The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Andrade, R. M.
Right arrow Articles by Subauste, C. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Andrade, R. M.
Right arrow Articles by Subauste, C. S.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
*Genetics Home Reference
The Journal of Immunology, 2003, 171: 6750-6756.
Copyright © 2003 by The American Association of Immunologists

CD154 Activates Macrophage Antimicrobial Activity in the Absence of IFN-{gamma} through a TNF-{alpha}-Dependent Mechanism 1

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

Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protection against certain intracellular pathogens can take place in the absence of IFN-{gamma} through mechanisms dependent on TNF-{alpha}. In this regard, patients with partial defect in IFN-{gamma} receptor 1 are not susceptible to toxoplasmosis. Thus, we used a model of Toxoplasma gondii infection to investigate whether CD154 modulates IFN-{gamma}-independent mechanisms of host protection. Human monocyte-derived macrophages 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 postinfection. CD154-bearing cells also induced antimicrobial activity in T. gondii-infected macrophages. CD154 enhanced macrophage anti-T. gondii activity independently of IFN-{gamma}. TNF-{alpha} mediated the effects of CD154 on macrophage anti-T. gondii activity. CD154 increased TNF-{alpha} production by T. gondii-infected macrophages, and neutralization of TNF-{alpha} inhibited the effect of CD154 on macrophage anti-T. gondii activity. These results demonstrate that CD154 triggers TNF-{alpha}-dependent antimicrobial activity in macrophages and suggest that CD154 regulates the mechanisms of host protection that take place when IFN-{gamma} signaling is deficient.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interferon-{gamma} is considered a major mediator of host protection against intracellular pathogens. However, there is increasing evidence pointing toward the presence of IFN-{gamma}-independent mechanisms of control of these infections. In this regard, either IFN-{gamma}-/- or IFN-{gamma}-receptor-/- (IFN-{gamma}R-/-) mice exhibit prolonged survival after infection with a virulent strain of Listeria monocytogenes if they are first infected with an attenuated strain or are treated with anti-IL-4 mAb or TNF-{alpha} (1, 2). IFN-{gamma}-/- mice infected with Leishmania donovani reduce parasite burden through the action of endogenous TNF-{alpha} (3). Mice deficient in IFN regulatory factor-1 gene exhibit IFN-{gamma}-independent mechanisms of resistance against Toxoplasma gondii (4). In addition, IFN-{gamma}-/- mice control secondary infection with Histoplasma capsulatum via a mechanism dependent on endogenous TNF-{alpha} (5). Together, these studies indicate that TNF-{alpha} may be central to the control of intracellular pathogens in the absence of IFN-{gamma} (2, 3, 5).

There is also indication that infections with certain intracellular pathogens can be controlled in humans with defective IFN-{gamma} signaling. Although patients with congenital deficiencies in IFN-{gamma}- and IL-12-mediated immune response are susceptible to disease caused by atypical Mycobacteria and Salmonella, diseases caused by other intracellular pathogens are uncommon (6). Indeed, patients with partial IFN-{gamma}R1 deficiency do not develop toxoplasmosis despite serological evidence of chronic infection with T. gondii (7). Thus, there is strong evidence for the existence of mechanisms of control of intracellular pathogens other than those mediated by IFN-{gamma}. However, the regulation of these mechanisms of host protection is not completely understood.

CD154 is a member of the TNF family that plays a pivotal role in the regulation of cellular and humoral immunity. CD154 is expressed as a membrane molecule (primarily on activated CD4+ T cells) and as a soluble protein (8, 9). Through its interaction with CD40, CD154 regulates many aspects of the immune response, including activation of APCs, priming of CD4+ and CD8+ T cells, stimulation of IL-12/IFN-{gamma} production, B cell proliferation, and Ig synthesis (10, 11, 12). The role of CD154 in orchestrating fundamental aspects of the immune response is likely to explain why defective CD154 signaling results in increased susceptibility to pathogens such as Leishmania, Pneumocystis carinii, T. gondii, Mycobacterium avium, Cryptosporidium parvum, Salmonella dublin, and Candida albicans (13, 14, 15, 16, 17, 18, 19, 20).

One of the consequences of CD154-mediated APC activation appears to be enhancement of macrophage anti-microbial activity. Studies in mice revealed that in the presence of IFN-{gamma}, signaling through CD40 induces macrophage anti-Leishmania major and anti-T. gondii activities (14, 16). Studies in humans have reported conflicting results about the effect of CD154 on macrophage anti-microbial activity. Although human monocyte-derived macrophages treated with CD154 impaired the intracellular growth of M. avium, CD154 had no effect on the replication of Mycobacterium tuberculosis within these host cells (18, 21). Thus, it is not clear whether CD154 can enhance macrophage antimicrobial activity in the absence of IFN-{gamma} 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 macrophages in conditions where IFN-{gamma} is lacking or deficient. T. gondii represents an excellent model for these studies because this pathogen is important in immunocompromised patients, macrophage activation is crucial for host protection against T. gondii (22, 23), and humans with defective IFN-{gamma} signaling are capable of controlling this pathogen (7). We report that CD154 activates human macrophages to exhibit anti-T. gondii activity in both the absence of IFN-{gamma} and the presence of concentrations of this cytokine insufficient to trigger full macrophage activation. In addition, we show that this effect of CD154 is mediated by TNF-{alpha}. These results suggest that CD154 regulates TNF-{alpha}-dependent mechanisms of host protection that operate when IFN-{gamma} signaling is deficient.


