CD40 signaling induces reciprocal outcomes in Leishmania-infected macrophages; roles of host genotype and cytokine milieu

We investigated the influence of CD40-CD40 ligand-mediated signaling on induction of microbicidal activity against Leishmania major in macrophages from resistant (B6) and susceptible (BALB) mouse strains. CD40 engagement induced leishmanicidal activity in resistant macrophages, but increased parasite replication in susceptible macrophages. CD40 engagement induced comparable TNF-alpha production in macrophages from both strains. However, increased IL-10 production was restricted to susceptible macrophages. Increased parasite replication in susceptible macrophages was prevented by a neutralizing anti-IL-10 antibody. In the presence of IFN-gamma, CD40 engagement induced Leishmania killing by macrophages from both strains. Therefore, the outcome of CD40 signaling on effector responses against L. major depends on host genotype and the cytokine milieu, and a source of IFN-gamma is required for a protective response.     

CD154 activates macrophage antimicrobial activity in the absence of IFN-gamma through a TNF-alpha-dependent mechanism

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.     

Suppressor of cytokine signaling 1 inhibits cytokine induction of CD40 expression in macrophages

CD40 is a type I membrane-bound molecule belonging to the TNFR superfamily that is expressed on various immune cells including macrophages and microglia. The aberrant expression of CD40 is involved in the initiation and maintenance of various human diseases including multiple sclerosis, arthritis, atherosclerosis, and Alzheimer's disease. Inhibition of CD40 signaling has been shown to provide a significant beneficial effect in a number of animal models of human diseases including the aforementioned examples. We have previously shown that IFN-gamma induces CD40 expression in macrophages and microglia. IFN-gamma leads to STAT-1alpha activation directly and up-regulation of NF-kappaB activity due to the secretion and subsequent autocrine signaling of TNF-alpha. However, TNF-alpha alone is not capable of inducing CD40 expression in these cells. Suppressor of cytokine signaling 1 protein (SOCS-1) is a cytokine-inducible Src homology 2-containing protein that regulates cytokine receptor signaling by inhibiting STAT-1alpha activation via a specific interaction with activated Janus kinase 2. Given the important role of CD40 in inflammatory events in the CNS as well as other organ systems, it is imperative to understand the molecular mechanisms contributing to both CD40 induction and repression. We show that ectopic expression of SOCS-1 abrogates IFN-gamma-induced CD40 protein expression, mRNA levels, and promoter activity. Additionally, IFN-gamma-induced TNF-alpha secretion, as well as STAT-1alpha and NF-kappaB activation, are inhibited in the presence of SOCS-1. We conclude that SOCS-1 inhibits cytokine-induced CD40 expression by blocking IFN-gamma-mediated STAT-1alpha activation, which also then results in suppression of IFN-gamma-induced TNF-alpha secretion and subsequent NF-kappaB activation.     

OX40 complexes with phosphoinositide 3-kinase and protein kinase B (PKB) to augment TCR-dependent PKB signaling

T lymphocyte activation requires signal 1 from the TCR and signal 2 from costimulatory receptors. For long-lasting immunity, growth and survival signals imparted through the Akt/protein kinase B (PKB) pathway in activated or effector T cells are important, and these can be strongly influenced by signaling from OX40 (CD134), a member of the TNFR superfamily. In the absence of OX40, T cells do not expand efficiently to Ag, and memory formation is impaired. How most costimulatory receptors integrate their signals with those from Ag through the TCR is not clear, including whether OX40 directly recruits PKB or molecules that regulate PKB. We show that OX40 after ligation by OX40L assembled a signaling complex that contained the adapter TNFR-associated factor 2 as well as PKB and its upstream activator phosphoinositide 3-kinase (PI3K). Recruitment of PKB and PI3K were dependent on TNFR-associated factor 2 and on translocation of OX40 into detergent-insoluble membrane lipid microdomains but independent of TCR engagement. However, OX40 only resulted in strong phosphorylation and functional activation of the PI3K-PKB pathway when Ag was recognized. Therefore, OX40 primarily functions to augment PKB signaling in T cells by enhancing the amount of PI3K and PKB available to the TCR. This highlights a quantitative role of this TNFR family second signal to supplement signal 1.     

