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The Journal of Immunology, 2004, 173: 1292-1297.
Copyright © 2004 by The American Association of Immunologists

Differential Induction of IL-1{beta} and TNF by CD40 Ligand or Cellular Contact with Stimulated T Cells Depends on the Maturation Stage of Human Monocytes1

Danielle Burger2, Nicolas Molnarfi, Lyssia Gruaz and Jean-Michel Dayer

Clinical Immunology Unit (Hans Wilsdorf Laboratory), Division of Immunology and Allergy, Department of Internal Medicine, University Hospital, Geneva, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cellular contact with stimulated T cells potently induces cytokine production in monocytes, a mechanism that is likely to be relevant to chronic inflammation. Although the identity of surface molecules involved in this process remains elusive, CD40 and its ligand, CD40L, are thought to be implicated, considering that they are expressed at the inflammatory site. To ascertain the involvement of CD40L, we compared the activation of three different types of human monocytic cells, i.e., freshly isolated monocytes, monocytes primed with IFN-{gamma} (IFN-{gamma}-macrophages), and THP-1 cells. These cells were activated by either membranes isolated from stimulated T cells (HUT-78 or T lymphocytes) to mimic cellular contact, soluble extracts from isolated membranes, or CD40L trimer (CD40LT). The production of TNF and IL-1{beta} was induced by membranes of stimulated T cells in the three types of target cells, whereas CD40LT induced TNF production in IFN-{gamma}-macrophages only. Similar results were obtained with soluble extracts of T cell membranes, demonstrating that the difference between membranes and CD40LT was not due to the particulate form of membranes. CD40LT induced neither transcript nor protein of cytokines in monocytes, whereas in IFN-{gamma}-macrophages, IL-1{beta} and TNF mRNA were observed, but only TNF was measured in cell supernatants. Finally, anti-CD40L Abs failed to inhibit TNF and IL-1{beta} production induced in IFN-{gamma}-macrophages by solubilized membranes, whereas TNF production induced by CD40LT was inhibited. These results demonstrate that CD40L is not required in monocyte activation by direct cellular contact with stimulated T cells, although soluble CD40LT induces the production of TNF in IFN-{gamma}-macrophages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD40L (CD154) is a member of the TNF family, which is expressed on activated T cells (1). It binds to CD40, a member of the TNFR family, and plays an important part in Th cell signal necessary for germinal center formation, isotype class switching, and production of Abs (for review, see Refs.1 and 2). In patients with rheumatoid arthritis and multiple sclerosis, CD40L expression is enhanced in T lymphocytes of both peripheral blood and the inflammatory site (3, 4, 5, 6). This suggests that CD40-CD40L interaction might play a part in the pathogenesis of these diseases. Because cellular interaction between stimulated T cells and monocyte-macrophages is an important process in inducing proinflammatory cytokines in the latter cells (for review, see Ref.7), it was tempting to hypothesize that CD40-CD40L interaction was involved in such a mechanism.

We previously demonstrated that direct contact with stimulated T cells was a potent stimulus of human monocytic cells and monocytes, which not only induces the production of IL-1 but also of other cytokines such as TNF, IL-6, IL-8, and metalloproteinases (8, 9, 10). We also observed that contact-mediated activation by stimulated T cells is as potent as optimal doses of LPS in inducing IL-1{beta} and TNF production in monocytes and cells of the monocytic lineage such as THP-1 cells (11). A dysregulated production of proinflammatory cytokines, such as IL-1 and TNF, plays an important part in the pathogenesis and persistence of chronic inflammatory diseases. Thus, the induction of cytokines by contact between stimulated T cells and monocytes might be relevant to the pathogenesis of such diseases.