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

Macrophages were generated by culturing monocytes in complete medium (CM) 3 containing human serum. Briefly, PBMC were isolated after buffy coats of heparinized blood from healthy volunteers (Hoxworth Blood Center, Cincinnati, OH) were centrifuged on Ficoll-Hypaque gradients (Amersham Pharmacia Biotech, Piscataway, NJ). Monocytes were purified by incubating PBMC with the following mAbs (all from BD PharMingen (San Jose, CA), except when indicated): anti-CD2, anti-CD3, anti-CD8, anti-CD19 (Coulter-Immunotech, Hialeah, FL), anti-CD56, and anti-CD66b (Coulter-Immunotech) (24). After addition of magnetic beads coated with anti-mouse IgG (Dynal, Great Neck, NY), rosetting cells were removed with a magnet. The resulting population was >92% CD14+ and contained <0.5% CD3+ T cells. Purified monocytes were incubated for 5 days in either eight-chamber tissue culture glass slides (Falcon; BD Biosciences, Franklin Lakes, NJ; 2 x 105 monocytes/chamber) or 96-well plates (Limbro; INC Biomedicals, Aurora, OH; 1 x 105 monocytes/well) using CM consisting of RPMI 1640 with 10% pooled human AB serum negative for anti-T. gondii Abs (Gemini Biological Products, Calabasas, CA). Macrophages were cultured in CM alone or CM plus recombinant CD154 trimer (3 µg/ml; gift from Immunex, Seattle, WA) for 5 days unless otherwise stated. In certain experiments, macrophages cultured in Teflon jars were stained with anti-CD40 FITC (BD PharMingen), followed by sorting into CD40+ and CD40- cells using a FACSVantage SE (BD PharMingen). The purity of these populations was >90%. Sorted cells were cultured with and without CD154 for 2 days. In some experiments either neutralizing mAbs against IFN-{gamma} (R&D Systems, Minneapolis, MN), TNF-{alpha} (R&D Systems), CD154 (Immunex), or isotype control (BD PharMingen) mAb (all at 10 µg/ml) were added during the 5 days of incubation or macrophages were incubated with IFN-{gamma} (either 500 pg/ml or 5 ng/ml; PeproTech, Rocky Hill, NJ) for 3 days. Cell recovery at the end of in vitro culture was not affected by CD154. Tissue culture reagents and parasite preparations lacked detectable levels of endotoxin (<0.015 endotoxin units/ml) using the Limulus amebocyte lysate assay (Sigma-Aldrich, St. Louis, MO).

T. gondii infection and parasite growth

Monolayers of monocyte-derived macrophages were washed before addition of T. gondii. Tachyzoites of the RH strain of T. gondii, obtained as previously described (25), were used to infect monolayers at a ratio of two parasites per macrophage. Parasite replication was assessed by light microscopy (eight-chamber tissue culture glass slide) and uptake of [3H]uracil (96-well plate) as previously described (26, 27). Briefly, monolayers were washed 1 h after addition of T. gondii to remove extracellular parasites. Thereafter, monolayers were either fixed and stained with Diff-Quick (Dade Diagnostics, Aguada, Puerto Rico), or monolayers were reincubated in fresh CM (without CD154 or cytokines), followed by fixation and staining 20 h after addition of T. gondii. The percentage of infected macrophages, the number of tachyzoites per infected macrophage, and the number of parasites per 100 macrophages in duplicate monolayers were determined by light microscopy by counting at least 200 macrophages/monolayer.

Unless otherwise stated, assays of [3H]uracil incorporation were performed by pulsing 96-well plates with 1 µCi 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 human CD154 or CD32 (gift from R. de Waal Malefyt, DNAX, Palo Alto, 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 macrophages had been incubated with T. gondii for 1 h and after removal of extracellular tachyzoites. At 28 h postinfection, these cells were pulsed with 1 µCi of [3H]uracil for 8 h. The integrity of the monolayers was confirmed microscopically before harvesting. Radioactivity was measured in a beta scintillation counter. Results are expressed as mean counts per minute of incorporation [3H]uracil of triplicate wells ± SEM.

Flow cytometry

After 5-d in vitro culture, monocyte-derived macrophages were incubated with human IgG (20 µg/ml; Sigma-Aldrich) for 10 min at room temperature, followed by addition of anti-CD40 FITC and anti-CD14-PE (Caltag, South San Francisco, CA) or isotype control mAbs. After 30-min incubation on ice, cells were washed and fixed with 1% paraformaldehyde. The expression of CD40 was analyzed using a FACSCalibur (BD PharMingen). Addition of recombinant CD154 to macrophages before staining caused an 18% inhibition of the percentage of CD40+ cells.

ELISA

Monolayers of monocyte-derived macrophages cultured in 96-well plates were washed before infection. Supernatants were collected 4 h postinfection and were used to measure the concentration of TNF-{alpha} by ELISA (Endogen, Cambridge, MA). The lower limit of detection of the assay was 9 pg/ml. Supernatants from monolayers were collected at the end of the 5-d culture and used to measure IFN-{gamma} levels by ELISA (Endogen). The lower limit of detection of the assay was 39 pg/ml.

Statistical analysis

Statistical significance was assessed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD154 activates anti-T. gondii activity of human macrophages

To determine whether CD154 modulates the anti-T. gondii activity of human macrophages, control and CD154-treated monocyte-derived macrophages were challenged with tachyzoites of the parasite. Table I shows that the percentage of infected macrophages and the numbers of parasites per infected macrophage and per 100 macrophages at 1 h postinfection were similar in both groups of macrophages. However, the number of tachyzoites per 100 macrophages at 20 h postinfection was significantly lower in CD154-treated macrophages than in control macrophages. The number of parasites per 100 macrophages was, on the average, 58.7 ± 4.4% lower in CD154-treated monolayers than in controls (p < 0.001; n = 9). This was accompanied by a 64.0 ± 8.7% decrease in the infection rate at 20 h compared with 1 h postinfection in CD154-treated monolayers (p < 0.00l; n = 4). The lower percentage of infected cells in the CD154-treated monolayers was not due to a preferential cell loss from these monolayers during washing steps before staining. Supernatants collected from CD154-treated and control monolayers revealed that the percentage of cells that detached was <1.5 and <3% for CD154-treated and control monolayers, respectively (data not shown). Studies of kinetics of macrophage activation revealed that incubation of macrophages with CD154 for 1 d enhanced anti-T. gondii activity, and this effect was maximal after 2 d (Fig. 1).


