CD154 Activates Antimicrobial Activity in the Absence of IFN- γ through a TNF- α-Dependent Mechanism

This information is current as Rosa M. Andrade, Matthew Wessendarp and Carlos S. of September 27, 2021. Subauste J Immunol 2003; 171:6750-6756; ; doi: 10.4049/jimmunol.171.12.6750 http://www.jimmunol.org/content/171/12/6750 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD154 Activates Macrophage Antimicrobial Activity in the Absence of IFN-␥ through a TNF-␣-Dependent Mechanism1

Rosa M. Andrade, Matthew Wessendarp, and Carlos S. Subauste2

Protection against certain intracellular pathogens can take place in the absence of IFN-␥ through mechanisms dependent on TNF-␣. In this regard, patients with partial defect in IFN-␥ receptor 1 are not susceptible to toxoplasmosis. Thus, we used a model of Toxoplasma gondii infection to investigate whether CD154 modulates IFN-␥-independent mechanisms of host protection. Hu- man -derived treated with recombinant CD154 exhibited increased anti-T. gondii activity. The number of tachyzoites per 100 macrophages at 20 h postinfection was lower in CD154-treated macrophages compared with controls. This was accompanied by a decrease in the percentage of infected cells in CD154-treated macrophages at 20 h compared with 1 h postin- fection. CD154-bearing cells also induced antimicrobial activity in T. gondii-infected macrophages. CD154 enhanced macrophage ␥ ␣ anti-T. gondii activity independently of IFN- . TNF- mediated the effects of CD154 on macrophage anti-T. gondii activity. CD154 Downloaded from increased TNF-␣ production by T. gondii-infected macrophages, and neutralization of TNF-␣ inhibited the effect of CD154 on macrophage anti-T. gondii activity. These results demonstrate that CD154 triggers TNF-␣-dependent antimicrobial activity in macrophages and suggest that CD154 regulates the mechanisms of host protection that take place when IFN-␥ signaling is deficient. The Journal of Immunology, 2003, 171: 6750Ð6756.

␥ ␥

nterferon- is considered a major mediator of host protection mediated by IFN- . However, the regulation of these mechanisms http://www.jimmunol.org/ against intracellular pathogens. However, there is increasing of host protection is not completely understood. I evidence pointing toward the presence of IFN-␥-independent CD154 is a member of the TNF family that plays a pivotal role mechanisms of control of these infections. In this regard, either in the regulation of cellular and humoral immunity. CD154 is ex- IFN-␥Ϫ/Ϫ or IFN-␥-receptorϪ/Ϫ (IFN-␥RϪ/Ϫ) mice exhibit pro- pressed as a membrane molecule (primarily on activated CD4ϩ T longed survival after infection with a virulent strain of Listeria cells) and as a soluble protein (8, 9). Through its interaction with monocytogenes if they are first infected with an attenuated strain or CD40, CD154 regulates many aspects of the immune response, are treated with anti-IL-4 mAb or TNF-␣ (1, 2). IFN-␥Ϫ/Ϫ mice including activation of APCs, priming of CD4ϩ and CD8ϩ T cells, infected with Leishmania donovani reduce parasite burden through stimulation of IL-12/IFN-␥ production, B cell proliferation, and Ig the action of endogenous TNF-␣ (3). Mice deficient in IFN regu- synthesis (10Ð12). The role of CD154 in orchestrating fundamen- by guest on September 27, 2021 latory factor-1 gene exhibit IFN-␥-independent mechanisms of re- tal aspects of the immune response is likely to explain why defec- sistance against Toxoplasma gondii (4). In addition, IFN-␥Ϫ/Ϫ tive CD154 signaling results in increased susceptibility to patho- mice control secondary infection with Histoplasma capsulatum via gens such as Leishmania, Pneumocystis carinii, T. gondii, a mechanism dependent on endogenous TNF-␣ (5). Together, Mycobacterium avium, Cryptosporidium parvum, Salmonella dub- these studies indicate that TNF-␣ may be central to the control of lin, and Candida albicans (13Ð20). intracellular pathogens in the absence of IFN-␥ (2, 3, 5). One of the consequences of CD154-mediated APC activation There is also indication that infections with certain intracellular appears to be enhancement of macrophage anti-microbial activity. pathogens can be controlled in humans with defective IFN-␥ sig- Studies in mice revealed that in the presence of IFN-␥, signaling naling. Although patients with congenital deficiencies in IFN-␥- through CD40 induces macrophage anti-Leishmania major and an- and IL-12-mediated immune response are susceptible to disease ti-T. gondii activities (14, 16). Studies in humans have reported caused by atypical Mycobacteria and Salmonella, diseases caused conflicting results about the effect of CD154 on macrophage anti- by other intracellular pathogens are uncommon (6). Indeed, pa- microbial