CD40-TRAF6 and autophagy-dependent anti-microbial activity in macrophages

A fundamental question in host-pathogen interaction is to determine if the immune system activates fusion with the lysosomes to eradicate pathogens. We recently reported that this task is accomplished by the interaction between CD40 expressed on macrophages and CD154 expressed on activated CD4+ T cells. CD40 stimulation of macrophages induces vacuole-lysosome fusion through autophagy and results in killing of the obligate intracellular pathogen Toxoplasma gondii. This response is independent of IFN-gamma, STAT1 and p47 GTPases. We now report that vacuole-lysosome fusion is dependent on synergy between TRAF6 signaling downstream of CD40 and TNF-alpha. These studies identified a new paradigm by which T cells eradicate an intracellular pathogen within macrophages.     

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 that can be restored by soluble CD40 ligand trimer.

Cell-mediated immunity that results in IL-12/IFN-gamma production is essential to control infections by intracellular organisms. Studies in animal models revealed contrasting results in regard to the importance of CD40-CD40 ligand (CD40L) signaling for induction of a type 1 cytokine response against these pathogens. We demonstrate that CD40-CD40L interaction in humans is critical for generation of the IL-12/IFN-gamma immune response against Toxoplasma gondii. Infection of monocytes with T. gondii resulted in up-regulation of CD40. CD40-CD40L signaling was required for optimal T cell production of IFN-gamma in response to T. gondii. Moreover, patients with hyper IgM (HIGM) syndrome exhibited a defect in IFN-gamma secretion in response to the parasite and evidence compatible with impaired in vivo T cell priming after T. gondii infection. Not only was IL-12 production in response to T. gondii dependent on CD40-CD40L signaling, but also, patients with HIGM syndrome exhibited deficient in vitro secretion of this cytokine in response to the parasite. Finally, in vitro incubation with agonistic soluble CD40L trimer enhanced T. gondii-triggered production of IFN-gamma and, through induction of IL-12 secretion, corrected the defect in IFN-gamma production observed in HIGM patients. Our results are likely to explain the susceptibility of patients with HIGM syndrome to infections by opportunistic pathogens.     

Stat1 as a component of tumor necrosis factor alpha receptor 1-TRADD signaling complex to inhibit NF-kappaB activation

Activated tumor necrosis factor alpha (TNF-alpha) receptor 1 (TNFR1) recruits TNFR1-associated death domain protein (TRADD), which in turn triggers two opposite signaling pathways leading to caspase activation for apoptosis induction and NF-kappaB activation for antiapoptosis gene upregulation. Here we show that Stat1 is involved in the TNFR1-TRADD signaling complex, as determined by employing a novel antibody array screening method. In HeLa cells, Stat1 was associated with TNFR1 and this association was increased with TNF-alpha treatment. TNFR1 signaling factors TRADD and Fas-associated death domain protein (FADD) were also found to interact with Stat1 in a TNF-alpha-dependent process. Our in vitro recombinant protein-protein interaction studies demonstrated that Stat1 could directly interact with TNFR1 and TRADD but not with FADD. Interaction between Stat1 and receptor-interacting protein (RIP) or TNFR-associated factor 2 (TRAF2) was not detected. Examination of Stat1-deficient cells showed an apparent increase in TNF-alpha-induced TRADD-RIP and TRADD-TRAF2 complex formation, while interaction between TRADD and FADD was unaffected. As a consequence, TNF-alpha-mediated I-kappaB degradation and NF-kappaB activation were markedly enhanced in Stat1-deficient cells, whereas overexpression of Stat1 in 293T cells blocked NF-kappaB activation by TNF-alpha. Thus, Stat1 acts as a TNFR1-signaling molecule to suppress NF-kappaB activation.     

Regulation of tumor necrosis factor-α expression by CD40 ligation in BV-2 microglial cells