View this table:
[in this window]
[in a new window]
  		Table I. CD154 enhances anti-T. gondii activity of human macrophagesa

 


View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 1. Kinetics of CD154-mediated stimulation of macrophage anti-T. gondii activity. Monocyte-derived macrophages were cultured for 5 d in either CM alone or CM to which CD154 (3 µg/ml) was added 1–5 d before T. gondii infection. Macrophages were washed and challenged with T. gondii for 1 h. Monolayers were examined by light microscopy 20 h after addition of T. gondii. The results shown are the mean ± SEM of duplicate monolayers from a representative experiment of three performed using macrophages from different donors.

 
Assessment of T. gondii growth by [3H]uracil uptake showed similar results and revealed that CD154 inhibited [3H]uracil incorporation in a dose-dependent manner (Fig. 2, A and B; p < 0.001). On the average, addition of CD154 (3 µg/ml) caused a 55.3 ± 2.2% reduction in [3H]uracil uptake compared with that in control macrophages (p < 0.0001; n = 10). The effect of CD154 was specific, as addition of a neutralizing anti-CD154 mAb resulted in 88.7 ± 5.0% inhibition of the effect of CD154 (p < 0.02; n = 2), whereas an isotype control mAb failed to alter the effect of CD154 (Fig. 2C). CD154 did not exhibit direct toxic activity against T. gondii, as incubation of tachyzoites with CD154 for 1 h at 37°C before addition to macrophages did not affect the parasite load in unstimulated macrophages at 20 h postinfection (data not shown). Taken together, CD154 stimulates anti-T. gondii activity in human macrophages.



View larger version (9K):
[in this window]
[in a new window]
  		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 µg/ml). Macrophages were washed and challenged with T. gondii. Monolayers were pulsed with 1 µCi of [3H]uracil for 8 h. Data are expressed as the mean counts per minute of [3H]uracil incorporation of triplicate wells ± 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 ± 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 µg/ml). The results of one representative experiment of two are shown.

 
While CD154 decreased the number of tachyzoites per 100 macrophages and the percentage of infected macrophages, the number of tachyzoites per infected macrophage was not affected (Table I). These results raised the possibility that only a subpopulation of macrophages responded to CD154. Thus, we examined the expression of CD40 on monocytes and monocyte-derived macrophages. Flow cytometric analysis revealed that 6.6 ± 0.5% of freshly isolated monocytes were CD40+ (n = 4). CD40 expression increased after 5 days of in vitro culture, resulting in percentages of CD40+ cells of 50.0 ± 8.0% in untreated macrophages and 47.9 ± 9.3% in CD154-treated macrophages. We examined the response of CD40+ and CD40- macrophages to CD154 to confirm that differential expression of CD40 was the explanation for the lack of response to CD154 by a subpopulation of macrophages. Fig. 3 shows that CD154 caused a profound decrease (79.0 ± 3.8% inhibition; p < 0.001; n = 3) in the parasite load in CD40+, but not in CD40-, macrophages. Thus, only CD40+ macrophages respond to CD154.



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 3. CD154 stimulates macrophage antimicrobial activity only in CD40+ macrophages. CD40+ and CD40- were isolated using FACSVantage SE and were cultured for 2 days in CM with or without CD154. Monolayers were examined by light microscopy 20 h after T. gondii challenge. Results are representative of one of three independent experiments.

 
CD154 is considered to exist primarily as a membrane molecule on activated CD4+ T cells. Thus, we determined whether CD154-bearing cells also stimulate antimicrobial activity of macrophages, and whether this effect could take place when CD154 interacts with macrophages already infected with an intracellular pathogen. Macrophages previously infected with T. gondii were exposed for 36 h to either L cells transfected with human CD154 or CD32 as a control. As shown in Fig. 4, addition of L-CD154 resulted in a significant decrease in [3H]uracil incorporation compared with either infected macrophages alone or infected macrophages incubated with L-CD32 (61.6 ± 4.0% inhibition; p < 0.003; n = 3). Moreover, a neutralizing anti-CD154 mAb caused significant inhibition of the effect of L-CD154 on T. gondii growth (68.7 ± 5.1% inhibition; p < 0.004). It is unlikely that the decrease in uracil incorporation was caused by preactivation of uninfected macrophages that subsequently became infected with T. gondii. Microscopic examination of culture slides set up in parallel revealed that there was no increase in the rate of infection and no evidence of destruction of macrophages during the 36 h of culture (data not shown). Therefore, cells that express CD154 on their membranes activate anti-T. gondii activity of human macrophages.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 4. CD154-bearing cells stimulate macrophage anti-T. gondii activity. Macrophages were infected with T. gondii for 1 h, and extracellular tachyzoites were removed by washing. Thereafter, gamma-irradiated L-CD32 or L-CD154 cells were added to infected macrophages, and [3H]uracil incorporation was assessed as described. Results are representative of one of three independent experiments.

 
CD154 acts independently of IFN-{gamma} and further stimulates macrophage anti-T. gondii activity when IFN-{gamma} signaling is suboptimal

In the next series of experiments we explored the mechanism of action of CD154. These experiments were conducted using recombinant CD154, as our goal was to identify the mechanism(s) by which CD154 alone stimulates macrophage antimicrobial activity, and CD154-bearing cells may express other factors that modulate macrophage function. We determined whether the effect of CD154 on macrophage anti-T. gondii activity is mediated by IFN-{gamma}. Incubation of macrophages with CD154 plus a neutralizing anti-IFN-{gamma} mAb resulted in similar inhibition of [3H]uracil uptake as in macrophages treated with CD154 alone or CD154 plus an isotype control mAb (Fig. 5A; p > 0.5; n = 3). At the concentration used, the anti-IFN-{gamma} mAb caused a 94.2 ± 10.9% inhibition of the anti-T. gondii activity produced by 5 ng/ml of IFN-{gamma} (p < 0.001; n = 3; Fig. 5A). Moreover, IFN-{gamma} was not detected (<39 pg/ml) in supernatants collected from CD154-treated macrophages. Together, CD154 stimulates macrophage anti-T. gondii activity independently of IFN-{gamma}.