activity. Although human monocyte-derived macrophages tients with partial IFN-␥R1 deficiency do not develop toxoplas- treated with CD154 impaired the intracellular growth of M. avium, mosis despite serological evidence of chronic infection with T. CD154 had no effect on the replication of Mycobacterium tuberculo- gondii (7). Thus, there is strong evidence for the existence of sis within these host cells (18, 21). Thus, it is not clear whether CD154 mechanisms of control of intracellular pathogens other than those can enhance macrophage antimicrobial activity in the absence of IFN-␥ and if this takes place in human macrophages. Using a model of T. gondii infection, we set out to determine whether CD154 modulates the antimicrobial activity of human Department of Internal Medicine, University of Cincinnati College of Medicine, Cin- macrophages in conditions where IFN-␥ is lacking or deficient. T. cinnati, OH 45267 gondii represents an excellent model for these studies because this Received for publication May 16, 2003. Accepted for publication October 2, 2003. pathogen is important in immunocompromised patients, macro- The costs of publication of this article were defrayed in part by the payment of page phage activation is crucial for host protection against T. gondii (22, charges. This article must therefore be hereby marked advertisement in accordance 23), and humans with defective IFN-␥ signaling are capable of with 18 U.S.C. Section 1734 solely to indicate this fact. controlling this pathogen (7). We report that CD154 activates hu- 1 This work was supported by National Institutes of Health Grant AI48406 (to C.S.S.). man macrophages to exhibit anti-T. gondii activity in both the 2 Address correspondence and reprint requests to Dr. Carlos S. Subauste, Department ␥ of Internal Medicine, University of Cincinnati College of Medicine, P.O. Box 670560, absence of IFN- and the presence of concentrations of this cyto- Cincinnati, OH 45267-0560. E-mail address: [email protected] kine insufficient to trigger full macrophage activation. In addition,

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 6751 we show that this effect of CD154 is mediated by TNF-␣. These ture, followed by addition of anti-CD40 FITC and anti-CD14-PE (Caltag, results suggest that CD154 regulates TNF-␣-dependent mecha- South San Francisco, CA) or isotype control mAbs. After 30-min incuba- nisms of host protection that operate when IFN-␥ signaling is tion on ice, cells were washed and fixed with 1% paraformaldehyde. The expression of CD40 was analyzed using a FACSCalibur (BD PharMingen). deficient. Addition of recombinant CD154 to macrophages before staining caused an 18% inhibition of the percentage of CD40ϩ cells. Materials and Methods Macrophage culture ELISA Macrophages were generated by culturing in complete medium Monolayers of monocyte-derived macrophages cultured in 96-well plates (CM)3 containing human serum. Briefly, PBMC were isolated after buffy were washed before infection. Supernatants were collected 4 h postinfec- coats of heparinized blood from healthy volunteers (Hoxworth Blood Cen- tion and were used to measure the concentration of TNF-␣ by ELISA ter, Cincinnati, OH) were centrifuged on Ficoll-Hypaque gradients (Am- (Endogen, Cambridge, MA). The lower limit of detection of the assay was ersham Pharmacia Biotech, Piscataway, NJ). Monocytes were purified by 9 pg/ml. Supernatants from monolayers were collected at the end of the 5-d incubating PBMC with the following mAbs (all from BD PharMingen (San culture and used to measure IFN-␥ levels by ELISA (Endogen). The lower Jose, CA), except when indicated): anti-CD2, anti-CD3, anti-CD8, anti- limit of detection of the assay was 39 pg/ml. CD19 (Coulter-Immunotech, Hialeah, FL), anti-CD56, and anti-CD66b (Coulter-Immunotech) (24). After addition of magnetic beads coated with Statistical analysis anti-mouse IgG (Dynal, Great Neck, NY), rosetting cells were removed Statistical significance was assessed by Student’s t test. with a magnet. The resulting population was Ͼ92% CD14ϩ and contained Ͻ0.5% CD3ϩ T cells. Purified monocytes were incubated for 5 days in either eight-chamber tissue culture glass slides (Falcon; BD Biosciences, Results 5 Franklin Lakes, NJ; 2 ϫ 10 monocytes/chamber) or 96-well plates (Lim- CD154 activates anti-T. gondii activity of human macrophages Downloaded from bro; INC Biomedicals, Aurora, OH; 1 ϫ 105 monocytes/well) using CM consisting of RPMI 1640 with 10% pooled human AB serum negative for To determine whether CD154 modulates the anti-T. gondii activity anti-T. gondii Abs (Gemini Biological Products, Calabasas, CA). Macro- of human macrophages, control and CD154-treated monocyte-de- phages