Although several T cell surface molecules have been involved in contact-mediated activation of monocyte-macrophages (for review, see Ref.11), the identity of the molecule that is required to trigger the engagement of other surface factors leading to monocyte-macrophage activation is still elusive. Controversial results were obtained concerning the involvement of CD40L. Indeed, CD40L was proved to be involved in T cell contact-mediated activation of murine and human macrophages (12, 13). However, when stimulated for 24 h, T lymphocytes isolated from CD40L-deficient mice triggered macrophage activation to a similar extent as T lymphocytes isolated from wild-type mice (12). Consequently, CD40-CD40L might be involved in contact activation of macrophages by T lymphocytes stimulated for short periods of time, contrary to T lymphocytes stimulated for long periods, because the latter cells cease to express CD40L beyond a certain time (14). A previous study of ours shows that, in cocultures of living human T cell clones and monocytic cells stimulated with IL-15 or IL-2, Th1 but not Th2 clones induced significant IL-1{beta} production in monocytes and THP-1 cells (15). Blockade of the CD40-CD40L interaction in cocultures of Th1 cells and monocytes resulted in the inhibition of IL-1{beta} production. However, the levels of CD40L expression did not differ in Th1 and Th2 cell clones, implying that one or more additional, unidentified molecules preferentially expressed by Th1 cells were involved in their capacity to induce IL-1{beta}. Furthermore, IFN-{beta}, which diminishes the ability of stimulated T lymphocytes to induce IL-1{beta} and TNF in monocytes, does not simultaneously modulate CD40L expression on the T cell surface (16). In many of our previous studies related to cell-cell contact, we used the T cell line HUT-78, which efficiently induces cytokine production in monocytes and THP-1 cells (9, 16, 17), but does not express CD40L mRNA in resting or activated conditions (18).

Because the evidence of CD40L triggering the production of IL-1{beta} and TNF was not obtained on monocytes but monocyte-derived macrophages, the present study was undertaken to determine whether membrane-associated CD40L was involved in cytokine induction in different monocytic cells and to compare its effects in various types of monocytic cells, i.e., THP-1 cells as a model for monocyte-derived macrophages (19) that we have been using previously (reviewed in Ref.7), freshly isolated monocytes, and IFN-{gamma}-macrophages. The technical approach consisted in the activation of monocytic cells by CD40L trimer (CD40LT),3 the most active form of CD40L (20), and membranes or soluble extracts from membranes isolated from stimulated T cells. The results show that only IFN-{gamma}-macrophages responded to CD40LT in terms of TNF production, whereas direct contact with stimulated T cells induced the production of both TNF and IL-1{beta} in the three types of target cells in a CD40L-independent way.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Streptomycin, penicillin, L-glutamine, PBS free of Ca2+ and Mg2+, and TRIzol reagent (Invitrogen Life Technologies, Paisley, U.K.); purified PHA (EY Laboratories, San Marco, CA); Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden); and FCS, RPMI 1640, PMA, PMSF, pepstatin A, leupeptin, iodoacetamide, neuraminidase, BSA, and polymyxin B sulfate (Sigma-Aldrich, St. Louis, MO) were purchased from the designated suppliers. Other reagents were of analytical grade or better.

Recombinant proteins and Abs

Recombinant human CD40L trimer/leucine zipper fusion protein (CD40LT), which has been described (20, 21), was given by Dr. E. Williamson (Immunex, Seattle, WA). Anti-CD40L mAb (clone 5C8) was provided by Dr. P. Lipsky (University of Texas, Dallas, TX). F(ab')2 were prepared as described (22) using immobilized papain (Sigma-Aldrich). IFN-{gamma} (5 x 106 IU/ml) was obtained from Biogen (Cambridge, MA).