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 5. CD154 acts independently of IFN-{gamma}, but cooperates with IFN-{gamma} in enhancing macrophage anti-T. gondii activity. A, Monocyte-derived macrophages were cultured with either CD154 or IFN-{gamma} in the presence of a neutralizing anti-IFN-{gamma} 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-{gamma} (500 pg/ml). The results of one representative experiments of three are shown.

 
Patients with partial IFN-{gamma}R1 deficiency control T. gondii infection (7). Thus, we studied the effect of CD154 on macrophage anti-T. gondii activity when IFN-{gamma} signaling was present, but deficient. Macrophages were incubated with suboptimal concentrations of CD154 (300 pg/ml) and IFN-{gamma} (500 pg/ml), either alone or in combination, before infection with T. gondii. Fig. 5B shows that whereas the growth of T. gondii was minimally affected by either CD154 or IFN-{gamma}, the combination of these two factors resulted in a significant inhibition in parasite growth (p < 0.01; n = 3). Thus, CD154 cooperates with IFN-{gamma} in stimulating the antimicrobial activity of human macrophages.

CD154 enhances human macrophage anti-T. gondiiactivity through a TNF-{alpha}-dependent mechanism

CD154 stimulates human monocytes to secrete monokines, including TNF-{alpha} (28, 29, 30), a cytokine that plays an important role in control of intracellular pathogens such as T. gondii (31, 32, 33). Thus, we ascertained whether TNF-{alpha} signaling mediates stimulation of macrophage anti-T. gondii activity caused by CD154. We began by studying the effect of CD154 on TNF-{alpha} production by T. gondii-infected macrophages. While uninfected, untreated macrophages did not secrete detectable amounts of TNF-{alpha}, incubation with T. gondii resulted in the production of low levels of this cytokine (Fig. 6). Incubation of uninfected macrophages with CD154 resulted in TNF-{alpha} secretion, whereas T. gondii infection of CD154-treated macrophages caused a significant further increase in TNF-{alpha} production (p < 0.03; n = 3). Secretion of TNF-{alpha} triggered by CD154 was not affect by a neutralizing mAb against IFN-{gamma} (data not shown).



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 6. CD154 stimulates TNF-{alpha} production by T. gondii-infected macrophages. Monocyte-derived macrophages (2.5 x 106/ml) were cultured in CM with or without CD154. Supernatants were collected 4 h after challenge with T. gondii and used to measure TNF-{alpha} concentrations by ELISA. Data are expressed as the mean ± SEM of three independent experiments.

 
To further determine whether TNF-{alpha} mediates the effect of CD154 on macrophage anti-T. gondii activity, control and CD154-treated macrophages were incubated with a neutralizing anti-TNF-{alpha} mAb. The anti-TNF-{alpha} mAb used does not cross-react with CD154 (34). Table II shows that whereas addition of an anti-TNF-{alpha} mAb to control macrophages did not affect T. gondii growth, this mAb increased the number of tachyzoites per 100 CD154-treated macrophages. Anti-TNF-{alpha} caused an 85.7 ± 4.7% inhibition of the effect of CD154 on T. gondii growth (n = 3; p < 0.05). Thus, CD154 stimulates anti-T. gondii activity in human macrophages at least in part through TNF-{alpha} signaling.


View this table:
[in this window]
[in a new window]
  		Table II. TNF-{alpha} mediates the effect of CD154 on anti-T. gondii activity of human macrophagesa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Macrophages are major effectors of control of intracellular pathogens. The induction of macrophage antimicrobial activity by IFN-{gamma} is considered crucial for protection against these organisms (22, 23). However, patients with defects in IFN-{gamma} signaling do not appear to be susceptible to infections other than atypical Mycobacteria and Salmonella (6). In addition, mice with defects in IFN-{gamma} signaling develop protective mechanisms against L. monocytogenes, L. donovani, H. capsulatum, and T. gondii (1, 2, 3, 4, 5). The aim of the present work was to identify IFN-{gamma}-independent mechanisms of induction of macrophage antimicrobial activity. We report that isolated CD154 signaling triggered anti-T. gondii activity in human macrophages without requiring the presence of IFN-{gamma}. Although CD154 induced macrophage anti-microbial activity in the absence of IFN-{gamma}, CD154 also stimulated this activity when IFN-{gamma} was present at concentrations inadequate to induce full macrophage activation. The latter observation suggests that CD154 may be an important mediator of protection against intracellular pathogens such as T. gondii in hosts with insufficient IFN-{gamma}-dependent signals, such as patients with partial IFN-{gamma}R1 deficiency who are not susceptible to toxoplasmosis (7). In addition, the evidence of cooperation between CD154 and IFN-{gamma} for activation of macrophage anti-T. gondii activity supports the idea that both molecules act in concert to control intracellular pathogens during the immune response in immunocompetent hosts (14, 16). TNF-{alpha} was a central mediator of the anti-T. gondii activity induced by CD154. CD154 stimulated TNF-{alpha} production by T. gondii-infected macrophages. In turn, TNF-{alpha} was pivotal for the expression of the enhanced antimicrobial state. These results demonstrate that CD154 is an important regulator of the effects of TNF-{alpha} on macrophage antimicrobial activity.

Defective CD154 signaling in humans and mice results in increased susceptibility to infections with intracellular pathogens, including T. gondii. We previously reported that patients with a congenital lack of functional CD154 (X-linked hyper-IgM syndrome (X-HIM)) have impaired in vitro production of IL-12 and IFN-{gamma} in response to T. gondii (35). We proposed this mechanism to explain the increased incidence of opportunistic infections in patients with this immunodeficiency (35). However, the fact that individuals with partial IFN-{gamma}R1 deficiency are not susceptible to toxoplasmosis suggests that there are additional mechanisms that account for the increased incidence of opportunistic infections in patients with X-HIM. Our previous work and this report indicate that impaired T cell priming (35) and defective macrophage activation are likely to contribute to the predisposition of patients with X-HIM to opportunistic infections.