were cultured in CM alone or CM plus recombinant CD154 trimer rived macrophages were challenged with tachyzoites of the para- (3 ␮g/ml; gift from Immunex, Seattle, WA) for 5 days unless otherwise site. Table I shows that the percentage of infected macrophages stated. In certain experiments, macrophages cultured in Teflon jars were and the numbers of parasites per infected macrophage and per 100 stained with anti-CD40 FITC (BD PharMingen), followed by sorting into CD40ϩ and CD40Ϫ cells using a FACSVantage SE (BD PharMingen). The macrophages at 1 h postinfection were similar in both groups of http://www.jimmunol.org/ purity of these populations was Ͼ90%. Sorted cells were cultured with and macrophages. However, the number of tachyzoites per 100 mac- without CD154 for 2 days. In some experiments either neutralizing mAbs rophages at 20 h postinfection was significantly lower in CD154- against IFN-␥ (R&D Systems, Minneapolis, MN), TNF-␣ (R&D Systems), treated macrophages than in control macrophages. The number of CD154 (Immunex), or isotype control (BD PharMingen) mAb (all at 10 Ϯ ␮g/ml) were added during the 5 days of incubation or macrophages were parasites per 100 macrophages was, on the average, 58.7 4.4% incubated with IFN-␥ (either 500 pg/ml or 5 ng/ml; PeproTech, Rocky Hill, lower in CD154-treated monolayers than in controls ( p Ͻ 0.001; NJ) for 3 days. Cell recovery at the end of in vitro culture was not affected n ϭ 9). This was accompanied by a 64.0 Ϯ 8.7% decrease in the by CD154. Tissue culture reagents and parasite preparations lacked detect- infection rate at 20 h compared with 1 h postinfection in CD154- Ͻ able levels of endotoxin ( 0.015 endotoxin units/ml) using the Limulus treated monolayers ( p Ͻ 0.00l; n ϭ 4). The lower percentage of amebocyte lysate assay (Sigma-Aldrich, St. Louis, MO). by guest on September 27, 2021 infected cells in the CD154-treated monolayers was not due to a T. gondii infection and parasite growth preferential cell loss from these monolayers during washing steps Monolayers of monocyte-derived macrophages were washed before addi- before staining. Supernatants collected from CD154-treated and tion of T. gondii. Tachyzoites of the RH strain of T. gondii, obtained as control monolayers revealed that the percentage of cells that de- previously described (25), were used to infect monolayers at a ratio of two tached was Ͻ1.5 and Ͻ3% for CD154-treated and control mono- parasites per macrophage. Parasite replication was assessed by light mi- layers, respectively (data not shown). Studies of kinetics of mac- croscopy (eight-chamber tissue culture glass slide) and uptake of [3H]uracil (96-well plate) as previously described (26, 27). Briefly, monolayers were rophage activation revealed that incubation of macrophages with washed 1 h after addition of T. gondii to remove extracellular parasites. CD154 for 1 d enhanced anti-T. gondii activity, and this effect was Thereafter, monolayers were either fixed and stained with Diff-Quick maximal after 2 d (Fig. 1). (Dade Diagnostics, Aguada, Puerto Rico), or monolayers were reincubated Assessment of T. gondii growth by [3H]uracil uptake showed in fresh CM (without CD154 or ), followed by fixation and staining 3 20 h after addition of T. gondii. The percentage of infected macrophages, the similar results and revealed that CD154 inhibited [ H]uracil incor- number of tachyzoites per infected macrophage, and the number of parasites poration in a dose-dependent manner (Fig. 2, A and B; p Ͻ 0.001). per 100 macrophages in duplicate monolayers were determined by light mi- On the average, addition of CD154 (3 ␮g/ml) caused a 55.3 Ϯ croscopy by counting at least 200 macrophages/monolayer. 2.2% reduction in [3H]uracil uptake compared with that in control Unless otherwise stated, assays of [3H]uracil incorporation were per- formed by pulsing 96-well plates with 1 ␮Ci of [3H]uracil (PerkinElmer, Boston, MA) 14 h postinfection and harvesting samples after 8 h. In certain experiments, gamma-irradiated (7500 rad) L cells transfected with either Table I. CD154 enhances anti-T. gondii activity of human a human CD154 or CD32 (gift from R. de Waal Malefyt, DNAX, Palo Alto, macrophages CA) were incubated with monolayers of infected macrophages at a ratio of 0.5 L cell/1 macrophage. L cells were added to monolayers after macro- % Infected M␾ Tg/Infected M␾ Tg/100 M␾ phages had been incubated with T. gondii for 1 h and after removal of extracellular tachyzoites. At 28 h postinfection, these cells were pulsed 1h with 1 ␮Ci of [3H]uracil for 8 h. The integrity of the monolayers was Control 28.19 Ϯ 1.54 1.15 Ϯ 0.04 32.1 Ϯ 1.91 confirmed microscopically before harvesting. Radioactivity was measured CD154 27.83 