoids from 3- to 10-year-old children were surgically dissected and brought to the laboratory at 4 °C, in a physiological solution containing 0.4O7o bovine serum albumin (BSA). 3.2. Isolation procedure of FDC We have previously described the isolation procedure of FDC [18]. In short, lymphoid follicles from tonsils dissected under the biomicroscope were submitted to four steps of enzyme treatment, using successively collagenase alone (0.05°7o; Boehringer: 2x15 min at 20°C) and a mixture of collagenase (0.05%), dispase (0.05%; Boehringer) and deoxyribonuclease (0.004°7o; Worthington: 2x15 min at 37°C). The freed cells were then separated by sedimentation at 1 x g in a MEM solution (Difco) containing 1.5°70 bovine serum albumin. After 30 min, the cells in the upper layer were mainly lymphocytes, macrophages and some rare FDC. The sedimented layer contained FDC organized in clusters mixed with free cells (lymphocytes and macrophages) or epithelial fragments. The population of FDC was 0.5 to 2%, whereas in the original suspension, their frequency is below 1 per 1000.

3.3. Preparation of the FDC-enriched population To remove monocytes and macrophages, the sedimented suspension containing FDC was incubated in Petri dishes (Falcon, 60 mm; Difco; 10.106 ceils per ml) for 60 to 90 min at 37 °C. The nonadhering cells were then gently recovered using 130

DMEM medium (Dulbecco, Difco) at 37 °C. This cell suspension contained FDC (0.5 to 2%), lymphocytes and some epithelial cells. Macrophages, as determined by non-specific esterase activity or by neutral red staining (final dilution 1/10000) represented less than 1% of the cells.

3.4. Preparation of lymphoid cell population Free cells collected from tonsils were filtered through a Sephadex G10 column (Pharmacia Fine Chemicals), pelleted and stored at 4°C until addition to the culture wells. Immunolabellings with anti-OKT3 (Ortho; 1/20) or anti-Leu MI0 (Pan B cells; Becton-Dickinson; 1/20) antibodies showed that about 25°70 of lymphoid cells were T ceils; the others were mainly B cells. Monocytes and macrophages were undetectable by non-specific esterase method.

3.5. Preparation of lymphoid cell population containing macrophages Free cells, collected from tonsils as above but without passage through the Sephadex column, contained T and B ceils (in similar proportions as in Section 3.4) but also about 2°7o monocytes or macrophages as shown by the non-specific esterase method. They were pelleted, resuspended in DMEM medium and stored at 4°C before use. 3.6. Cell cultures Cultures were set up in triplicate (at least) in 96well flat-bottomed microtest plates (Nunc). The cells (105 cells per well) were kept in a final volume of 200 #1 of RPMI 1640 supplemented with 10°70 calf serum and different mitogens (phytohemagglutinin 1%, P H A from Bacto Difco or pokeweed mitogen 1°70, PWM from Gibco). Cultures were incubated at 37 °C in a 5°70 CO2 enriched atmosphere for 1, 3, 5 and 7 days. Thereafter the microtest plates were centrifuged and the supernatants collected and stored at - 3 0 ° C until use. 3.7. Radioimmunoassay (R.LA.) Using IL-1/3 (Cistron Biotechnology), IL-2 (IRE Medgenix), IFN-y (IRE Medgenix) and TNF (IRE

M e d g e n i x ) the c o n t e n t s o f these cytokines in the sup e r n a t a n t s were d e t e r m i n e d a c c o r d i n g to a n R I A m e t h o d d e s c r i b e d by Reuter, Ketelslegers, H e n d r i c k a n d F r a n c h i m o n t [22].

4.2. IL-2 or IFN-q/

IL-4 in the culture s u p e r n a t a n t was m e a s u r e d by the c a p a c i t y to induce the expression o f Fcereceptors (FceR) on B cells, as d e s c r i b e d previously [231.

We d e t e c t e d no I D 2 n o r I F N - 7 in the s u p e r n a t a n t s o f the different cell p o p u l a t i o n s (B+T, B + T + M , B + T + F D C ) f r o m three different patients, even w h e n t h o s e p o p u l a t i o n s were c u l t u r e d in presence o f either P H A o r P W M . In a d d i t i o n a l experiments, we a d d e d anti-IL-2r e c e p t o r a n t i b o d y (1/20; serotex) in o r d e r to b l o c k the a d s o r b a n c e o f IL-2 o n cells, b u t even t h e n no IL-2 n o r IFN-3, was detected.

3.9. Bio-assay for IL-6

4.3. TNF

IL-6 activity was d e t e r m i n e d by the s t i m u l a t i o n o f I g M p r o d u c t i o n in h u m a n B l y m p h o b l a s t o i d cell line, S K W 6 - C L 4 [13] o r by its g r o w t h factor activity o n FTD1 cells [29].