Monocytes and THP-1 cells

The human monocytic cell line THP-1, derived from a patient with acute monocytic leukemia (23), was obtained from the American Type Culture Collection (Manassas, VA). Monocytes were isolated from buffy coats of blood from healthy volunteers as previously described (17). Briefly, monocytes purified from PBMC isolated by Ficoll-Paque density gradient were washed in PBS and suspended (50 x 106 cells/ml) into RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 µg/ml streptomycin, 50 U/ml penicillin, 2 mM L-glutamine, and 1 µg/ml polymyxin B sulfate (medium). Cell suspension was incubated for 40 min at 4°C with gentle agitation. Monocyte aggregates were sedimented on ice for 10 min, overlaid on FCS, and incubated on ice for 10 more min. Monocytes contained in the FCS lower layer were further depleted of remaining T and NK cells by rosetting with neuraminidase-treated SRBC and Ficoll-Paque density centrifugation. Monocyte purity routinely consisted of >90% CD14+ cells, <1% CD3+ cells, and <1% CD19+ cells as assessed by flow cytometry. To avoid activation by endotoxin, polymyxin B was added to all solutions during the isolation procedure (1 µg/ml) and experiments (5 µg/ml). IFN-{gamma}-primed monocytes, referred to as IFN-{gamma}-macrophages, were prepared by treating purified monocytes with 500 U/ml IFN-{gamma}. After 7 days, IFN-{gamma}-macrophages were washed twice in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 µg/ml streptomycin, 50 U/ml penicillin, and 2 mM L-glutamine, and activated as indicated. IFN-{gamma} treatment resulted in the down-modulation of CD14 and the enhancement of CD163 expression at the surface of IFN-{gamma}-macrophages.

T cell stimulation and membrane isolation

HUT-78, a human T cell line (24), was obtained from the American Type Culture Collection. Cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 µg/ml streptomycin, 50 IU/ml penicillin, and 2 mM L-glutamine in 5% CO2-air-humidified atmosphere at 37°C. T lymphocytes were obtained from buffy coats of healthy donors as previously described (8), and contained 94–98% CD2+, 83–94% CD3+, and <2% CD14+ as assessed by flow cytometry. HUT-78 cells (2 x 106 cells/ml) and T lymphocytes were stimulated for 6 and 48 h, respectively, by PHA (1 µg/ml) and PMA (5 ng/ml). To avoid interferences by soluble products from T cells, membranes of stimulated T cells were used as monocyte activator, the latter preparations displaying similar activity to fixed, stimulated T cells (9, 17). Plasma membranes of stimulated (msHUT, msT) or unstimulated (musHUT, musT) HUT-78 cells and T lymphocytes, respectively, were prepared as previously described (25). When required, isolated membranes were solubilized with 16 mM CHAPS for 1 h at 4°C and then centrifuged for 30 min at 20,000 rpm in a JA20.1 rotor (Beckman Instruments, Fullerton, CA). The supernatant was dialyzed in PBS containing 8 mM CHAPS and kept at –20°C before use. CHAPS extract of msHUT and musHUT was referred to as CEsHUT and CEusHUT, respectively, and that of msT and musT as CEsT and CEusT. Proteins were measured by the method of Bradford (26) and cytokines as described below. IL-1{beta} was not detected in membrane preparations. TNF was present at 713 pg/mg of proteins in msHUT, 551 pg/mg of proteins in musHUT, 294 pg/mg of proteins in CEsHUT, 68 pg/mg of proteins in CEusHUT, and 8.3 ng/mg of proteins in msT, 65 pg/mg of proteins in musT, 4.2 ng/mg of proteins in CEsT, and <4 pg/mg of proteins in CEusT.

Cytokine production

THP-1 cells, monocytes, or IFN-{gamma}-macrophages (50 x 103 cells/well/200 µl) were activated with the indicated stimulus in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 50 µg/ml streptomycin, 50 U/ml penicillin, 2 mM L-glutamine, and 5 µg/ml polymyxin B sulfate (medium) in 96-well plates (Costar, Corning, NY) and cultured for 48 h or as otherwise stated. The production of cytokines was measured in culture supernatants by commercially available enzyme immunoassay: IL-1{beta} (Immunotech, Marseille, France) and TNF (Quantikine; R&D Systems, Minneapolis, MN).

mRNA

IFN-{gamma}-macrophages or monocytes (5 x 106 cells/well/500 µl) were cultured in medium in 24-well plates (Costar) for 1 h with the indicated stimulus. Total RNA was isolated with TRIzol and analyzed by a commercially available RNase protection assay system kit with hck2 template set (BD Pharmingen, San Diego, CA) to which an antisense riboprobe for TNF was added as previously described (17).