CD154 is expressed as a membrane glycoprotein on activated CD4+ T cells. However, cleavage of membrane CD154 results in the release of a soluble form of the protein (8, 9). We report that soluble trimeric recombinant CD154 and CD154-bearing cells stimulated anti-T. gondii activity of macrophages. Although both forms of CD154 have been reported to be biologically active (9, 36), membrane CD154 is considered to be more effective at triggering CD40 signal transduction (37). Thus, segregation of receptor and counter-receptors into supramolecular clusters (38) in the immunological synapse may promote optimal signaling by causing CD40 clustering (39). The superior capacity of membrane CD154 to induce CD40 signaling may explain why, in contrast to our studies using CD154-bearing cells, the addition of recombinant CD154 to macrophages already infected with T. gondii did not reproducibly induce anti-T. gondii activity (R. M. Andrade and C. S. Subauste, unpublished observations). The lack of induction of macrophage antimicrobial activity after isolated CD40 signaling reported in other studies may be explained by the use of suboptimal CD40 signaling and/or by differences in the requirements for generation of macrophage antimicrobial activity depending on the pathogen used to infect macrophages. In support of the latter possibility is the fact that patients with defective IFN-{gamma}/IL-12 signaling are susceptible to Mycobacteria and Salmonella, whereas disease caused by other intracellular pathogens, such as T. gondii, L. monocytogenes, and Legionella, is uncommon (6, 7).

The present study revealed that endogenous TNF-{alpha} is crucial for the induction of anti-T. gondii activity in human macrophages stimulated with CD154. Consistent with the role of CD154 as a stimulator of monokine secretion (28, 29, 30) is our demonstration that CD154 enhanced TNF-{alpha} production by T. gondii-infected human macrophages. Noteworthy are reports of the poor capacity of T. gondii to induce TNF-{alpha} production by macrophages (40, 41). Thus, CD154 may play an important role in enhancing the secretion of this cytokine in response to T. gondii. There is precedent for the role of TNF-{alpha} as an autocrine regulator of macrophage antimicrobial activity. Endogenous TNF-{alpha} mediates the effects of IFN-{gamma} on macrophage microbiostatic activity (41). Importantly, we now show that CD154 provides an alternative mechanism for activation of the TNF-{alpha} signaling and macrophage antimicrobial activity that take place in the absence of IFN-{gamma}.

TNF-{alpha} is pivotal for control of intracellular pathogens such as T. gondii (31, 32, 33, 42), Mycobacteria (43), Leishmania (3, 44), L. monocytogenes (2, 45, 46), and H. capsulatum (5, 47). The fact that CD154 activated TNF-{alpha}-dependent antimicrobial activity of macrophages suggests that CD154 is an important regulator of TNF-{alpha}-mediated mechanisms of host protection. However, TNF-{alpha} alone is unable to activate anti-T. gondii activity in macrophages (40, 41, 48). Our results raise the possibility that the CD40-CD154 interaction may act not only by stimulating TNF-{alpha} production by infected macrophages, but also by making these cells responsive to TNF-{alpha}. It is interesting to note that signal transduction triggered by the different members of the TNF receptor (TNFR) family (including TNFRI, TNFRII, and CD40) is similar, but not identical (49). Indeed, these differences are likely to explain the intracellular convergence of independent signaling triggered by members of this family of receptors (50). Thus, signals through TNFR and CD40 may act in concert to induce a state of increased macrophage antimicrobial activity. The fact that TNF-{alpha} mediates the enhanced anti-T. gondii activity in macrophages stimulated with CD154 may explain why both CD154-/- and TNFR-/- mice show similar susceptibility to T. gondii and develop toxoplasmic encephalitis (16, 31, 33).

Activated macrophages use a variety of effector mechanisms to impair the growth or kill intracellular pathogens. These mechanisms include the production of reactive oxygen intermediates and reactive nitrogen intermediates, tryptophan degradation, iron sequestration, and delivery of antimicrobial polypeptides into phagosomes (51). The fact that treatment with CD154 decreases the percentage of infected macrophages suggests that this molecule induces toxoplasmacidal activity in human macrophages. Studies to identify the effector mechanism(s) activated by CD154 are currently underway in our laboratory.

Many intracellular pathogens, including T. gondii, infect not only macrophages, but also cells of nonhemopoietic origin. Thus, effector mechanisms for control of these pathogens must be operative at both levels. Indeed, a study using bone marrow chimeras revealed that the lack of TNFR in either hemopoietic or nonhemopoietic compartments resulted in the inability to control chronic T. gondii infection and the development of toxoplasmic encephalitis (52). These results suggest that the effect of TNF-{alpha} on nonhemopoietic cells in the brain is particularly important for protection against an intracellular pathogen. Of relevance to these results is the demonstration that CD40 is expressed not only on professional APCs, but also on nonhemopoietic cells, including endothelial cells, neurons, fibroblasts, and epithelial and muscle cells (53, 54, 55, 56, 57). Thus, it becomes important to determine whether CD40 signaling also impairs the growth of intracellular pathogens within these cells and if such activity is dependent on TNF-{alpha}.

Endogenous TNF-{alpha} as an important mediator of host protection in animals with impaired IFN-{gamma} signaling (2, 3, 5). Work in humans also supports the role of TNF-{alpha} in control of certain intracellular pathogens in individuals with inadequate IFN-{gamma} signals. Although IFN-{gamma} is unable to activate anti-T. gondii activity of macrophages from patients with partial IFN-{gamma}R1 defect, addition of exogenous TNF-{alpha} results in control of T. gondii growth (7). Our results raise the possibility that CD154 may be one of the factors that activates TNF-{alpha}-mediated mechanisms of host protection that become especially important in conditions associated with impaired IFN-{gamma} response. Further studies of the regulation of macrophage activation by CD154 and TNF-{alpha} are likely to improve our understanding of the gamut of mechanisms of control of intracellular pathogens and help explain the contrasts in the spectrum of opportunistic infections associated with various immunodeficiencies in humans.


