The role of CD40L in T cell-dependent nitric oxide production by murine macrophages

Transplant Immunology 8 Ž2000. 195᎐202

The role of CD40L in T cell-dependent nitric oxide production by murine macrophages Adam W. Bingaman, Thomas C. Pearson, Christian P. LarsenU The Carlos and Marguerite Mason Transplantation Biology Research Center, Department of Surgery, Emory Uni¨ ersity School of Medicine, Atlanta, GA, 30322, USA Received 18 July 2000; accepted 14 August 2000

Abstract Upon activation, T cells express CD40L, a member of the TNF cytokine superfamily, which serves as a ligand for CD40 on antigen presenting cells, including dendritic cells, B cells, and macrophages. While initial studies on the function of CD40 focused its role in the regulation of B cell activation, more recent studies have indicated that CD40 ligation may be critical for the initiation of T cell-dependent macrophage activation, including stimulation of nitric oxide production. However, the relative contribution of the CD40 pathway in macrophage nitric oxide production during T-dependent immune responses remains unclear. We have found that while CD40 ligation of macrophages can stimulate nitric oxide production, disruption of CD40 signaling during a T cell-mediated alloimmune response has no appreciable effect on nitric oxide production. If the T cell alloimmune response is restricted to CD4 cells, CD40L blockade has only a minimal effect on nitric oxide production. Rather, IFN␥, produced by alloactivated T cells, seems to be a necessary ‘first’ signal for nitric oxide production, while TNF␣ and CD40L each provide independent ‘second’ signals. Finally, we demonstrate that CD40L stimulates macrophage NO production independent from autocrine TNF␣ stimulation. These results suggest that macrophage nitric oxide production during a T-dependent immune response requires IFN␥ production by CD4 cells whereas TNF␣ and CD40L can each provide important functionally overlapping ‘second’ signals to costimulate nitric oxide production, though neither is required. 䊚 2000 Elsevier Science B.V. All rights reserved. Keywords: Costimulatory molecules; Nitric oxide; Macrophages; Transplantation

1. Introduction Nitric oxide ŽNO. plays an important role in the initiation and development of various inflammatory and immune responses w1,2x. In recent years, NO has

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Corresponding author. Emory University, Transplantation Immunology Laboratory, Suite 5105, WMB, 1639 Pierce Drive, Atlanta, GA 30322, USA. Tel.: q1-404-727-8466r727-8464; fax: q1-404-7273660. E-mail addresses: tpearson@ emory.org Ž T.C. Pearson . , [email protected] ŽC.P. Larsen..

been implicated as one of the major mechanisms of defense against intracellular infections including tuberculosis, leishmaniasis, schistosomiasis, and trypanosomiasis w3᎐6x. In addition, NO has been implicated as an important effector molecule in rejection of organ allografts w7x. In contrast, in other circumstances NO appears to down regulate immune responses. For example, NO production decreases lymphocyte proliferation during a mixed leukocyte reaction and inhibits development of allospecific cytotoxic T lymphocytes in a sponge matrix allograft model, suggesting possible immunosuppressive effects of this molecule w8᎐10x.