Ligation of CD40 has been shown to induce/stimulate the expression of tumor necrosis factor-alpha (TNF-alpha) in microglial cells. This study delineates the mechanism by which CD40 ligation regulates the expression of TNF-alpha in BV-2 microglial cells. There was very little induction of TNF-alpha by ligation of CD40 alone by either cross-linking antibodies against CD40 or recombinant CD40 ligand (CD154). The absence of any increase in TNF-alpha production by CD40 ligation alone even in CD40-overexpressed BV-2 microglial cells suggest that signal transduced by the ligation of CD40 alone is not sufficient for strong induction of TNF-alpha. However, CD40 ligation markedly induced the production of TNF-alpha as well as the expression of TNF-alpha mRNA in interferon-gamma (IFN-gamma)-stimulated BV-2 glial cells. Ligation of CD40 in CD40-overexpressed cells markedly enhanced the expression of TNF-alpha in the presence of IFN-gamma. To understand the mechanism of CD40 ligation-mediated induction/stimulation of TNF-alpha, we investigated the role of nuclear factor-kappaB (NF-kappaB) and C/EBPbeta. IFN-gamma alone was able to induce the activation of NF-kappaB as well as C/EBPbeta. However, CD40 ligation alone in the presence or absence of CD40 overexpression induced the activation of only NF-kappaB and not that of C/EBPbeta, suggesting that the activation of NF-kappaB alone by CD40 ligation is not sufficient to induce the expression of TNF-alpha and that the activation of C/EBPbeta is also necessary for strong induction of TNF-alpha. Consistently, a dominant-negative mutant of p65 (Delta(p65)) and that of C/EBPbeta (DeltaC/EBPbeta) inhibited the expression of TNF-alpha in BV-2 microglial cells stimulated with the combination of IFN-gamma and CD40 ligand. Taken together, these studies suggest that activation of both NF-kappaB and C/EBPbeta is important for strong induction of TNF-alpha and that CD40 ligation regulates the expression of TNF-alpha by modulating the activation of only NF-kappaB but not that of C/EBPbeta.     

Tumor necrosis factor-alpha triggers antitoxoplasmal activity of IFN-gamma primed macrophages

IFN-gamma is an important mediator of cellular resistance against microbial pathogens and tumor cells due in part to its potent capacity to activate macrophages for enhanced cytotoxicity. The present study demonstrates that TNF-alpha regulates the expression of enhanced antimicrobial activity by triggering IFN-gamma primed macrophages to kill or inhibit intracellular Toxoplasma gondii. Resident mouse macrophages stimulated with rIFN-gamma at levels up to 2500 U/ml failed to display enhanced antitoxoplasmal activity when cultured in vitro under low endotoxin conditions (less than 10 pg/ml), but were triggered by addition of small amounts of LPS (0.1 ng/ml). A similar requirement for LPS as a second signal necessary to trigger antitoxoplasmal activity was observed when IFN-gamma was administered to mice in vivo. The essential nature of this triggering step allowed us to explore the role of cytokines that act as endogenous regulators of macrophage activation. rTNF-alpha, although unable to confer antitoxoplasmal activity when used alone to treat macrophages, was capable of triggering IFN-gamma-primed macrophages cultured under low endotoxin conditions. The ability of TNF-alpha to trigger IFN-gamma-primed macrophages was blocked by rabbit anti-TNF-alpha polyclonal antisera but was not affected by polymyxin B indicating that TNF-alpha triggering was not due to contamination with LPS. Collectively, these findings demonstrate that TNF-alpha performs an important regulatory role in the expression of enhanced anti-microbial activity by IFN-gamma-primed macrophages.     

Paradoxical anti-inflammatory actions of TNF-alpha: inhibition of IL-12 and IL-23 via TNF receptor 1 in macrophages and dendritic cells

IL-12 and TNF-alpha are central proinflammatory cytokines produced by macrophages and dendritic cells. Disregulation of TNF-alpha is associated with sepsis and autoimmune diseases such as rheumatoid arthritis. However, new evidence suggests an anti-inflammatory role for TNF-alpha. TNF-alpha-treated murine macrophages produced less IL-12p70 and IL-23, after stimulation with IFN-gamma and LPS. Frequency of IL-12p40-producing macrophages correspondingly decreased as measured by intracellular cytokine staining. IL-12p40 production was also inhibited in dendritic cells. TNFR1 was established as the main receptor involved in IL-12p40 regulation, because IL-12p40 levels were not affected by TNF-alpha in TNFR1(-/-)-derived macrophages. Macrophages activated during Listeria monocytogenes infection were more susceptible to inhibition by TNF-alpha than cells from naive animals, which suggests a regulatory role for TNF-alpha in later stages of infection. This nonapoptotic anti-inflammatory regulation of IL-12 and IL-23 is an important addition to the multitude of TNF-alpha-induced responses determined by cell-specific receptor signaling.     

Differential regulation of CD40-mediated TNF receptor-associated factor degradation in B lymphocytes

Engagement of CD40 on murine B cells by its ligand CD154 induces the binding of TNFR-associated factors (TRAFs) 1, 2, 3, and 6, followed by the rapid degradation of TRAFs 2 and 3. TRAF degradation occurs in response to signaling by other TNFR superfamily members, and is likely to be a normal regulatory component of signaling by this receptor family. In this study, we found that receptor-induced TRAF degradation limits TRAF2-dependent CD40 signals to murine B cells. However, TRAFs 1 and 6 are not degraded in response to CD40 engagement, despite their association with CD40. To better understand the mechanisms underlying differential TRAF degradation, mixed protein domain TRAF chimeras were analyzed in murine B cells. Chimeras containing the TRAF2 zinc (Zn) domains induced effective degradation, if attached to a TRAF domain that binds to the PXQXT motif of CD40. However, the Zn domains of TRAF3 and TRAF6 could not induce degradation in response to CD40, regardless of the TRAF domains to which they were attached. Our data indicate that TRAF2 serves as the master regulator of TRAF degradation in response to CD40 signaling, and this function is dependent upon both the TRAF Zn domains and receptor binding position.     

TNF receptor-associated factor 6 is an essential mediator of CD40-activated proinflammatory pathways in monocytes and macrophages

The interaction between CD40 and its ligand, CD154, has been shown to play a role in the onset and maintenance of inflammatory disease. Contributing to this process is the ability of CD40 to signal monocyte and macrophage inflammatory cytokine production. We have shown that this event is dependent on Src family tyrosine kinase activity and the subsequent activation of ERK1/2. To address the role of TNFR-associated factor (TRAF) family members in facilitating this signaling pathway, we transfected a CD40-deficient macrophage cell line with wild-type human CD40, or with CD40 containing disrupted TRAF binding sites. Ligation of either wild-type CD40, or a CD40 mutant unable to bind TRAF2/3/5, resulted in the stimulation of inflammatory cytokine production. However, ligation of a CD40 mutant lacking a functional TRAF6 binding site did not initiate inflammatory cytokine production, and this mutant was found to be defective in CD40-mediated activation of ERK1/2, as well as IkappaB kinase (IKK) and NF-kappaB. Likewise, introduction of a dominant-negative TRAF6 into a wild-type (CD40(+)) macrophage cell line resulted in abrogation of CD40-mediated induction of inflammatory cytokine synthesis. Finally, treatment of monocytes with a cell-permeable peptide corresponding to the TRAF6-binding motif of CD40 inhibited CD40 activation of ERK1/2, IKK, and inflammatory cytokine production. These data demonstrate that TRAF6 acts as a critical adapter of both the Src/ERK1/2 and IKK/NF-kappaB proinflammatory signaling pathways in monocytes and macrophages.     

CMRF-35-like molecule 3 preferentially promotes TLR9-triggered proinflammatory cytokine production in macrophages by enhancing TNF receptor-associated factor 6 ubiquitination

TLRs are critical innate immune sensors in the induction of proinflammatory cytokines to eliminate invading pathogens. However, the mechanisms for the full activation of TLR-triggered innate immune response need to be fully understood. The murine CMRF-35-like molecule (CLM)-3 is a representative of CLM family belonging to the Ig superfamily. Considering that CLM-3 is selectively expressed in macrophages and the roles of CLM members in innate immune response remain unclear, in this study we investigated the role of CLM-3 in the regulation of TLR-triggered innate response. We found that CLM-3 was an endosome/lysosome-localized molecule, and was downregulated in macrophages by stimulation with TLR9 ligand, but not TLR4 and TLR3 ligands. Interestingly, CLM-3 selectively promoted production of TNF-α and IL-6 in macrophages triggered by TLR9, but not TLR4 or TLR3. CLM-3 enhanced activation of MAPKs and NF-κB pathways in TLR9-triggered macrophages. Furthermore, CLM-3-transgenic mice were generated, and CLM-3 expression was confirmed by mAb against CLM-3 that we prepared. Accordingly, the macrophages derived from CLM-3-transgenic mice were more sensitive to TLR9 ligand stimulation, with more pronounced production of TNF-α, IL-6, and increased activation of MAPKs and NF-κB pathways. Moreover, ubiquitination of TNFR-associated factor 6, a crucial signaling transducer of TLR-triggered MAPKs and NF-κB activation, was found to be significantly promoted by CLM-3 in macrophages. Collectively, the endosome/lysosome-localized CLM-3 can promote full activation of TLR9-triggered innate responses by enhancing TNFR-associated factor 6 ubiquitination and subsequently activating MAPKs and NF-κB.     