T h e presence o f T N F was tested in the s u p e r n a tants used for the search o f IL-2 o r I F N - 7 (Table 2). In P H A - s t i m u l a t e d cultures, T N F was m a i n l y f o u n d in the F D C - e n r i c h e d p o p u l a t i o n s a n d n o t at all in the B + T p o p u l a t i o n s . W h e n a n t i - I L - 2 - r e c e p t o r a n t i b o d i e s were a d d e d to these cultures, an increase o f T N F p r o d u c t i o n was m e a s u r e d in m o s t c o n d i t i o n s even in the B + T p o p u lation supernatants. W h e n P W M was used as mitogen, T N F was also p r o d u c e d by the B + T + M p o p u l a t i o n .

3.8. Measurement of lL-4

4.

Results

4.1. IL-13 S u p e r n a t a n t s were collected f r o m 24-h cultures o f l y m p h o i d p o p u l a t i o n s a l o n e ( B + T ) , e n r i c h e d in m a c r o p h a g e s (B + T + M) o r in F D C (B + T + F D C ) a n d a n a l y s e d for their c o n t e n t in IL-13. A s shown in Table 1, the c o n c e n t r a t i o n o f IL-13 was v a r i a b l e a n d seemed to d e p e n d on the d o n o r , the s t i m u l a t i o n a n d the cell c o m p o s i t i o n . I n one case ( p a t i e n t No. 5), the F D C - c o n t a i n i n g p o p u l a t i o n p r o d u c e d clearly m o r e IL-1 t h a n the B + T + M p o p u l a t i o n . TABLE 1 Concentrations of IL-13 expressed (ng/ml) in the supernatants of different tonsillar cell populations incubated 24 h in RPMI medium supplemented with 10% FCS. Patient No.

Condition B+T

B+T+M

B+T+FDC

1

+ PHA +PWM + PWM +PWM + PWM +PWM +PWM

<0.005 < 0.005 <0.005 0.760 <0.005 <0.005 <0.005 <0.005

<0.005 < 0.005 <0.005 0.670 <0.005 0.919 <0.005 <0.005

2 3 4 5 6

<0.005 < 0.005 <0.005 N.T. a N.T. N.T. N.T. N.T.

aN.T.; not tested.

4.4. 1L-4 I n d u c t i o n o f FceR (CD23) expression o n n o r m a l h u m a n p e r i p h e r a l B cells was a n a l y z e d by FACS (Tab l e 3). T h e expression o f FceR o n B cells, represented by the m e a n fluorescent intensity, was dosed e p e n d e n t . This was o b s e r v e d after the s t i m u l a t i o n with v a r y i n g a m o u n t s o f IL-4 (0.8 U / m l to TABLE 2 Concentrations of TNF (pg/ml) in the supernataJats of different tonsillar cell populations incubated 24 h in RPMI medium supplemented with 10% FCS. Patient Condition No.

B+T B + T + M

B+T+FDC

7

<25 80 <25 <25 65 <25 <25

115 115 55 115 100 175 130

8 9

+PHA + PHA + anti-IL-2R +PWM +PHA + P H A + anti-IL-2R +PHA + PHA + anti-IL-2R

<25 <25 70 <25 65 115 165

131

TABLE 3

TABLE 4

Induction of FceR (CD23) on human peripheral blood mononuclear cells cultured with serial dilutions of purified IL-4 solution (A) or 2507o of sample supernatants (B) of tonsillar cell populations cultured in RPMI medium supplemented with 1007o FCS and 1070 PWM.

Concentration of IL-6 expressed (U/ml using the CL4 assay) in the supernatants of different tonsillar cell populations incubated in RPMI medium supplemented with 1007o FCS and 1070 PWM.

A

10

3,IL-4 (U/ml)

Mean intensity a 15.9 33.6 36.7 35.3 31.3

0.8

3.1 12.5 50 250 B

Patient No.

Day

B+ T

B+T +M

B + T + FDC

10

1 3 1

10.90) 10.9 12.7

10.4 11.6 11.6

11.1 12.1 11.7

Patient No.

Day

13

11

aMean intensity: after 48 h co-culture, washed cells were stained with biotinylated anti-CD23 antibody (gift of Dr. Suemura) and analyzed by FACS.

250 U/ml). With this indirect method the IL-4 amount in all the tonsillar supernatants could be estimated as being below 0.8 U/ml.