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Similarly, recent data indicate that NO may play a protective role in the development of transplant arteriosclerosis, a manifestation of chronic rejection, in a murine cardiac allograft model w11x. Thus, the regulation of NO production appears to be critical to the host during a microbial infection or during an alloimmune response. The gene responsible for high output NO production by murine macrophages during inflammation and infection is the inducible nitric oxide synthase ŽNOS2. gene. Numerous cytokines can stimulate NOS2-dependent NO production, often acting in synergistic pairs. IFN␥ alone has been shown to stimulate low levels of NO production by thioglycollate elicited peritoneal macrophages w12,13x. In contrast, TNF␣ and LPS stimulate high levels of NO secretion only in the presence of IFN␥ w14,15x. More recent data have suggested that CD40rCD40L interactions play a dominant role in stimulating cell mediated responses involving macrophages including stimulation of NO production w16᎐19x. While it is clear from these studies that CD40 ligation of murine peritoneal macrophages can stimulate NO production in the presence of IFN␥, it is unclear whether this is a critical and necessary interaction, or whether other redundant pathways exist during a T-dependent immune response. Furthermore, since ligation of CD40 on the cell surface of macrophages has been shown to stimulate TNF␣ secretion w20x, it is not clear whether the CD40 signal stimulates NO production directly or merely through autocrine stimulation mediated by TNF␣. In this report we have investigated the requirements for murine macrophage NO production during T celldependent immune responses. We show that IFN␥ is a necessary first signal to stimulate T-dependent NO production by macrophages. TNF␣ and CD40 ligands can each provide independent second signals that potentiate the signal delivered by IFN␥, but are unable to stimulate NO production alone or in combination with each other. Additionally, we show that while CD40 ligation can stimulate TNF␣ secretion by macrophages, it provides a co-stimulus for NO production that is not dependent on an autocrine TNF-dependent mechanism. These results indicate that during T-dependent immune responses, ligation of CD40 on macrophages is neither critical nor required for NO production, but rather serves as a redundant signal that is independent from TNF receptor signaling.

2. Objective To determine the role of the CD40 costimulatory pathway on T cell-dependent macrophage nitric oxide production.

3. Materials and methods 3.1. Animals Male 6᎐8-week-old C57BLr6, DBAr2, C3HrHeJ, and C3HrHeJ ␤ 2 Myry mice were purchased from The Jackson Laboratory ŽBar Harbor, ME .. B6,129 CD40Lyry mice were obtained from Immunex Inc. ŽSeattle, WA. w21x and bred as homozygotes under sterile conditions at Emory University. 3.2. Abs and reagents Polyclonal goat anti-mouse TNF␣ ŽND50 0.2᎐0.4 ␮grml in a neutralizing bioassay using murine L929 cells. was obtained from Rq D Systems Inc ŽMinneapolis, MN.. Rat anti-mouse IFN␥ was obtained from Pharmingen, Inc. ŽSan Diego, CA.. Anti-CD40L monoclonal antibody was obtained from Bristol-Myers Squibb Pharmaceutical Research Institute ŽPrinceton, NJ.. Soluble recombinant CD40L ŽrCD40L. fusion protein was obtained from Immunex Inc. Recombinant mouse Žrm. IFN␥ and rm TNF␣ were obtained from Pharmingen Inc. 3.3. Medium Cells were cultured in RPMI 1640 media ŽMediatech, Washington, DC. supplemented with 10% FBS ŽHyClone, Logan, UT., 100 IU penicillin-streptomycin ŽMediatech., and 5 = 10y5 M 2-ME ŽSigma, St. Louis, MO.. 3.4. Preparation of murine peritoneal macrophages Murine peritoneal macrophages were obtained by lavage 5 days after intra-peritoneal injection of 1 ml of 4% Brewer’s thioglycollate ŽSigma. solution. 5 = 10 5 cellsrwell were cultured in round bottomed 96 well plates in media at 37⬚C, 5% CO 2 for 2 h. Plates were then vigorously rinsed three times with warm media to remove non-adherent cells. 3.5. Preparation of alloacti¨ ated T cells An enriched population of dendritic cells was obtained by collecting transiently adherent cells from murine splenocytes as previously described w22x. T cells were obtained by nylon wool passage of murine splenocytes. CD4 cells were obtained by incubating nylon wool passaged T cells with rat anti-mouse CD8 supernatant ŽTIB 105. ŽATCC, Rockville, MD. followed by anti-rat magnetic beads ŽBiosource International, Camarillo, CA. Ž20:1 beadrtarget ratio.. Adequacy of depletion Ž- 1% CD8q. was confirmed on a FACScan flow cytometer ŽBecton Dickinson, Braintree, MA. us-

A.W. Bingaman et al. r Transplant Immunology 8 (2000) 195᎐202

ing PE conjugated antibodies ŽRt IgG 2a, anti-CD4, anti-CD8, Pharmingen.. Enriched dendritic cells were cocultured with allogeneic nylon wool passaged T cells Ž1:10. in media for 4 days at 37⬚C, 5% CO 2 . Cells were then collected, washed, and viable cells counted using trypan blue exclusion.