   Acknowledgments
 
We thank William Fanslow and Elaine Thomas for making recombinant CD154 and anti-CD154 mAb available for our studies, and Rene de Waal Malefyt for providing us with L cells transfected with CD32 and CD154.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI48406 (to C.S.S.). Back

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, Cincinnati, OH 45267-0560. E-mail address: carlos.subauste{at}uc.edu Back

3 Abbreviations used in this paper: CM, complete medium; TNFR, TNF receptor; X-HIM, X-linked hyper-IgM syndrome. Back

Received for publication May 16, 2003. Accepted for publication October 2, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Harty, J. T., M. J. Bevan. 1995. Specific immunity to Listeria monocytogenes in the absence of IFN-{gamma}. Immunity 3:109.[Medline]
  2. Szalay, G., C. H. Label, C. Blum, S. H. E. Kaufmann. 1996. IL-4 neutralization or TNF-{alpha} treatment ameliorate disease by an intracellular pathogen in IFN-{gamma} receptor deficient mice. J. Immunol. 157:4746.[Abstract]
  3. Taylor, A. P., H. W. Murray. 1997. Intracellular antimicrobial activity in the absence of interferon-{gamma}: effect of interleukin-12 in experimental visceral leishmaniasis in interferon-{gamma} gene-disrupted mice. J. Exp. Med. 185:1231.[Abstract/Free Full Text]
  4. Khan, I. A., T. Matsuura, S. Fonseka, L. H. Kasper. 1996. Production of nitric oxide (NO) is not essential for protection against Toxoplasma gondii infection in IRF-1-/- mice. J. Immunol. 156:636.[Abstract]
  5. Zhou, P., G. Miller, R. A. Seder. 1998. Factors involved in regulating primary and secondary immunity to infection with Histoplasma capsulatum: TNF-{alpha} plays a critical role in maintaining secondary immunity in the absence of IFN-{gamma}. J. Immunol. 160:1359.[Abstract/Free Full Text]
  6. Ottenhoff, T. H. M., D. Kumararatne, J. L. Casanova. 1998. Novel human immunodeficiencies reveal the essential role of type-1 cytokines in immunity to intracellular bacteria. Immunol. Today 19:491.[Medline]
  7. Janssen, R., A. van Wengen, E. Verhard, T. de Boer, T. Zomerdijk, T. H. M. Ottenhoff, J. T. van Dissel. 2002. Divergent role for TNF-{alpha} in IFN-{gamma}-induced killing of Toxoplasma gondii and Salmonella typhimurium contributes to selective susceptibility of patients with partial IFN-{gamma} receptor 1 deficiency. J. Immunol. 169:3900.[Abstract/Free Full Text]
  8. Graf, D., S. Muller, U. Korthauer, C. van Kooten, C. Weise, R. A. Kroczek. 1995. A soluble form of TRAP (CD40 ligand) is rapidly released after T cell activation. Eur. J. Immunol. 25:1749.[Medline]
  9. Pietravalle, F., S. Lecoanet-Henchoz, H. Blasey, J. P. Aubry, G. Elson, M. D. Edgerton, J. Y. Bonnefoy, J. F. Gauchat. 1996. Human native soluble CD40L is a biologically active trimer, processed inside microsomes. J. Biol. Chem. 271:5965.[Abstract/Free Full Text]
  10. Durie, F. H., T. M. Foy, S. R. Masters, J. D. Laman, R. J. Noelle. 1994. The role of CD40 in the regulation of humoral and cell-mediated immunity. Immunol. Today 15:406.[Medline]
  11. Grewal, I. S., R. A. Flavell. 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16:111.[Medline]
  12. van Kooten, C., J. Banchereau. 2000. CD40-CD40 ligand. J. Leukocyte Biol. 67:2.[Abstract]
  13. Campbell, K. A., P. J. Ovendale, M. K. Kennedy, W. C. Fanslow, S. G. Reed, C. R. Maliszewski. 1996. CD40 ligand is required for protective cell-mediated immunity to Leishmania major. Immunity 4:283.[Medline]
  14. Kamanaka, M., P. Yu, T. Yasui, K. Yoshida, T. Kawabe, T. Horii, T. Kishimoto, H. Kikutani. 1996. Protective role of CD40 in Leishmania major infection at two distinct phases of cell-mediated immunity. Immunity 4:275.[Medline]
  15. Wiley, J. A., A. G. Harmsen. 1995. CD40 ligand is required for resolution of Pneumocystis carinii pneumonia in mice. J. Immunol. 155:3525.[Abstract]
  16. Reichmann, G., W. Walker, E. N. Villegas, L. Craig, G. Cai, J. Alexander, C. A. Hunter. 2000. The CD40/CD40 ligand interaction is required for resistance to toxoplasmic encephalitis. Infect. Immun. 68:1312.[Abstract/Free Full Text]
  17. Cosyns, M., S. Tsirkin, M. Jones, R. Flavell, H. Kikutani, A. R. Hayward. 1998. Requirement for CD40-CD40 ligand interaction for elimination of Cryptosporidium parvum from mice. Infect. Immun. 66:603.[Abstract/Free Full Text]
  18. Hayashi, T., S. P. Rao, P. R. Meylan, R. S. Kornbluth, A. Catanzaro. 1999. Role of CD40 ligand in Mycobacterium avium infection. Infect. Immun. 67:3558.[Abstract/Free Full Text]
  19. Marriott, I., E. K. Thomas, K. L. Bost. 1999. CD40-CD40 ligand interactions augment survival of normal mice, but not CD40 ligand knockout mice, challenged orally with Salmonella dublin. Infect. Immun. 67:5253.[Abstract/Free Full Text]
  20. Netea, M. G., J. W. M. van der Meer, I. Verschueren, B. J. Kullberg. 2002. CD40/CD40 ligand interactions in the host defense against disseminated Candida albicans infection: the role of macrophage-derived nitric oxide. Eur. J. Immunol. 32:1455.[Medline]
  21. Larkin, R., C. D. Benjamin, Y. M. Hsu, Q. Li, L. Zukowski, R. F. Silver. 2002. CD40 ligand (CD154) does not contribute to lymphocyte-mediated inhibition of virulent Mycobacterium tuberculosis within human monocytes. Infect. Immun. 70:4716.[Abstract/Free Full Text]