Ϯ 1.8 1.19 Ϯ 0.04 35.6 Ϯ 2.3 in a beta scintillation counter. Results are expressed as mean counts per 20 h minute of incorporation [3H]uracil of triplicate wells Ϯ SEM. Control 27.85 Ϯ 2.02 3.83 Ϯ 0.35 101.6 Ϯ 7.87 CD154 10.02 Ϯ 1.39 3.48 Ϯ 0.08 34.56 Ϯ 5.81 Flow cytometry a Monocyte-derived macrophages (M␾) were cultured in CM alone or in CM plus After 5-d in vitro culture, monocyte-derived macrophages were incubated CD154 (3 ␮g/ml) as described in Materials and Methods. Macrophages were washed with human IgG (20 ␮g/ml; Sigma-Aldrich) for 10 min at room tempera- and challenged with T. gondii for 1 h. Monolayers were examined by light micros- copy 1 and 20 h after addition of T. gondii. Results shown are the means Ϯ SEM of four independent experiments using macrophages from different donors. Experiments 3 Abbreviations used in this paper: CM, complete medium; TNFR, TNF receptor; using five additional donors in which macrophages were evaluated only at 20 h X-HIM, X-linked hyper-IgM syndrome. postinfection yielded similar results. 6752 CD154, TNF-␣, AND MACROPHAGE ANTI-MICROBIAL ACTIVITY

FIGURE 1. KineticsofCD154-mediatedstimulationofmacrophageanti- FIGURE 3. CD154 stimulates macrophage antimicrobial activity only T. gondii activity. Monocyte-derived macrophages were cultured for5din in CD40ϩ macrophages. CD40ϩ and CD40Ϫ were isolated using FACS- either CM alone or CM to which CD154 (3 ␮g/ml) was added 1Ð5 d before Vantage SE and were cultured for 2 days in CM with or without CD154. T. gondii infection. Macrophages were washed and challenged with T. Monolayers were examined by light microscopy 20 h after T. gondii chal- gondii for 1 h. Monolayers were examined by light microscopy 20 h after lenge. Results are representative of one of three independent experiments. addition of T. gondii. The results shown are the mean Ϯ SEM of duplicate Downloaded from monolayers from a representative experiment of three performed using macrophages from different donors. CD40ϩ and CD40Ϫ macrophages to CD154 to confirm that dif- ferential expression of CD40 was the explanation for the lack of macrophages ( p Ͻ 0.0001; n ϭ 10). The effect of CD154 was response to CD154 by a subpopulation of macrophages. Fig. 3 specific, as addition of a neutralizing anti-CD154 mAb resulted in shows that CD154 caused a profound decrease (79.0 Ϯ 3.8% in- ϩ

88.7 Ϯ 5.0% inhibition of the effect of CD154 ( p Ͻ 0.02; n ϭ 2), hibition; p Ͻ 0.001; n ϭ 3) in the parasite load in CD40 , but not http://www.jimmunol.org/ whereas an isotype control mAb failed to alter the effect of CD154 in CD40Ϫ, macrophages. Thus, only CD40ϩ macrophages respond (Fig. 2C). CD154 did not exhibit direct toxic activity against T. to CD154. gondii, as incubation of tachyzoites with CD154 for1hat37¡C CD154 is considered to exist primarily as a membrane molecule before addition to macrophages did not affect the parasite load in on activated CD4ϩ T cells. Thus, we determined whether CD154- unstimulated macrophages at 20 h postinfection (data not shown). bearing cells also stimulate antimicrobial activity of macrophages, Taken together, CD154 stimulates anti-T. gondii activity in human and whether this effect could take place when CD154 interacts macrophages. with macrophages already infected with an intracellular pathogen. While CD154 decreased the number of tachyzoites per 100 mac- Macrophages previously infected with T. gondii were exposed for rophages and the percentage of infected macrophages, the number 36 h to either L cells transfected with human CD154 or CD32 as by guest on September 27, 2021 of tachyzoites per infected macrophage was not affected (Table I). a control. As shown in Fig. 4, addition of L-CD154 resulted in a These results raised the possibility that only a subpopulation of significant decrease in [3H]uracil incorporation compared with ei- macrophages responded to CD154. Thus, we examined the expres- ther infected macrophages alone or infected macrophages incu- sion of CD40 on monocytes and monocyte-derived macrophages. bated with L-CD32 (61.6 Ϯ 4.0% inhibition; p Ͻ 0.003; n ϭ 3). Flow cytometric analysis revealed that 6.6 Ϯ 0.5% of freshly iso- Moreover, a neutralizing anti-CD154 mAb caused significant in- lated monocytes were CD40ϩ (n ϭ 4). CD40 expression increased hibition of the effect of L-CD154 on T. gondii growth (68.7 Ϯ after 5 days of in vitro culture, resulting in percentages of CD40ϩ 5.1% inhibition; p Ͻ 0.004). It is unlikely that the decrease in cells of 50.0 Ϯ 8.0% in untreated macrophages and 47.9 Ϯ 9.3% uracil incorporation was caused by preactivation of uninfected in CD154-treated macrophages. We examined the response of macrophages that subsequently became infected with T. gondii.