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in a total volume of 800 ␮l. At the indicated time points, supernatants were harvested from the bottom chamber for nitrite assay and replaced with fresh media. In subsequent experiments, 5 = 10 5 C57BLr6 thioglycollate elicited peritoneal macrophages were cultured in media with 5 = 10 5 primed DBAr2 T cells in 96 well plates in a volume of 150 ␮l.

3.6. Culture of macrophages and alloacti¨ ated T cells 3.7. Nitrite assay In initial experiments described in Fig. 1, 5 = 10 C57BLr6 thioglycollate elicited peritoneal macrophages were cultured in media with 5 = 10 5 primed DBAr2 T cells in the upper well of 0.4 ␮m pore size transwell chambers ŽCorning Costar, Cambridge, MA. 5

The concentration of nitrite in supernatants was used as a measure of NO production w23,24x. Nitrite concentration was assayed from culture supernatants Ž50 ␮lrsample. using the Griess reagent Ž1% sulfani-

Fig. 1. CD40L is not required for T-dependent macrophage NO production. In Exp. 1 Ža. 5 = 10 5 C57BLr6 ŽH-2 b . thioglycollate elicited peritoneal macrophages were cocultured with 5 = 10 5 primed DBAr2 ŽH-2 d . allogeneic T cells in the upper well of 0.4 ␮m pore size transwell chambers in the presence or absence of anti-CD40L antibody Ž50 ␮grml.. Supernatants from the lower chamber were harvested every 24 h for 4 days and replaced with fresh media. Supernatants were assayed for nitrite production using the Griess reagent. Results are plotted at each time period against a standard curve with known concentrations of nitrite. Addition of anti-CD40L had no significant effect on NO production at any time point tested. In Exp. 2 Žb. DBAr2 T cells were depleted of CD8 cells by incubation with rat anti-mouse CD8 ŽTIB 105. followed by incubation with anti-rat magnetic beads. Flow cytometry confirmed - 1% CD8 cells after depletion. CD4 cells were then primed in a 4-day mixed lymphocyte reaction as described in Section 2 and cocultured with C57BLr6 peritoneal macrophages as in Exp. 1A. Supernatants were harvested at the indicated time periods and assayed for nitrite. Addition of anti-CD40L significantly inhibited NO production at 24- and 48-h time points Ž P - 0.01.. Experimental groups were done in triplicate. Data shown are from one of two experiments with similar results.

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lamider0.1% naphthylethylediaminer2.5% H 3 PO4 . ŽSigma.. The absorbance was recorded at 550 nm using a Spectramax 250 microplate reader ŽMolecular Devices, Sunnyvale, CA.. A freshly prepared standard curve with known concentrations of nitrite was prepared with each assay to convert absorbance into ␮M nitrite. 3.8. Cytokine assay TNF␣ cytokine concentrations were assayed from cell culture supernatants using a TNF␣ cytokine ELISA kit ŽGenzyme, Cambridge, MA. according to the manufacturer’s instructions. 3.9. Statistical analysis Results are depicted as mean values "S.D. When error bars are not apparent in a figure, bars appear within the data point. Statistical analysis was carried out using the t-test for independent means to calculate P values.

4. Results 4.1. CD40 ligand is not required for T cell-dependent nitric oxide production As an initial approach to explore the role of the CD40rCD40L pathway in T cell-dependent macrophage NO production, thioglycollate elicited C57BLr6 ŽB6, H-2 b . peritoneal macrophages were cultured in the presence or absence of allogeneic DBAr2 ŽH-2 d . T cells which had been primed in a 4-day mixed leukocyte reaction with B6 dendritic cells. Supernatants were harvested at 24-h intervals and assayed for nitrite production ŽFig. 1a.. Cultures containing both activated T cells and macrophages produced large amounts of NO within the first 20 h. In contrast, neither macrophages nor activated T cells alone produced detectable nitrite, establishing the T-dependence of NO production in this system. Surprisingly, addition of antiCD40L antibody had no effect on NO production at any time point, even at concentrations of 50 ␮grml. As an alternative approach to explore the role of the CD40 pathway, we stimulated macrophages with sensitized T cells from allogeneic CD40Lyry mice or wild type mice. As suggested by the previous experiment, macrophage NO production was similar after stimulation with either CD40Lyry or wild type T cells Žnot shown.. These results suggest that activated T cells do not require CD40L to initiate or stimulate macrophage NO production. Previous studies have demonstrated the importance of CD40 ligand on T-dependent macrophage NO pro-

duction using CD4 cells. Our initial studies were performed using unseparated activated T cells containing both CD4 and CD8 cells. Therefore, we performed similar experiments to study the role of CD40L in CD4q T cell-dependent NO production ŽFig. 1b.. Allogeneic T cells were depleted of CD8 cells before priming in a 4-day mixed leukocyte reaction. Alloactivated CD4 cells were then cocultured with allogeneic macrophages in the presence or absence of anti-CD40L antibody. Culture of macrophages with alloactivated CD4 cells resulted in no NO production in either experimental group in the first 12 h of culture. By 24 h, low levels of NO were detectable, with peak levels produced by 48 h. Surprisingly, addition of anti-CD40L resulted in only a modest decrease in NO production at 24 and 48 h. Similar results were obtained using alloactivated T cells from ␤ 2 Myry mice, which lack CD8 cells Žnot shown., suggesting that possible low level CD8 contamination in the CD8 depleted T cell experiments did not have a significant effect on the results. These results indicate that CD40L may contribute to CD4q T cell-dependent NO production, but is neither critical nor required. 4.2. Role of IFN␥ , TNF␣ , and CD40 ligand in nitric oxide production Since CD40 ligand did not appear to be the major regulator of T-dependent NO production by macrophages in this experimental system, we investigated the role of other CD4q T cell inflammatory cytokines which are produced during an alloimmune response. As an initial approach, macrophages were cultured with alloactivated CD4 cells in the presence or absence of neutralizing antibodies to IFN␥, TNF␣ , or CD40L ŽFig. 2.. Blockade of IFN␥ almost completely eliminated NO production, whereas blockade of TNF␣ or CD40L only partially inhibited NO production. Simultaneous blockade of both TNF␣ and CD40L significantly diminished NO production more than either antibody alone Ž P- 0.001.. These data confirm that IFN␥ is a critical regulator of CD4-dependent macrophage NO production, and suggest that TNF␣ and CD40L costimulate high levels of NO only in the presence of IFN␥. Moreover, neither TNF␣ nor CD40L appear to be required for CD4-dependent NO production. As an alternative approach to explore the role of CD4q T cell-derived inflammatory mediators, we stimulated macrophages with recombinant IFN␥, TNF␣ , agonistic soluble CD40L, or combinations of these molecules ŽFig. 3.. IFN␥ alone stimulated very low levels of macrophage NO production. In contrast, neither TNF␣ nor CD40L alone stimulated any detectable NO production. As suggested in the previous experiment, combinations of IFN␥ with either TNF␣

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Fig. 2. Effects of neutralizing anti-TNF␣ , anti-IFN␥, and anti-CD40L on CD4-dependent macrophage NO production; 5 = 10 5 C57BLr6 thioglycollate elicited peritoneal macrophages were cocultured with 5 = 10 5 primed DBAr2 CD4 cells in the presence or absence of neutralizing anti-TNF␣ Ab Ž20 ␮grml., anti-IFN␥ Ab Ž12.5 ␮grml., or anti-CD40L Ab Ž50 ␮grml. for 48 h. Supernatants were harvested and assayed for nitrite. Experimental groups were done in triplicate. Data shown are from one of three experiments with similar results.