  22. Deckert-Schluter, M., A. Rang, D. Weiner, S. Huang, O. D. Wiestler, H. Hof, D. Schluter. 1996. Interferon-{gamma} receptor-deficiency renders mice highly susceptible to toxoplasmosis by decreased macrophage activation. Lab. Invest. 75:827.[Medline]
  23. Dighe, A. S., D. Campbell, C. S. Hsieh, S. Clarke, D. R. Greaves, S. Gordon, K. M. Murphy, R. D. Schreiber. 1995. Tissue-specific targeting of cytokine unresponsiveness in transgenic mice. Immunity 3:657.[Medline]
  24. Subauste, C. S., R. de Waal Malefyt, F. Fuh. 1998. Role of CD80 (B7.1) and CD86 (B7.2) in the immune response to an intracellular pathogen. J. Immunol. 160:1831.[Abstract/Free Full Text]
  25. Subauste, C. S., J. Y. Chung, D. Do, A. H. Koniaris, C. A. Hunter, J. G. Montoya, S. Porcelli, J. S. Remington. 1995. Preferential activation and expansion of human peripheral blood {gamma}{delta} T cells in response to Toxoplasma gondii in vitro and their cytokine production and cytotoxic activity against T. gondii-infected cells. J. Clin. Invest. 96:610.
  26. McLeod, R., J. S. Remington. 1979. A method to evaluate the capacity of monocytes and macrophages to inhibit multiplication of an intracellular pathogen. J. Immunol. Methods 27:19.[Medline]
  27. Murray, H. W., B. Y. Rubin, S. M. Carriero, A. M. Harris, E. A. Jaffee. 1985. Human mononuclear phagocyte anti-protozoal mechanisms: oxygen-dependent vs oxygen-independent activity against intracellular Toxoplasma gondii. J. Immunol. 134:1982.[Abstract]
  28. Alderson, M. R., R. J. Armitage, T. W. Tough, L. Strockbine, W. C. Fanslow, M. K. Spriggs. 1993. CD40 expression by human monocytes: regulation by cytokines and activation of monocytes by the ligand for CD40. J. Exp. Med. 178:669.[Abstract/Free Full Text]
  29. Caux, C., C. Massacrier, B. Banbervliet, B. Dubois, C. van Kooten, I. Durand, J. Banchereau. 1994. Activation of human dendritic cells through CD40 cross-linking. J. Exp. Med. 180:1263.[Abstract/Free Full Text]
  30. Kiener, P. A., P. Moran-Davis, B. M. Rankin, A. F. Wahl, A. Aruffo, D. Hollenbaugh. 1995. Stimulation of CD40 with purified soluble gp39 induces proinflammatory responses in human monocytes. J. Immunol. 155:4917.[Abstract]
  31. Deckert-Schluter, M., H. Bluethmann, A. Rang, H. Hof, D. Schluter. 1998. Crucial role of TNF receptor type 1 (p55), but not of TNF receptor type 2 (p75), in murine toxoplasmosis. J. Immunol. 160:3427.[Abstract/Free Full Text]
  32. Johnson, L. L.. 1992. A protective role for endogenous tumor necrosis factor in Toxoplasma gondii infection. Infect. Immun. 60:1979.[Abstract/Free Full Text]
  33. Yap, G. S., T. Scharton-Kersten, H. Charest, A. Sher. 1998. Decreased resistance of TNF receptor p55- and p75-deficient mice to chronic toxoplasmosis despite normal activation of inducible nitric oxide synthase in vivo. J. Immunol. 160:1340.[Abstract/Free Full Text]
  34. Cho, C. S., M. L. Cho, S. Y. Min, W. U. Kim, J. M. D. , S. S. Lee, S. H. Park, J. Choe, H. Y. Kim. 2000. CD40 engagement on synovial fibroblast up-regulates production of vascular endothelial growth factor. J. Immunol. 164:5055.[Abstract/Free Full Text]
  35. Subauste, C. S., M. Wessendarp, R. U. Sorensen, L. Leiva. 1999. CD40-CD40 ligand interaction is central to cell-mediated immunity against Toxoplasma gondii: patients with hyper IgM syndrome have a defective type-1 immune response which can be restored by soluble CD40L trimer. J. Immunol. 162:6690.[Abstract/Free Full Text]
  36. Ludewig, B., V. Henn, J. M. Schroeder, D. Graf, R. A. Kroczek. 1996. Induction, regulation, and function of soluble TRAP (CD40 ligand) during interaction of primary CD4+ CD45RA+ T cells with dendritic cells. Eur. J. Immunol. 26:3137.[Medline]
  37. Fanslow, W. C., S. Srinivasan, R. Paxton, M. G. Gibson, M. K. Spriggs, R. J. Armitage. 1994. Structural characteristics of CD40 ligand that determine biological functions. Semin. Immunol. 6:267.[Medline]
  38. van der Merwe, P. A., S. J. Davis, A. S. Shaw, M. Dustin. 2000. Cytoskeletal polarization and redistribution of cell-surface molecules during T cell antigen recognition. Semin. Immunol. 12:5.[Medline]
  39. Pullen, S. S., M. E. Labadia, R. H. Ingraham, S. M. McWhirter, D. S. Everdeen, T. Alber, J. J. Crute, M. R. Kehry. 1999. High-affinity interactions of tumor necrosis factor receptor-associated factors (TRAFs) and CD40 require TRAF trimerization and CD40 multimerization. Biochemistry. 38:10168.[Medline]
  40. Sibley, L. D., L. B. Adams, Y. Fukutomi, J. L. Krahenbuhl. 1991. Tumor necrosis factor-{alpha} triggers antitoxoplasmal activity of IFN-{gamma} primed macrophages. J. Immunol. 147:2340.[Abstract]
  41. Langermans, J. A. M., M. E. B. van der Hulst, P. H. Nibbering, P. S. Hiemstra, L. Fransen, R. van Furth. 1992. IFN-{gamma}-induced L-arginine-dependent toxoplasmastatic activity in murine peritoneal macrophages is mediated through endogenous tumor necrosis factor-{alpha}. J. Immunol. 148:568.[Abstract]
  42. Chang, H. R., G. E. Grau, J. C. Pechere. 1990. Role of TNF and IL-1 in infection with Toxoplasma gondii. Immunology 69:33.[Medline]