FIGURE 2. CD154 stimulates anti-T. gondii activity of human macrophages. A, Monocyte-derived macrophages were cultured in CM alone or CM plus CD154 (3 ␮g/ml). Macrophages were washed and challenged with T. gondii. Monolayers were pulsed with 1 ␮Ci of [3H]uracil for 8 h. Data are expressed as the mean counts per minute of [3H]uracil incorporation of triplicate wells Ϯ SEM. Results are representative of one of 10 independent experiments using different donors. B, Dose-dependent stimulation of macrophage anti-T. gondii activity. Macrophages were incubated with increasing concentrations of CD154. The results are shown as inhibition of [3H]uracil incorporation compared with untreated macrophages and represent the mean Ϯ SEM of four individual experiments. C, The effect of CD154 on macrophage anti-T. gondii activity is specific. Monocyte-derived macrophages were cultured in CM alone or in CM plus CD154 (300 ng/ml) with either a neutralizing anti-CD154 or isotype control mAbs (10 ␮g/ml). The results of one representative experiment of two are shown. The Journal of Immunology 6753

FIGURE 4. CD154-bearing cells stimulate macrophage anti-T. gondii FIGURE 6. CD154 stimulates TNF-␣ production by T. gondii-infected activity. Macrophages were infected with T. gondii for 1 h, and extracel- macrophages. Monocyte-derived macrophages (2.5 ϫ 106/ml) were cul- lular tachyzoites were removed by washing. Thereafter, gamma-irradiated tured in CM with or without CD154. Supernatants were collected 4 h after 3 L-CD32 or L-CD154 cells were added to infected macrophages, and [ H]u- challenge with T. gondii and used to measure TNF-␣ concentrations by racil incorporation was assessed as described. Results are representative of ELISA. Data are expressed as the mean Ϯ SEM of three independent one of three independent experiments. experiments. Downloaded from

Microscopic examination of culture slides set up in parallel re- Patients with partial IFN-␥R1 deficiency control T. gondii in- vealed that there was no increase in the rate of infection and no fection (7). Thus, we studied the effect of CD154 on macrophage evidence of destruction of macrophages during the 36 h of culture anti-T. gondii activity when IFN-␥ signaling was present, but de- (data not shown). Therefore, cells that express CD154 on their ficient. Macrophages were incubated with suboptimal concentra- membranes activate anti-T. gondii activity of human macrophages. tions of CD154 (300 pg/ml) and IFN-␥ (500 pg/ml), either alone or http://www.jimmunol.org/ in combination, before infection with T. gondii. Fig. 5B shows that ␥ CD154 acts independently of IFN- and further stimulates whereas the growth of T. gondii was minimally affected by either ␥ macrophage anti-T. gondii activity when IFN- signaling is CD154 or IFN-␥, the combination of these two factors resulted in suboptimal a significant inhibition in parasite growth ( p Ͻ 0.01; n ϭ 3). Thus, In the next series of experiments we explored the mechanism of CD154 cooperates with IFN-␥ in stimulating the antimicrobial ac- action of CD154. These experiments were conducted using recom- tivity of human macrophages. binant CD154, as our goal was to identify the mechanism(s) by which CD154 alone stimulates macrophage antimicrobial activity, CD154 enhances human macrophage anti-T. gondii activity ␣ by guest on September 27, 2021 and CD154-bearing cells may express other factors that modulate through a TNF- -dependent mechanism macrophage function. We determined whether the effect of CD154 CD154 stimulates human monocytes to secrete monokines, includ- on macrophage anti-T. gondii activity is mediated by IFN-␥. In- ing TNF-␣ (28Ð30), a that plays an important role in cubation of macrophages with CD154 plus a neutralizing anti- control of intracellular pathogens such as T. gondii (31Ð33). Thus, IFN-␥ mAb resulted in similar inhibition of [3H]uracil uptake as in we ascertained whether TNF-␣ signaling mediates stimulation of macrophages treated with CD154 alone or CD154 plus an isotype macrophage anti-T. gondii activity caused by CD154. We began control mAb (Fig. 5A; p Ͼ 0.5; n ϭ 3). At the concentration used, by studying the effect of CD154 on TNF-␣ production by T. gon- the anti-IFN-␥ mAb caused a 94.2 Ϯ 10.9% inhibition of the anti- dii-infected macrophages. While uninfected, untreated macro- T. gondii activity produced by 5 ng/ml of IFN-␥ ( p Ͻ 0.001; n ϭ phages did not secrete detectable amounts of TNF-␣, incubation 3; Fig. 5A). Moreover, IFN-␥ was not detected (Ͻ39 pg/ml) in with T. gondii resulted in the production of low levels of this supernatants collected from CD154-treated macrophages. To- cytokine (Fig. 6). Incubation of uninfected macrophages with gether, CD154 stimulates macrophage anti-T. gondii activity in- CD154 resulted in TNF-␣ secretion, whereas T. gondii infection of dependently of IFN-␥. CD154-treated macrophages caused a significant further increase