Flow cytometry

The expression of CD40L and CD40 on T cells and monocytes was assessed by flow cytometry as previously described (16, 17) with 5C8 anti-CD40L and anti-CD40 (DakoCytomation, Carpinteria, CA) Abs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD40LT induces the production of TNF in IFN-{gamma}-macrophages but not in freshly isolated monocytes and THP-1 cells

To compare the effect of cellular contact and CD40LT on cytokine induction in different types of monocytic cells, THP-1 cells, monocytes, and IFN-{gamma}-macrophages were activated by either CD40LT or membranes isolated from HUT-78 cells stimulated (msHUT) or not (musHUT) by PHA/PMA, the latter mimicking direct cellular contact. Because preliminary data and previous studies showed that the amount of msHUT required to stimulate THP-1 cells and IFN-{gamma}-macrophages was 10 times higher than that to activate monocytes, THP-1 cells and IFN-{gamma}-macrophages were activated with 60 µg/ml proteins of msHUT or musHUT, whereas monocytes were activated with 6 µg/ml proteins of msHUT or musHUT. As shown in Fig. 1, musHUT did not induce cytokine production in monocytic cells. The production of cytokines in either IFN-{gamma}-macrophages or THP-1 cells was ~10 times lower than in monocytes, although 10 times lower doses of msHUT were used to activate the latter (Fig. 1). CD40LT was inefficient in inducing TNF and IL-1{beta} in either freshly isolated human monocytes or THP-1 cells (Fig. 1, A–D). However, in IFN-{gamma}-macrophages, CD40LT triggered the production of TNF but not of IL-1{beta} (Fig. 1, E and F). In contrast, msHUT triggered the production of both cytokines in the three types of monocytic cells. Together, these results suggest that CD40L was not involved in IL-1{beta} induction in the three types of monocytic cells used in this study upon activation by msHUT, although it induced TNF production in IFN-{gamma}-macrophages.



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  		FIGURE 1. Induction of TNF and IL-1{beta} production in THP-1 cells, monocytes, and IFN-{gamma}-macrophages. THP-1 cells (A and B), monocytes (C and D), and IFN-{gamma}-macrophages (E and F) were cultured (50 x 103 cells/200 µl/well) with the indicated stimulus for 48 h. Freshly isolated human blood monocytes were activated by 6 µg/ml proteins of msHUT or musHUT, 7 µg/ml proteins of CEsHUT or CEusHUT, or 3 µg/ml CD40LT. THP-1 cells and IFN-{gamma}-macrophages (i.e., isolated peripheral blood monocytes primed with 500 U/ml IFN-{gamma} for 7 days) were activated with 60 µg/ml proteins of msHUT or musHUT, 70 µg/ml proteins of CEsHUT or CEusHUT, or 3 µg/ml CD40LT. TNF (A, C, and E) and IL-1{beta} (B, D, and F) were measured in supernatants as described in Materials and Methods.

 
Because macrophages might be activated by particles such as those formed by isolated plasma membranes, to assess whether the difference between membranes and CD40LT was due to the particulate form of msHUT, the latter were solubilized with CHAPS (see Materials and Methods). As shown in Fig. 1, CEusHUT (7 µg/ml proteins) did not trigger TNF and IL-1{beta} production in any of the target cells. In contrast, CEsHUT triggered TNF and IL-1{beta} production in isolated monocytes, although to different extents than msHUT, CEsHUT being more potent than msHUT in inducing IL-1{beta} (Fig. 1). This demonstrates that the particulate form of membranes was not required for the activation of monocytes, and that soluble extracts from membranes can be used to induce cytokine production.

Solubilized membranes from both HUT-78 cells and T lymphocytes, but not CD40LT, induce IL-1{beta} in monocytes and IFN-{gamma}-macrophages

Because CD40L is not expressed in HUT-78 cells (18) but might be expressed by freshly isolated T lymphocytes upon stimulation, solubilized membranes from both types of T cells were compared in terms of induction of TNF and IL-1{beta} production in THP-1 cells, monocytes, and IFN-{gamma}-macrophages. CEsT and CEsHUT induced TNF and IL-1{beta} production in all target cells (Fig. 2). This did not apply to CD40LT, which, once more, induced TNF production only in IFN-{gamma}-macrophages. These results imply that CD40L, which might be present in CEsT, could be involved in the production of TNF but not that of IL-1{beta} in IFN-{gamma}-macrophages.



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  		FIGURE 2. Comparison of TNF and IL-1{beta} induction in THP-1 cells, monocytes, and IFN-{gamma}-macrophages by CD40LT and solubilized membranes from stimulated HUT-78 cells and isolated T lymphocytes. A and B, THP-1 cells (50 x 103 cells/200 µl/well) were cultured for 48 h in the presence or absence of CD40LT (3 µg/ml), CEsHUT (70 µg proteins/ml), or CEsT (70 µg proteins/ml). C and D, Monocytes (50 x 103 cells/200 µl/well) were cultured for 48 h in the presence or absence of CD40LT (3 µg/ml), CEsHUT (7 µg/ml proteins), or CEsT (7 µg/ml proteins). E and F, IFN-{gamma}-macrophages (50 x 103 cells/200 µl/well) were cultured for 48 h in the presence or absence of CD40LT, CEsHUT, or CEsT (at the same doses as for THP-1 cells). TNF (A, C, and E) and IL-1{beta} (B, D, and F) were measured in supernatants as described in Materials and Methods.

 
CD40L is not involved in the induction of TNF and IL-1{beta} in IFN-{gamma}-macrophages upon cell-cell contact

To assess the involvement of CD40L in the induction of cytokines by CEsHUT and CEsT, IFN-{gamma}-macrophages were activated by CD40LT, CEsHUT, or CEsT in the presence or absence of anti-CD40L blocking Abs. The Ab 5C8, which blocks the binding of CD40L to CD40, activated the production of TNF and IL-1{beta} in IFN-{gamma}-macrophages (not shown). This was likely to be due to cross-linking of FcRs by anti-CD40L Abs bound to the trimeric form of CD40LT; indeed, in the absence of CD40LT, 5C8 did not activate cytokine production. To avoid nonspecific effects of anti-CD40L Ab, we generated F(ab')2 from 5C8 Ab. As shown in Fig. 3A, F(ab')2 from 5C8 inhibited the CD40LT induction of TNF production in IFN-{gamma}-macrophages. F(ab')2 of 5C8 did not affect TNF and IL-1{beta} production triggered by either CEsHUT or CEsT (Fig. 3, B and C). Because TNF levels induced by CEsT and CD40LT were similar in this experiment, it is likely that the lack of inhibition was not due to an excessive activation of IFN-{gamma}-macrophages by CEsT whose other activating factors could putatively overcome the effect of CD40L. In the absence of stimulus, neither F(ab')2 of 5C8 nor control IgG displayed any effect (Fig. 3D). These results correlated with CD40L and CD40 expression at the surface of T lymphocytes, monocytes, and IFN-{gamma}-macrophages, as determined by flow cytometry (Figs. 4 and 5).



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  		FIGURE 3. CD40L is not required for the induction of TNF and IL-1{beta} production upon contact-mediated activation of IFN-{gamma}-macrophages. IFN-{gamma}-macrophages (50 x 103 cells/200 µl/well) were cultured for 48 h in the presence of CD40LT (3 µg/ml) (A), CEsHUT (70 µg/ml) (B), or CEsT (70 µg/ml) (C), or absence of stimulus (D) with or without 10 µg/ml anti-CD40L F(ab')2 of 5C8 Ab (5C8F), or 10 µg/ml control IgG1. Culture supernatants were analyzed for their content in the indicated cytokine.