  43. Kindler, V., A. P. Sappiano, G. E. Grau, P. F. Piguet, P. Vassali. 1989. The inducing role of tumor necrosis factor in the development of bacterial granulomas during BCG infection. Cell 56:731.[Medline]
  44. Liew, F. Y., C. Parkinson, S. Millot, A. Severn, M. Carrier. 1990. Tumour necrosis factor (TNF-{alpha}) in leishmaniasis. I. TNF-{alpha} mediates host protection against cutaneous leishmaniasis. Immunology 69:570.[Medline]
  45. Nakane, A., T. Minagawa, K. Kato. 1988. Endogenous tumor necrosis factor (cachectin) is essential to host resistance against Listeria monocytogenes infection. Infect. Immun. 56:2563.[Abstract/Free Full Text]
  46. Havell, E. A.. 1989. Evidence that tumor necrosis factor has an important role in antibacterial resistance. J. Immunol. 143:2894.[Abstract]
  47. Allendoerfer, R., G. S. Deepe, Jr. 1998. Blockade of endogenous TNF-{alpha} exacerbates primary and secondary pulmonary histoplasmosis by differential mechanisms. J. Immunol. 160:6072.[Abstract/Free Full Text]
  48. de Tito, E. H., J. R. Catterall, J. S. Remington. 1986. Activity of recombinant tumor necrosis factor on Toxoplasma gondii and Trypanosoma cruzi. J. Immunol. 137:1342.[Abstract]
  49. Locksley, R. M., N. Killeen, M. J. Lenardo. 2001. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104:487.[Medline]
  50. Lazdins, J., M. Grell, M. R. Walker, K. Woods-Cook, P. Scheurich, K. Pfizenmaier. 1997. Membrane tumor necrosis factor (TNF) induced cooperative signaling of TNFR60 and TNFR80 favors induction of cell death rather than virus production in HIV-infected T cells. J. Exp. Med. 185:81.[Abstract/Free Full Text]
  51. Reiner, N. E.. 1994. Altered cell signaling and mononuclear phagocyte deactivation during intracellular infection. Immunol. Today 15:374.[Medline]
  52. Yap, G., A. Sher. 1999. Effector cells of both nonhematopoietic and hematopoietic origin are required for interferon (IFN)-{gamma}- and tumor necrosis factor (TNF)-{alpha}-dependent host resistance to the intracellular pathogen, Toxoplasma gondii. J. Exp. Med. 189:1083.[Abstract/Free Full Text]
  53. Karmann, K., C. C. W. Hughes, J. Schechner, W. C. Fanslow, J. S. Pober. 1995. CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression. Proc. Natl. Acad. Sci. USA 92:4342.[Abstract/Free Full Text]
  54. Tan, J., T. Town, T. Mori, D. Obregon, Y. Wu, A. DelleDonne, A. Rojani, F. Crawford, R. A. Flavell, M. Mullan. 2002. CD40 is expressed and functional on neuronal cells. EMBO J. 21:643.[Medline]
  55. Yellin, M. J., S. Winikoff, S. M. Fortune, D. Baum, M. K. Crow, S. Lederman, L. Chess. 1995. Ligation of CD40 on fibroblasts induces CD54 (ICAM-1) and CD106 (VCAM-1) upregulation, IL-6 production and proliferation. J. Leukocyte Biol. 58:209.[Abstract]
  56. Galy, A. H. M., H. Spits. 1992. CD40 is functionally expressed on human thymic epithelial cells. J. Immunol. 149:775.[Abstract]
  57. Mach, F., U. Schonbeck, G. K. Sukhova, T. Bourcier, J. Y. Bonnefoy, J. S. Pober, P. Libby. 1997. Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells and macrophages: implications for CD40-CD40 ligand signaling in atherosclerosis. Proc. Natl. Acad. Sci. USA 94:1931.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
A. Dupoux, J. Cartier, S. Cathelin, R. Filomenko, E. Solary, and L. Dubrez-Daloz
cIAP1-dependent TRAF2 degradation regulates the differentiation of monocytes into macrophages and their response to CD40 ligand
Blood, January 1, 2009; 113(1): 175 - 185.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
C. S. Subauste and M. Wessendarp
CD40 Restrains In Vivo Growth of Toxoplasma gondii Independently of Gamma Interferon
Infect. Immun., March 1, 2006; 74(3): 1573 - 1579.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Danese, M. Sans, F. Scaldaferri, A. Sgambato, S. Rutella, A. Cittadini, J. M. Pique, J. Panes, J. A. Katz, A. Gasbarrini, et al.
TNF-{alpha} Blockade Down-Regulates the CD40/CD40L Pathway in the Mucosal Microcirculation: A Novel Anti-Inflammatory Mechanism of Infliximab in Crohn's Disease
J. Immunol., February 15, 2006; 176(4): 2617 - 2624.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. M. Andrade, M. Wessendarp, J.-A. C. Portillo, J.-Q. Yang, F. J. Gomez, J. E. Durbin, G. A. Bishop, and C. S. Subauste
TNF Receptor-Associated Factor 6-Dependent CD40 Signaling Primes Macrophages to Acquire Antimicrobial Activity in Response to TNF-{alpha}
J. Immunol., November 1, 2005; 175(9): 6014 - 6021.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
R. M. Andrade, J.-A. C. Portillo, M. Wessendarp, and C. S. Subauste
CD40 Signaling in Macrophages Induces Activity against an Intracellular Pathogen Independently of Gamma Interferon and Reactive Nitrogen Intermediates
Infect. Immun., May 1, 2005; 73(5): 3115 - 3123.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Andrade, R. M.
Right arrow Articles by Subauste, C. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Andrade, R. M.
Right arrow Articles by Subauste, C. S.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
*Genetics Home Reference

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS