Defective microarchitecture of the spleen marginal zone and impaired response to a thymus-independent type 2 antigen in mice lacking scavenger receptors MARCO and SR-A

The macrophage scavenger receptor macrophage receptor with a collagenous structure (MARCO) is expressed in mice by the marginal zone macrophages of the spleen and by macrophages of the medullary cords of lymph nodes, as well as the peritoneal macrophages. MARCO is a relative of scavenger receptor A (SR-A), the more widely expressed prototypic member of the scavenger receptor family. In the present study, we found that genetic ablation of MARCO leads to changes in the organization of the splenic marginal zone, and causes a significant reduction in the size of the resident peritoneal macrophage population, possibly due to changes in adhesion and migration capacity. In mice lacking both MARCO and SR-A these effects are even more apparent. During ontogeny, the appearance and organization of the MARCO-expressing cells in the spleen precedes the appearance of other receptors on macrophages in the marginal zone, such as SIGNR1 and Siglec-1. In the absence of MARCO, a clear delay in the organization of the marginal zone was observed. Similar findings were seen when the reappearance of the various subsets from precursors was studied after depleting macrophages from the adult spleen by a liposome treatment. When challenged with a pneumococcal polysaccharide vaccine, a T-independent type 2 Ag for which an intact marginal zone is crucial, the knockout mice exhibited a clearly impaired response. These findings suggest that both MARCO and SR-A, in addition to being important scavenger receptors, could be involved in the positioning and differentiation of macrophages, possibly through interaction with endogenous ligands.     

Reduced expression of STAT4 and IFN-gamma in macrophages from BALB/c mice

BALB/c mice have been shown to easily induce Th2 type responses in several infection models. In this study, to examine the mechanisms of Th2 dominant responses in BALB/c mice, we assessed several macrophage functions using C3H/HeN, C57BL/6, and BALB/c mouse strains. Peritoneal macrophages from three strains of mice equally produced IL-12 by stimulation with LPS plus IFN-gamma. However, IFN-gamma production in response to IL-12 or IL-12 plus IL-18 was much lower in macrophages from BALB/c mice than other strains. IFN-gamma produced by activated macrophages induced IL-12R mRNA expression in T cells and macrophages themselves depending on their amount of IFN-gamma; namely, macrophages from BALB/c mice induced lower expression of IL-12R. Intracellular levels of STAT4 were much lower in macrophages from BALB/c mice. However, other STATs, such as STAT1 or STAT6, were expressed similarly in the three mouse strains. STAT4 and IFN-gamma production by other cell types such as T cells and B cells were equal in C3H/HeN and BALB/c mice. These results indicate that macrophages from Th2-dominant BALB/c mice have different functional characters compared with other mouse strains; that is, STAT4 expression and IFN-gamma production are reduced, which is one of the causes to shift to Th2-type responses.     

Interleukin-10 (IL-10) inhibits the induction of nitric oxide synthase by interferon-gamma in murine macrophages.

A murine macrophage cell line, J774, expresses high levels of the enzyme nitric oxide synthase (NOS) and produces large amounts of nitric oxide (NO) when activated with recombinant interferon (IFN)-gamma and a low concentration of LPS (10 ng/ml). Both the expression of NOS and the production of NO were inhibited by recombinant IL-10 in a dose-dependent manner. The inhibition was effective only when the cells were pretreated with IL-10; addition of IL-10 at the same time or after IFN-gamma activation was without effect. These results demonstrate that IL-10, a product of Th2 (helper T lymphocyte 2) cells, can antagonise the function of IFN-gamma, a product of Th1 cells, by modulating the mechanism of synthesis of nitric oxide in the macrophages.     

Scavenger receptor A (SR-A) is required for LPS-induced TLR4 mediated NF-κB activation in macrophages

Recent evidence suggests that the macrophage scavenger receptor class A (SR-A, aka, CD204) plays a role in the induction of innate immune and inflammatory responses. We investigated whether SR-A will cooperate with Toll-like receptors (TLRs) in response to TLR ligand stimulation. Macrophages (J774/a) were treated with Pam2CSK4, (TLR2 ligand), Polyinosinic:polycytidylic acid (Poly I:C) (TLR3 ligand), and Lipopolysaccharides (LPS) (TLR4 ligand) for 15 min in the presence or absence of fucoidan (the SR-A ligand). The levels of phosphorylated IκBα (p-IκBα) were examined by Western blot. We observed that Poly I:C and LPS alone, but not Pam2CSK4 or fucoidan increased the levels of p-IκBα. However, LPS-induced increases in p-IκBα levels were further enhanced when presence of the fucoidan. Immunoprecipitation and double fluorescent staining showed that LPS stimulation promotes SR-A association with TLR4 in the presence of fucoidan. To further confirm our observation, we isolated peritoneal macrophages from SR-A deficient (SR-A(-/-)), TLR4(-/-) and wild type (WT) mice, respectively. The peritoneal macrophages were treated with LPS for 15min in the presence and absence of fucoidan. We observed that LPS-stimulated TNFα and IL-1β production was further enhanced in the WT macrophages, but did not in either TLR4(-/-) or SR-A(-/-) macrophages, when fucoidan was present. Similarly, in the presence of fucoidan, LPS-induced IκBα phosphorylation, NF-κB binding activity, and association between TLR4 and SR-A were significantly enhanced in WT macrophages compared with LPS stimulation alone. The data suggests that SR-A is needed for LPS-induced inflammatory responses in macrophages.     