FIGURE 5. CD154 acts independently of IFN-␥, but cooperates with IFN-␥ in enhancing macrophage anti-T. gondii activity. A, Monocyte-derived macrophages were cultured with either CD154 or IFN-␥ in the presence of a neutralizing anti-IFN-␥ or isotype control mAbs. T. gondii growth was assessed by [3H]uracil incorporation. Results shown are representative of one of three independent experiments. B, Monocyte-derived macrophages were cultured with suboptimal concentrations of either CD154 (300 pg/ml) or IFN-␥ (500 pg/ml). The results of one representative experiments of three are shown. 6754 CD154, TNF-␣, AND MACROPHAGE ANTI-MICROBIAL ACTIVITY

Table II. TNF-␣ mediates the effect of CD154 on anti-T. gondii activity Defective CD154 signaling in humans and mice results in in- of human macrophagesa creased susceptibility to infections with intracellular pathogens, including T. gondii. We previously reported that patients with a % Infected M␾ Tg/infected M␾ Tg/100 M␾ congenital lack of functional CD154 (X-linked hyper-IgM syn- Control 22.31 Ϯ 0.50 3.34 Ϯ 0.05 74.58 Ϯ 1.12 drome (X-HIM)) have impaired in vitro production of IL-12 and Control ϩ mIgG 20.03 Ϯ 1.28 3.22 Ϯ 0.01 64.33 Ϯ 4.26 IFN-␥ in response to T. gondii (35). We proposed this mechanism Control ϩ @TNF-␣ 17.94 Ϯ 0.54 3.48 Ϯ 0.05 62.46 Ϯ 1.55 to explain the increased incidence of opportunistic infections in CD154 10.88 Ϯ 0.62 3.29 Ϯ 0.29 35.95 Ϯ 5.16 patients with this immunodeficiency (35). However, the fact that ϩ Ϯ Ϯ Ϯ CD154 mIgG 12.96 1.66 3.08 0.38 39.35 0.18 individuals with partial IFN-␥R1 deficiency are not susceptible to CD154 ϩ @TNF-␣ 24.27 Ϯ 2.69 2.90 Ϯ 0.40 68.97 Ϯ 5.36 toxoplasmosis suggests that there are additional mechanisms that a Monocyte-derived macrophages (M␾) were cultured in CM alone or in CM plus account for the increased incidence of opportunistic infections in CD154 (3 ␮g/ml) with or without a neutralizing anti-TNF-␣ or a control mAb (10 ␮g/ml) as described in Materials and Methods. Macrophages were washed and chal- patients with X-HIM. Our previous work and this report indicate lenged with T. gondii for 1 h. Monolayers were examined by light microscopy 20 h that impaired T cell priming (35) and defective macrophage acti- after addition of T. gondii. Results shown are the mean Ϯ SEM of duplicate mono- layers from a representative experiment of three performed using macrophages from vation are likely to contribute to the predisposition of patients with different donors. X-HIM to opportunistic infections. CD154 is expressed as a membrane glycoprotein on activated CD4ϩ T cells. However, cleavage of membrane CD154 results in the release of a soluble form of the protein (8, 9). We report that in TNF-␣ production ( p Ͻ 0.03; n ϭ 3). Secretion of TNF-␣ soluble trimeric recombinant CD154 and CD154-bearing cells Downloaded from triggered by CD154 was not affect by a neutralizing mAb against stimulated anti-T. gondii activity of macrophages. Although both IFN-␥ (data not shown). forms of CD154 have been reported to be biologically active (9, To further determine whether TNF-␣ mediates the effect of 36), membrane CD154 is considered to be more effective at trig- CD154 on macrophage anti-T. gondii activity, control and CD154- gering CD40 signal transduction (37). Thus, segregation of recep- treated macrophages were incubated with a neutralizing anti- tor and counter-receptors into supramolecular clusters (38) in the ␣ ␣ TNF- mAb. The anti-TNF- mAb used does not cross-react with immunological synapse may promote optimal signaling by causing http://www.jimmunol.org/ CD154 (34). Table II shows that whereas addition of an anti- CD40 clustering (39). The superior capacity of membrane CD154 TNF-␣ mAb to control macrophages did not affect T. gondii to induce CD40 signaling may explain why, in contrast to our growth, this mAb increased the number of tachyzoites per 100 studies using CD154-bearing cells, the addition of recombinant CD154-treated macrophages. Anti-TNF-␣ caused an 85.7 Ϯ 4.7% CD154 to macrophages already infected with T. gondii did not inhibition of the effect of CD154 on T. gondii growth (n ϭ 3; p Ͻ reproducibly induce anti-T. gondii activity (R. M. Andrade and 0.05). Thus, CD154 stimulates anti-T. gondii activity