 


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  		FIGURE 4. Expression of CD40 in monocytes, IFN-{gamma}-macrophages, and THP-1 cells. Monocytes (A), IFN-{gamma}-macrophages (B), and THP-1 cells (C) were analyzed for CD40 expression by flow cytometry as described in Materials and Methods.

 


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  		FIGURE 5. Expression of CD40L in HUT-78 cells and isolated T lymphocytes. Unstimulated (A) and stimulated (B) HUT-78 cells, and unstimulated and 6- or 48-h-stimulated T lymphocytes (C) were analyzed for CD40L expression by flow cytometry.

 
Although the expression of low or undetectable levels of stimulating factors or receptors might be sufficient to induce/transduce signals, we assessed the expression of CD40 and CD40L at the surface of monocytic and lymphocytic cells used in this study. CD40 was expressed at the surface of 63 ± 11% (mean of three experiments ± SD) of monocytes and to a higher level on 97 ± 5% of IFN-{gamma}-macrophages but not on THP-1 cells (Fig. 4). The expression of CD40L was not observed in HUT-78 cells, whether unstimulated or stimulated (Fig. 5, A and B), or in T lymphocytes stimulated for 48 h (C). However, CD40L was up-regulated at the surface of T lymphocytes stimulated for 6 h (Fig. 5C). These results demonstrate that CD40L expression was very low in both types of stimulated T cells used in this study, i.e., PHA/PMA-stimulated HUT-78 cells and T lymphocytes stimulated for 48 h, consistent with the lack of or minor involvement of this factor in cell-cell contact activation of monocyte-macrophages. Furthermore, although CD40 was expressed on monocytes, the latter cells were not activated by CD40LT.

CD40LT induces TNF and IL-1{beta} transcript expression in IFN-{gamma}-macrophages but not in monocytes

Considering that the production of proinflammatory cytokines is controlled at several levels (i.e., transcriptional, translational, posttranslational, and/or transcript stability), and because monocytes expressed significant levels of CD40 (Fig. 5), we ascertained whether CD40LT would induce cytokine mRNA in monocytes, despite the lack of cytokine production, and compared it to IFN-{gamma}-macrophages. CD40LT did not induce TNF or IL-1{beta} transcript expression nor the production of proteins in monocytes (Fig. 6, A, B, and E). However, in IFN-{gamma}-macrophages, CD40LT induced the expression of TNF and IL-1{beta} mRNA (Fig. 6, C and D) without triggering the production of IL-1{beta} protein (F). As expected, both CEsHUT and CEsT induced the expression of TNF and IL-1{beta} mRNA in monocytes and in IFN-{gamma}-macrophages (Fig. 6). This result confirms that CD40LT did not induce cytokine expression and production in freshly isolated monocytes, thus ruling out the possibility that CD40L is involved in cell-cell contact activation of monocytes by stimulated T cells.