Roles of the Kinase TAK1 in TRAF6-Dependent Signaling by CD40 and Its Oncogenic Viral Mimic, LMP1

The Epstein-Barr virus (EBV)-encoded protein latent membrane protein 1 (LMP1) is essential for EBV-mediated B cell transformation and plays a critical role in the development of post-transplant B cell lymphomas. LMP1 also contributes to the exacerbation of autoimmune diseases such as systemic lupus erythematosus (SLE). LMP1 is a functional mimic of the tumor necrosis factor receptor (TNFR) superfamily member CD40, and relies on TNFR-associated factor (TRAF) adaptor proteins to mediate signaling. However, LMP1 activation signals to the B cell are amplified and sustained compared to CD40 signals. We previously demonstrated that LMP1 and CD40 use TRAF molecules differently. Although associating with CD40 and LMP1 via separate mechanisms, TRAF6 plays a significant role in signal transduction by both. It is unknown whether TRAF6 mediates CD40 versus LMP1 functions via distinct or shared pathways. In this study, we tested the hypothesis that TRAF6 uses the kinase TAK1 to trigger important signaling pathways following both CD40 and LMP1 stimulation. We determined that TAK1 was required for JNK activation and interleukin-6 (IL-6) production mediated by CD40 and LMP1, in both mouse and human B cells. Additionally, TRAF3 negatively regulated TRAF6-dependent, CD40-mediated TAK1 activation by limiting TRAF6 recruitment. This mode of regulation was not observed for LMP1 and may contribute to the dysregulation of LMP1 compared to CD40 signals.     

Apoptosis mediated by the TNF-related cytokine and receptor families

T lymphocytes use several specialized mechanisms to induce apoptotic cell death. The tumor necrosis factor (TNF)-related family of membrane-anchored and secreted ligands represent a major mechanism regulating cell death and cell survival. These ligands also coordinate differentiation of tissue to defend against intracellular pathogens and regulate development of lymphoid tissue. Cellular responses are initiated by a corresponding family of specific receptors that includes two distinct TNFR (TNFR60 and TNFR80), Fas (CD95), CD40, p75NTF, and the recently identified lymphotoxin β-receptor (LTβR), among others. The MHC-encoded cytokines, TNF and LTα, form homomeric trimers, whereas LTβ assembles into heterotrimers with LTα, creating multimeric ligands with distinct receptor specificities. The signal transduction cascade is initiated by transmembrane aggregation (clustering) of receptor cytoplasmic domains induced by binding to their multivalent ligands. The TRAF family of Zn RING/finger proteins bind to TNFR80; CD40 and LTβR are involved in induction NFκB and cell survival. TNFR60 and Fas interact with several distinct cytosolic proteins sharing the “death domain” homology region. TNF binding to TNFR60 activates a serine protein kinase activity and phosphoproteins are recruited to the receptor forming a multicomponent signaling complex. Thus, TNFRs use diverse sets of signaling molecules to initiate and regulate cell death and survival pathways. © 1996 Wiley-Liss, Inc.     

A signal through OX40 (CD134) allows anergic, autoreactive T cells to acquire effector cell functions

To study mechanisms of peripheral self-tolerance, we injected small numbers of naive CD4(+) TCR-transgenic T cells into mice expressing the MHC/peptide ligand under the control of an MHC class II promoter. The donor T cells expand rapidly to very large numbers, acquire memory markers, and go out into tissues, but the animals remain healthy, and the accumulated T cells are profoundly anergic to restimulation with Ag in vitro. Provision of a costimulatory signal by coinjection of an agonist Ab to OX40 (CD134), a TNFR family member expressed on activated CD4 T cells, results in death of the mice within 12 days. TCR-transgenic T cells recovered at 5 days from anti-OX40-treated mice have a unique phenotype: they remain unresponsive to Ag in vitro, but they are larger, more granular, and strongly IL-2R positive. Some spontaneously secrete IFN-gamma directly ex vivo, and the majority make IFN-gamma in response to PMA and ionomycin. Although they are anergic by conventional tests requiring Ag recognition, they respond vigorously to cytokines, proliferating in response to IL-2, and secreting IFN-gamma when TCR signaling is bypassed with IL-12 and IL-18. We conclude that the costimulatory signal through OX40 allows otherwise harmless, proliferating, autoreactive T cells to acquire effector cell functions. The ability of these T cells to respond to cytokines by synthesizing additional inflammatory cytokines without a TCR signal may drive the fatal pathogenic process in vivo.     