in human C. S. Subauste, unpublished observations). The lack of induction macrophages at least in part through TNF-␣ signaling. of macrophage antimicrobial activity after isolated CD40 signaling reported in other studies may be explained by the use of subopti- Discussion mal CD40 signaling and/or by differences in the requirements for by guest on September 27, 2021 Macrophages are major effectors of control of intracellular patho- generation of macrophage antimicrobial activity depending on the gens. The induction of macrophage antimicrobial activity by pathogen used to infect macrophages. In support of the latter pos- IFN-␥ is considered crucial for protection against these organisms sibility is the fact that patients with defective IFN-␥/IL-12 signal- (22, 23). However, patients with defects in IFN-␥ signaling do not ing are susceptible to Mycobacteria and Salmonella, whereas dis- appear to be susceptible to infections other than atypical Myco- ease caused by other intracellular pathogens, such as T. gondii, L. bacteria and Salmonella (6). In addition, mice with defects in monocytogenes, and Legionella, is uncommon (6, 7). IFN-␥ signaling develop protective mechanisms against L. mono- The present study revealed that endogenous TNF-␣ is crucial for cytogenes, L. donovani, H. capsulatum, and T. gondii (1Ð5). The the induction of anti-T. gondii activity in human macrophages aim of the present work was to identify IFN-␥-independent mech- stimulated with CD154. Consistent with the role of CD154 as a anisms of induction of macrophage antimicrobial activity. We re- stimulator of monokine secretion (28Ð30) is our demonstration port that isolated CD154 signaling triggered anti-T. gondii activity that CD154 enhanced TNF-␣ production by T. gondii-infected hu- in human macrophages without requiring the presence of IFN-␥. man macrophages. Noteworthy are reports of the poor capacity of Although CD154 induced macrophage anti-microbial activity in T. gondii to induce TNF-␣ production by macrophages (40, 41). the absence of IFN-␥, CD154 also stimulated this activity when Thus, CD154 may play an important role in enhancing the secre- IFN-␥ was present at concentrations inadequate to induce full mac- tion of this cytokine in response to T. gondii. There is precedent for rophage activation. The latter observation suggests that CD154 the role of TNF-␣ as an autocrine regulator of macrophage anti- may be an important mediator of protection against intracellular microbial activity. Endogenous TNF-␣ mediates the effects of pathogens such as T. gondii in hosts with insufficient IFN-␥-de- IFN-␥ on macrophage microbiostatic activity (41). Importantly, pendent signals, such as patients with partial IFN-␥R1 deficiency we now show that CD154 provides an alternative mechanism for who are not susceptible to toxoplasmosis (7). In addition, the ev- activation of the TNF-␣ signaling and macrophage antimicrobial idence of cooperation between CD154 and IFN-␥ for activation of activity that take place in the absence of IFN-␥. macrophage anti-T. gondii activity supports the idea that both mol- TNF-␣ is pivotal for control of intracellular pathogens such as T. ecules act in concert to control intracellular pathogens during the gondii (31Ð33, 42), Mycobacteria (43), Leishmania (3, 44), L. immune response in immunocompetent hosts (14, 16). TNF-␣ was monocytogenes (2, 45, 46), and H. capsulatum (5, 47). The fact a central mediator of the anti-T. gondii activity induced by CD154. that CD154 activated TNF-␣-dependent antimicrobial activity of CD154 stimulated TNF-␣ production by T. gondii-infected mac- macrophages suggests that CD154 is an important regulator of rophages. In turn, TNF-␣ was pivotal for the expression of the TNF-␣-mediated mechanisms of host protection. However, TNF-␣ enhanced antimicrobial state. These results demonstrate that alone is unable to activate anti-T. gondii activity in macrophages CD154 is an important regulator of the effects of TNF-␣ on mac- (40, 41, 48). Our results raise the possibility that the CD40-CD154 rophage antimicrobial activity. interaction may act not only by stimulating TNF-␣ production by The Journal of Immunology 6755 infected macrophages, but also by making these cells responsive to 2. Szalay, G., C. H. Label, C. Blum, and S. H. E. Kaufmann. 1996. IL-4 neutral- ␣ TNF-␣. It is interesting to note that signal transduction triggered ization or TNF- treatment ameliorate disease by an intracellular pathogen in IFN-␥ receptor deficient mice. J. Immunol. 157:4746. by the different members of the TNF receptor (TNFR) family (in- 3. Taylor, A. P., and H. W. Murray. 