or CD40L greatly enhanced NO production, however, combination of TNF␣ and CD40L in the absence of IFN␥ elicited no detectable NO production. Thus, these data provide further evidence that IFN␥ is an obligatory mediator of CD4q T cell-dependent macrophage NO production, whereas TNF␣ and CD40L each stimulate high levels of NO only in the presence of IFN␥. 4.3. CD40L does not mediate NO production through autocrine TNF␣ stimulation Prior studies have shown that ligation of CD40 on

Fig. 3. Effects of recombinant IFN␥, TNF␣ , or CD40L on macrophage NO production. 5 = 10 5 C57BLr6 thioglycollate elicited peritoneal macrophages were cultured in the presence or absence of rm TNF␣ Ž500 urml., rm IFN␥ Ž100 urml., or soluble recombinant CD40L ŽrCD40L. Ž1 ␮grml. in different experimental combinations for 48 h. Supernatants were harvested and assayed for nitrite. Experimental groups were done in triplicate. Data shown are from one of three experiments with similar results.

the cell surface of human monocytes stimulates TNF␣ release w17x. Therefore, we investigated whether the effects of the CD40 pathway on NO production might be mediated through an autocrine TNF␣-dependent pathway. For this, macrophages were stimulated with TNF␣ or CD40L in addition to recombinant IFN␥ in the presence or absence of neutralizing anti-TNF␣ antibody. After 48 h of culture, supernatants were assayed for nitrite production ŽFig. 4a.. As previously demonstrated, TNF␣ or CD40L in the presence of IFN␥ stimulated high levels of NO production. The addition of anti-TNF␣ antibody in the presence of IFN␥ and TNF␣ almost completely eliminated NO production. In contrast, anti-TNF␣ had significantly less effect on inhibition of NO production in the presence of IFN␥ and soluble CD40L Ž P- 0.001.. This result indicated that either CD40L stimulated NO production via a TNF␣-independent mechanism, or that the amount of TNF␣ stimulated by CD40L exceeded the neutralizing capacity of the anti-TNF␣ antibody. To distinguish between these possibilities, we quantified the amount of TNF␣ present in the cultures by ELISA ŽFig. 4b.. As expected, macrophages cultured in the presence of IFN␥ and CD40L produced some TNF␣ Ž413 pgrml., however, cultures containing IFN␥ and recombinant TNF␣ contained much higher levels of TNF␣ Ž2723 pgrml.. Thus, these data suggest that in the presence of IFN␥, CD40L stimulates macrophage NO production independent from TNF␣ stimulation.

5. Discussion In this study, we have investigated the requirements for macrophage NO production during T cell-depen-

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Fig. 4. CD40L stimulates macrophage NO production via TNF␣-independent mechanisms. 5 = 10 5 C57BLr6 thioglycollate elicited peritoneal macrophages were cultured with rm IFN␥ Ž100 urml. and rm TNF␣ Ž500 urml. or rCD40L Ž1 ␮grml. in the presence or absence of neutralizing antibody to TNF␣ Ž20 ␮grml.. After 48 h, supernatants were harvested and assayed for Ža. nitrite using the Griess reagent and for Žb. TNF␣ concentration by ELISA. Experimental groups were done in triplicate. Data shown are from one of two experiments with similar results.

dent alloimmune responses. Previous reports have demonstrated that ligation of CD40 on macrophages stimulates production of numerous cytokines, including IL1␤, IL6, IL8, IL12, TNF␣ , and NO in the presence of IFN␥ w18,20,25x. These reports and others have suggested that CD40 ligation is critical for these functions w18,19x. In addition, we have previously demonstrated that CD40L blockade significantly prolongs murine cardiac allograft survival, and is associated with decreased levels of NOS2 transcript within the allograft w26x, further suggesting that the CD40 pathway is critical for macrophage NO production in vivo. Consistent with previous reports, we have found that IFN␥ is a major regulator of NO production by macrophages w11᎐13x. Surprisingly, however, in our studies CD40 ligation did not play an obligatory role in the stimulation of NO production. Rather, CD40L appears to deliver a redundant ‘second’ signal that is qualitatively overlapping in function with TNF␣. These results are in apparent conflict with previous studies suggesting that CD40L plays a dominant role in T-de-