Characterization of a CD40-dominant inhibitory receptor mutant.

CD40 is an important mediator of immune and inflammatory responses. It is a costimulatory molecule for B cell proliferation and survival. Blockade of CD40 has been shown to induce tolerance and its role in other pathogenic conditions has led to the proposal that CD40 inhibition could be valuable therapeutically. As a first step to this end, we have characterized a CD40-dominant negative receptor. This inhibitory mutant lacks the identified CD40 signaling domains. It inhibits both cotransfected and endogenous CD40 activation of NF-kappaB. This mutant is specific, as it does not affect TNF or latent membrane protein 1 signaling. Its potential usefulness is illustrated by its ability to inhibit the CD40 ligand-stimulated increases of HLA and CD54 expression, molecules involved in Ag recognition and lymphocyte recruitment leading to organ rejection. The inhibitory mutant has no TNFR-associated factor 2-binding capabilities and inhibits the recruitment of TNFR-associated factor 2 to the CD40 signaling complex after stimulation. These studies show that the CD40 inhibitory receptor molecule is effective, specific, and useful both for research and potentially as a clinical tool. And furthermore, it is likely that similar dominant inhibitory receptors can be generated for all of the members of the TNFR superfamily.     

Divergent response to LPS and bacteria in CD14-deficient murine macrophages

Gram-negative bacteria and the LPS constituent of their outer membranes stimulate the release of inflammatory mediators believed to be responsible for the clinical manifestations of septic shock. The GPI-linked membrane protein, CD14, initiates the signaling cascade responsible for the induction of this inflammatory response by LPS. In this paper, we report the generation and characterization of CD14-null mice in which the entire coding region of CD14 was deleted. As expected, LPS failed to elicit TNF-alpha and IL-6 production in macrophages taken from these animals, and this loss in responsiveness is associated with impaired activation of both the NF-kappaB and the c-Jun N-terminal mitogen-activated protein kinase pathways. The binding and uptake of heat-killed Escherichia coli, measured by FACS analysis, did not differ between CD14-null and wild-type macrophages. However, in contrast to the findings with LPS, whole E. coli stimulated similar levels of TNF-alpha release from CD14-null and wild-type macrophages at a dose of 10 bioparticles per cell. This effect was dose dependent, and at lower bacterial concentrations CD14-deficient macrophages produced significantly less TNF-alpha than wild type. Approximately half of this CD14-independent response appeared to be mediated by CD11b/CD18, as demonstrated by receptor blockade using neutrophil inhibitory factor. An inhibitor of phagocytosis, cytochalasin B, abrogated the induction of TNF-alpha in CD14-deficient macrophages by E. coli. These data indicate that CD14 is essential for macrophage responses to free LPS, whereas other receptors, including CD11b/CD18, can compensate for the loss of CD14 in response to whole bacteria.     

Role of TNF receptor-associated factor 2 in the activation of IgM secretion by CD40 and CD120b

TNFR-associated factors (TRAFs) participate in the signaling of many TNFR family members, including CD40, CD120a (TNFR1), and CD120b (TNFR2). Previously, we found that a dominant-negative TRAF2 molecule inhibits CD40-mediated Ab secretion by the mouse B cell line CH12.LX. However, disruption of the TRAF2 binding site in the cytoplasmic domain of CD40 does not diminish the ability of CD40 to stimulate Ab secretion, nor is this mutation able to circumvent the inhibition of Ab secretion by dominant-negative TRAF2. Here we demonstrate that CD40-induced TNF stimulates IgM production through CD120b and that CD120b signaling is required for optimal CD40-induced IgM secretion. Furthermore, although both CD40 and CD120b can bind TRAF2, TRAF2-dependent CD40 signals cannot substitute for TRAF2-dependent CD120b signals in the activation of IgM secretion. Our results indicate a potentially important role for CD120b in the activation of IgM secretion and that TRAF2 is used by CD40 and CD120b in distinct ways.