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  		FIGURE 6. Induction of TNF and IL-1{beta} transcripts in monocytes and IFN-{gamma}-macrophages. A and B, Monocytes (5 x 106 cells/500 µl/well) were cultured for 3 h in the presence or absence of CD40LT (3 µg/ml), CEsHUT (7 µg/ml proteins), or CEsT (7 µg/ml proteins). C and D, IFN-{gamma}-macrophages (5 x 106 cells/500 µl/well) were cultured in the presence or absence of CD40LT (3 µg/ml), CEsHUT (70 µg/ml proteins), or CEsT (70 µg/ml proteins). Total RNA was isolated by TRIzol and analyzed by RNase protection assay. Autoradiography of the gel (A and C); densitometry analysis of A and C, normalized with GAPDH (B and D). E and F, Cytokine production in the same experiments after activation of either monocytes or IFN-{gamma}-macrophages by the indicated stimulus for 48 h.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that CD40LT induces TNF production in IFN-{gamma}-macrophages, but not in freshly isolated monocytes or THP-1 cells. Furthermore, the present data demonstrate that CD40L is not required for the induction of IL-1{beta} and TNF production in freshly isolated monocytes, THP-1 cells, or IFN-{gamma}-macrophages upon contact with stimulated T cells. Additionally, cells of the monocytic lineage display different degrees of sensitivity to activation by direct cell-cell contact with stimulated T cells, which depend on the maturation stage of the target cells, freshly isolated monocytes being more sensitive than THP-1 cells or IFN-{gamma}-macrophages. This suggests either possibility: 1) THP-1 cell and IFN-{gamma}-macrophage receptors for the surface activating factor(s) on stimulated T cells were down-regulated upon cell differentiation/maturation; 2) the usage of signaling pathways varied as a function of the monocytic cell type. This might also be the case for monocytes that expressed CD40 but failed to respond to CD40LT activation.

The premise that macrophages derived from monocytes by priming with different agents or macrophage cell lines respond differentially to various stimuli has been observed for a long time (27, 28). This might be due to a differential usage of signaling pathways in these cells (29, 30). It is thus not surprising that THP-1 cells, blood monocytes, and IFN-{gamma}-macrophages display differential responses to stimuli used in this study. Although all three types of cells were activated by msHUT, msT, and their soluble extracts (CEsHUT and CEsT), higher doses were required to activate THP-1 cells and IFN-{gamma}-macrophages than for activating blood monocytes. Indeed, a dose of CD40LT that induced TNF in IFN-{gamma}-macrophages, which displayed low sensitivity to stimulus, did not induce TNF or other cytokines in highly sensitive monocytes. This further confirms that CD40L was not required for cytokine induction by contact with stimulated T cells.

In accordance with the present results, it was previously demonstrated that, although monocytes express CD40, they needed to be primed by GM-CSF, IL-3, or IFN-{gamma} to respond to activation by CD40L (31). Furthermore, to display optimal activation ability, CD40L requires cross-linking, i.e., either expressed at the surface of transfected cells (31) or trimerized (21). In this context, it was previously demonstrated that, when expressed at the surface of transfected cells, CD40L activated human monocytes primed with GM-CSF, IL-3, or IFN-{gamma}, to produce TNF and IL-6 (31), in accordance with the present data. Because contact between stimulated T cells and monocytes induces cytokine production in the latter cells (8) and stimulated T lymphocytes may express CD40L, the role of CD40-CD40L interaction in this mechanism has been investigated. When T cells were stimulated for 6 h, they induced the expression of pro-IL-1{beta} in adherent monocytes via engagement and cross-linking of CD40 (32), but the latter study did not demonstrate that pro-IL-1{beta} was processed to mature form and secreted. These results are in agreement with the present data, because adherent monocytes do not have the same characteristics as freshly isolated blood monocytes. Indeed, in this study, we demonstrate that, although the protein production was not detected in cell culture supernatants, CD40LT induced the expression of IL-1{beta} transcript in IFN-{gamma}-macrophages. This is also consistent with a study showing that, in IFN-{gamma}-macrophages, cellular contact with a transfectant cell line expressing CD40L enhanced the production of IL-1{beta} around six times that observed on a control cell line transfected with an empty vector (28). Thus, CD40L, when expressed in stimulated T cells might play a part in increased cytokine production in macrophages (including IL-1{beta}), although, according to the present results, it is not required for the latter mechanism. Furthermore, we observed that the levels of expression of TNF and IL-1{beta} mRNA that CD40LT induced in IFN-{gamma}-macrophages were similar to those observed with CEsT (Fig. 4C). However, IL-1{beta} production occurred only when IFN-{gamma}-macrophages were activated by CEsT. The premise that CD40LT failed to trigger IL-1{beta} protein production is reminiscent of the observation that stimuli such as the complement component C5a, hypoxia, adherence to surfaces, and clotting of blood, all induced the synthesis of large amounts of IL-1{beta} mRNA in monocytic cells without significant translation into the IL-1{beta} protein (for review, see Ref.33). This further emphasizes that distinctive mechanisms rule the translation and production of TNF and IL-1{beta}.

It has been demonstrated that, although monocytes do not express CD40 mRNA in the absence of priming, they express low levels of CD40 at their surface (31). In this study, CD40 was detected at the surface of 63% of freshly isolated monocytes, and its expression was enhanced when cells were primed for 7 days with IFN-{gamma} (IFN-{gamma}-macrophages). The fact that cytokine production was not induced by CD40LT in monocytes might be due to the level of CD40 expression being too low for signal transduction, the threshold being surpassed after priming with IFN-{gamma}. Another possibility is that transduction pathway usage and cytokine production depend on cell differentiation/maturation stage (34, 35) or the route of monocyte differentiation (28). This is reminiscent of previous results showing that human alveolar macrophages and lung tissue macrophages display distinct responses to activation by msT (36). Indeed, msT strongly induced the production of matrix metalloproteinases-1 (MMP-1), MMP-9, and tissue inhibitor of MMP-1 exclusively in lung tissue macrophages but not in alveolar macrophages, indicating that the activating capacity is not only related to the difference in phenotype of mononuclear phagocytes, but that it might also depend on cell localization.

In conclusion, this study rules out the possibility that CD40L is a major stimulus involved in the induction of cytokines in monocyte-macrophages upon cellular contact with stimulated T cells. This does not answer the question of the identity of the surface factors involved in this process. Our studies and others have shown that LFA-1 (CD11a/CD18) and CD69 play a role in the activation of human monocytic cells by stimulated T cells (8, 37). The latter data were confirmed in a study showing that IL-15 induces synovial T cells from rheumatoid arthritis patients to activate the production of TNF by macrophages. This effect was inhibited by Abs to CD69, LFA-1, and ICAM-1 (38). Other studies have shown that cytokine production is induced in monocytes by soluble CD23 (39, 40). In monocytes, the counterligands for CD23 are CD11b/CD18 and CD11c/CD18 rather than CD21 (41). We demonstrated that cross-linking of {beta}2-integrin induces IL-1{beta} in freshly isolated monocytes (42); however, blocking Abs to {beta}2-integrins inhibited but did not abolish IL-1{beta} induction (8, 9). Although some of the above molecules as well as CD40L might be involved to some extent in contact-mediated monocyte activation by T cells, the factor(s) to first engage monocytes in the activation processes in turn leading to cytokine production, remain to be identified.


   Acknowledgments
 
We thank Mrs. R. Rehm for skillful reading of the manuscript.


   Footnotes
 
1 This work was supported by Grant 3200-068286.02 from the Swiss National Science Foundation and a grant from the Swiss Society for Multiple Sclerosis. Back

2 Address correspondence and reprint requests to Dr. Danielle Burger, Clinical Immunology Unit (Hans Wilsdorf Laboratory), Division of Immunology and Allergy, University Hospital, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. E-mail address: danielle.burger{at}hcuge.ch Back

3 Abbreviations used in this paper: CD40LT, CD40L trimer; msHUT, membranes of stimulated HUT-78 cells; musHUT, membranes of unstimulated HUT-78 cells; msT, membranes of stimulated T lymphocytes; musT, membranes of unstimulated T lymphocytes; CEsHUT, CHAPS extract of msHUT; CEusHUT, CHAPS extract of musHUT; CEsT, CHAPS extract of msT; CEusT, CHAPS extract of musT; MMP, matrix metalloproteinase. Back

Received for publication February 26, 2004. Accepted for publication May 7, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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