pendent macrophage activation and NO production w18,19x. We believe that methodological differences account for these apparent discrepancies and that our current studies are compatible with and extend these earlier observations. Stout et al. have demonstrated a clear defect in macrophage NO production stimulated by T cells from CD40L deficient mice w18x. These studies were performed using naive CD4q T cells or membrane preparations derived from CD4q T cells that had been activated with immobilized anti-CD3. In contrast, our studies were performed using CD4q T cells that were activated with transiently adherent allogeneic splenic DC. We believe that these differences in the T cell populations are important. It is now clear that CD40L plays a pivotal role in providing optimal delivery of costimulation during T cell activation w27,28x. Therefore, studies demonstrating an impaired ability of naive CD40Lyry CD4q T cells to elicit NO production w18x may reflect the inability of the CD40Lyry cells to induce equivalent costimulatory molecule expression andror IL12 production by the APC population, thus diminishing production of T cell cytokines such as IFN␥ and TNF␣. This decrease in IFN␥ and TNF␣ , in addition to the lack of CD40 signaling, might explain sub-optimal macrophage NO production stimulated by naive allogeneic CD4 cells. In other studies, addition of fixed activated CD4 cells from wild type or CD40Lyry mice were used in combination with IFN␥ to stimulate macrophage NO production w16,18x. This model, however, restricts the response of macrophages to T cell membrane bound molecules. Therefore, in the absence of any soluble second signal, such as TNF␣ , it is perhaps not surprising that fixed 6-h activated CD40Lyry CD4 cells did not stimulate significant macrophage NO production compared with fixed activated wild type CD4 cells. In contrast, in our model CD4q T cells were activated with transiently adherent splenic dendritic cells which express high levels of costimulatory molecules w22,29x. In this setting, when CD4q T cells are intact and fully activated, CD40L appears to play only a minor role as a redundant ‘second’ signal in stimulating macrophage NO production. Thus, it may be necessary to re-examine the importance of the CD40 pathway in the induction of other T-dependent macrophage effector molecules independent from its effects in regulating the activation of CD4q T cells. It remains unclear why recipients of cardiac allografts treated with anti-CD40L have decreased levels of intra-graft NOS2 transcript expression compared with untreated Žcontrol. recipients w26x. It is possible, as in the studies using naive T cells, that CD40L blockade prevented full T cell activation, thus diminishing the expression of cytokines which regulate NOS2 expression. This appears unlikely to be the sole explanation since T cell cytokine transcripts for IL2 and IFN␥ were

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not decreased. However, decreased TNF␣ transcript expression remains a possible explanation since this transcript was not analyzed in this earlier study. An alternative possibility is that CD40 ligand may provide an important survival signal for macrophages during an immune response w17,30,31x. Therefore, recipients treated with anti-CD40L may have decreased NOS2 transcript levels within cardiac allografts due to shortened macrophage survival, rather than lack of CD40 stimulation of NO production. Future studies investigating the role of the CD40 pathway in macrophage survival in vivo will help clarify these findings. Finally, we have shown that the effects of the CD40 pathway on NO production are not solely mediated through an autocrine TNF␣-dependent pathway. Thus, CD40L provides a TNF-independent mechanism for NO production during CD4-dependent immune responses. These data may explain earlier findings demonstrating that while macrophages from TNFRp55yry mice do not produce NO in the presence of IFN␥ and TNF␣ , they can produce readily detectable levels of NO in the setting of parasitic or mycobacterial infection in vivo w32,33x. It is possible that in the absence of TNFR signaling, CD40LrCD40 interactions are critical for NO production in vivo. In summary, our data indicate that during intact CD4-dependent immune responses, IFN␥ provides an obligatory ‘first’ signal for NO production, whereas ligation of CD40 or TNF receptors on the cell surface of macrophages delivers a redundant rather than requisite ‘second’ activation signal for NO production.

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