1997. Intracellular antimicrobial activity in the cluding TNFRI, TNFRII, and CD40) is similar, but not identical absence of -␥: effect of -12 in experimental visceral leish- maniasis in interferon-␥ gene-disrupted mice. J. Exp. Med. 185:1231. (49). Indeed, these differences are likely to explain the intracellular 4. Khan, I. A., T. Matsuura, S. Fonseka, and L. H. Kasper. 1996. Production of nitric convergence of independent signaling triggered by members of oxide (NO) is not essential for protection against Toxoplasma gondii infection in Ϫ Ϫ this family of receptors (50). Thus, signals through TNFR and IRF-1 / mice. J. Immunol. 156:636. 5. Zhou, P., G. Miller, and R. A. Seder. 1998. Factors involved in regulating pri- CD40 may act in concert to induce a state of increased macrophage mary and secondary immunity to infection with Histoplasma capsulatum: TNF-␣ antimicrobial activity. The fact that TNF-␣ mediates the enhanced plays a critical role in maintaining secondary immunity in the absence of IFN-␥. anti-T. gondii activity in macrophages stimulated with CD154 may J. Immunol. 160:1359. Ϫ/Ϫ Ϫ/Ϫ 6. Ottenhoff, T. H. M., D. Kumararatne, and J. L. Casanova. 1998. Novel human explain why both CD154 and TNFR mice show similar immunodeficiencies reveal the essential role of type-1 cytokines in immunity to susceptibility to T. gondii and develop toxoplasmic encephalitis intracellular bacteria. Immunol. Today 19:491. (16, 31, 33). 7. Janssen, R., A. van Wengen, E. Verhard, T. de Boer, T. Zomerdijk, T. H. M. Ottenhoff, and J. T. van Dissel. 2002. Divergent role for TNF-␣ in Activated macrophages use a variety of effector mechanisms to IFN-␥-induced killing of Toxoplasma gondii and Salmonella typhimurium con- impair the growth or kill intracellular pathogens. These mecha- tributes to selective susceptibility of patients with partial IFN-␥ receptor 1 defi- ciency. J. Immunol. 169:3900. nisms include the production of reactive oxygen intermediates and 8. Graf, D., S. Muller, U. Korthauer, C. van Kooten, C. Weise, and R. A. Kroczek. reactive nitrogen intermediates, tryptophan degradation, iron se- 1995. A soluble form of TRAP (CD40 ligand) is rapidly released after T cell questration, and delivery of antimicrobial polypeptides into phago- activation. Eur. J. Immunol. 25:1749. 9. Pietravalle, F., S. Lecoanet-Henchoz, H. Blasey, J. P. Aubry, G. Elson, somes (51). The fact that treatment with CD154 decreases the per- M. D. Edgerton, J. Y. Bonnefoy, and J. F. Gauchat. 1996. Human native soluble Downloaded from centage of infected macrophages suggests that this molecule CD40L is a biologically active trimer, processed inside microsomes. J. Biol. induces toxoplasmacidal activity in human macrophages. Studies Chem. 271:5965. 10. Durie, F. H., T. M. Foy, S. R. Masters, J. D. Laman, and R. J. Noelle. 1994. The to identify the effector mechanism(s) activated by CD154 are cur- role of CD40 in the regulation of humoral and cell-mediated immunity. Immunol. rently underway in our laboratory. Today 15:406. Many intracellular pathogens, including T. gondii, infect not 11. Grewal, I. S., and R. A. Flavell. 1998. CD40 and CD154 in cell-mediated im- munity. Annu. Rev. Immunol. 16:111. only macrophages, but also cells of nonhemopoietic origin. Thus, 12. van Kooten, C., and J. Banchereau. 2000. CD40-CD40 ligand. J. Leukocyte Biol. http://www.jimmunol.org/ effector mechanisms for control of these pathogens must be oper- 67:2. 13. Campbell, K. A., P. J. Ovendale, M. K. Kennedy, W. C. Fanslow, S. G. Reed, and ative at both levels. Indeed, a study using bone marrow chimeras C. R. Maliszewski. 1996. CD40 ligand is required for protective cell-mediated revealed that the lack of TNFR in either hemopoietic or nonhe- immunity to Leishmania major. Immunity 4:283. mopoietic compartments resulted in the inability to control chronic 14. Kamanaka, M., P. Yu, T. Yasui, K. Yoshida, T. Kawabe, T. Horii, T. Kishimoto, and H. Kikutani. 1996. Protective role of CD40 in Leishmania major infection at T. gondii infection and the development of toxoplasmic encepha- two distinct phases of cell-mediated immunity. Immunity 4:275. litis (52). These results suggest that the effect of TNF-␣ on non- 15. Wiley, J. A., and A. G. Harmsen. 1995. CD40 ligand is required for resolution of hemopoietic cells in the brain is particularly important for protec- Pneumocystis carinii pneumonia in mice. J. Immunol. 155:3525. 16. Reichmann, G., W. Walker, E. N. Villegas, L. Craig, G. Cai, J. 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