4.5. II-6 The supernatants of most of the cultures contained measurable amounts of I1_,-6(Tables 3 and 4); FDC-enriched populations appeared to secrete more IL-6 than the B + T + M populations; B + T did not produce IL-6, or did so only at very low levels. This IL-6 secretion increased with time, especially for the B +T+ FDC populations (Tables 4 and 5). PWM and P H A induced the production of IL-6 in the B +T+ M population and particularly in the B + T + F D C population. P M A had no or an inhibitory effect on IL-6 secretion. 5. Discussion

In the present work, we analyzed the secretion of cytokines by cell populations prepared from tonsillar lymph follicles. IL-6 and TNF were found in the supernatants of cell cultures stimulated with P H A or PWM. IL-13 was occasionally detected; IL-2, IL-4 and IFN--/were not present in significant levels. 132

0.4 0.3

0.4 0.3

1

0.3

3.6

3 5 7 I 3 6 1 3 7

12

B+ T + M

3

1

11

B+ T

1.0 2.0 8.0 0.4 1.4 N.T. N.T. < 1.0 < 1.0

B + T + FDC 3.1 12.5 47 16.5 66.0 141.0 5.4 10.9 44.0 N.T. 15.0 54.0

0.6 87.0 348.0 4.7 6.3 N.T. N.T. 2.0 3.0

TABLE 5. Concentration of IL-6 expressed (U/ml using the FTD l assay) in the supernatants of different tonsillar cell populations incubated in RPMI medium supplemented with 10°70 FCS. Patient Day Condition No. 14 15 16 17

1

-

1

-

B+T

<2 4

B+T+M

<2 6

B+T+FDC

54 173

1

-

<2

<2

1

-

<2

<2

<2

53

1 1 1 3 3 3 3

+PHA + P H A + PMA +PWM +PHA + P H A + PMA +PWM

<2 <4 <2 <2 <2 <2 <2

4 4 <2 <2 13.2 12 3.7

13.2 12 3.7 90 96 23 123

The detected cytokines were mostly found in the supernatants of FDC-containing cell suspensions. Since the FDC-clusters mainly contain g.c. lymphoid cells [27], we can suggest that these cytokines are produced in the germinal centers and play a part in the regulatory mechanisms directing the evolution of the lymphoid cell population. IL-2 and IFN--y are apparently not produced; this could mean that the T cells secreting those bioactive molecules do not play a major role inside the g.c. The g.c. T cells are indeed peculiar: they are CD4 positive, are potentially cytolytic and do not secrete IL-2

[16, 30]. In the mouse, two different CD4 positive T cell populations could be distinguished: one secreting IL-4, the other secreting IL-2 and IFN-3, [2]; perhaps in the near future such a distinction will also be possible on human CD4-positive cells. The role of TNF in g.c., as well as its origin in our culture system, are unknown. Most probably, TNF is produced by stimulated T ceils and some B cells [24]. Since TNF secretion was found in the FDCenriched cultures, we have to test the FDC capacity either to produce TNF or to induce its production by other cells. Moreover, we have no clear explanation concerning the stimulatory action on TNF production due to the anti-IL-2-receptor antibodies. IL-4 was only detected in very low concentrations in the supernatants of our cell cultures. However, it cannot be excluded that IL-4 production occurs but is followed by an immediate uptake by B cells and/or T cells. This IL-4 may affect other metabolic events than FceR expression (for example, cell proliferation and isotype switch [24]). Interestingly, in our culture, FDC are highly CD23-positive, whereas the lymphoid cells are negative. We have to determine the biological role of this CD23 expression by FDC. IL-6, discovered by distinct laboratories and previously called hybridoma growth factor [1], IFN/32 [28], B cell stimulatory factor [11], B cell differentiation factor [11] etc., is secreted by accessory cells. Macrophages do not produce high amounts of IL-6 in our conditions. However, IL-6 was mainly found in the FDC-enriched populations. It is thus likely that FDC themselves produce IL-6, first because the level of contaminating monocytes and macrophages is low and the BTM produce less IL-6, and secondly because lectin-activated B or T cells do not produce detectable levels of IL-6. Fibroblasts can also form IL-6, but they are only occasionally encountered in the cell suspensions prepared from lymph follicles. One can propose that some lymphoid cells are able to secrete IL-6 when stimulated by accessory cells (for example FDC). T cells then would be candidates, especially since Van Snick and co-workers [29] observed IL-6 secretion by T hybridoma cells. We are investigating techniques to detect the ceils responsible for the secretion of TNF, IL-1 and IL-6 in our culture systems. But we also have to detect other bioactive substances produced by the g.c. cells specifically stimulated by antigens, and to determine the relationships existing between these products, in

order to comprehend the mechanisms leading to an equilibrated germinal center reaction.

Acknowledgements Tonsils were kindly provided by Drs. R. Boniver, R. Daele (Liege), J. Wada, T. Arai and M. Ohhashi (Tsukuba). B cell line SKW6-CL4 was the generous gift of Drs. Hirano and Kishimoto (Osaka). Financial support was received from the Fonds de la Recherche Scientifique M~dicale, Belgique and the Ministry of Education, Science and Culture, Japan.

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