Interleukin 4 induces the expression of inducible nitric oxide synthase in eosinophils

We investigated the effects of the Th2-like cytokines IL-4 and IL-13 and of IL-10 on the induction of iNOS and NO production in rat eosinophils. Addition of mIL-4 to the eosinophil culture increased iNOS activity and nitrite production but did not improve the stimulatory effect of IFN-gamma and LPS. In contrast to eosinophils, addition of mIL-4 to macrophage cultures inhibited the iNOS expression and nitrite production induced by IFN-gamma plus LPS. Addition of mIL-13 to the eosinophil cultures did not significantly change iNOS activity and nitrite production in cells stimulated or not with IFN-gamma plus LPS. On the other hand, IL-13 inhibited iNOS activity in IFN-gamma plus LPS-stimulated macrophages. In the presence of IL-10, iNOS activity in non-stimulated eosinophil or macrophage cultures was not significantly altered, but the enzyme expression was inhibited in IFN-gamma plus LPS-stimulated eosinophils or macrophages. The production of nitrite by eosinophils stimulated by IFN-gamma plus LPS was inhibited by the presence of IL-10 in the medium. In conclusion, eosinophils might exhibit differential modulation of the L-arginine/iNOS pathway depending on the profile of Th2 cytokines produced during allergic diseases. IL-4 appears to be an important Th2 cytokine involved in the induction of the L-arginine/iNOS pathway in eosinophils.     

Interferon gamma priming is not critical for IL-12 production of murine spleen cells

Interferon gamma (IFN-gamma) priming is considered to be critical for interleukin 12 (IL-12) production of murine macrophages and human monocytes by lipopolysaccharide (LPS) stimulation. In our present experiments, freshly prepared spleen cells (f-spleen cells) were confirmed not to produce detectable level of IL-12 by LPS stimulation, although they produced significant amount of IL-12 by the stimulation with LPS plus IFN-gamma. However, the stimulation only with LPS induced IL-12 production of spleen cells preincubated in the absence of IFN-gamma. Findings on IL-12 p40 mRNA accumulation were consistent with their IL-12 production. Essentially the same results were obtained using spleen cells from IFN-gamma deficient mice. In the presence of anti-IL-10, f-spleen cells produced IL-12 upon LPS stimulation, indicating that the failure of f-spleen cells in IL-12 production is caused by IL-10 produced by themselves upon LPS stimulation. In addition, f-spleen cells produced IL-12 upon CD40 ligand stimulation, and the production was hardly affected by the presence of IFN-gamma or preincubation. These results indicate that IFN-gamma priming is not critical for IL-12 production of spleen cells stimulated with LPS or CD40 ligand, although IFN-gamma enhances the production, especially, in response to LPS stimulation.     

Scavenger Receptors SR-AI/II and MARCO limit pulmonary dendritic cell migration and allergic airway inflammation

The class A scavenger receptors (SR-A) MARCO and SR-AI/II are expressed on lung macrophages (MPhis) and dendritic cells (DCs) and function in innate defenses against inhaled pathogens and particles. Increased expression of SR-As in the lungs of mice in an OVA-asthma model suggested an additional role in modulating responses to an inhaled allergen. After OVA sensitization and aerosol challenge, SR-AI/II and MARCO-deficient mice exhibited greater eosinophilic airway inflammation and airway hyperresponsiveness compared with wild-type mice. A role for simple SR-A-mediated Ag clearance ("scavenging") by lung MPhis was excluded by the observation of a comparable uptake of fluorescent OVA by wild-type and SR-A-deficient lung MPhis and DCs. In contrast, airway instillation of fluorescent Ag revealed a significantly higher traffic of labeled DCs to thoracic lymph nodes in SR-A-deficient mice than in controls. The increased migration of SR-A-deficient DCs was accompanied by the enhanced proliferation in thoracic lymph nodes of adoptively transferred OVA-specific T cells after airway OVA challenge. The data identify a novel role for SR-As expressed on lung DCs in the down-regulation of specific immune responses to aeroallergens by the reduction of DC migration from the site of Ag uptake to the draining lymph nodes.     

IL-4 pretreatment selectively enhances cytokine and chemokine production in lipopolysaccharide-stimulated mouse peritoneal macrophages

Although well recognized for its anti-inflammatory effect on gene expression in stimulated monocytes and macrophages, IL-4 is a pleiotropic cytokine that has also been shown to enhance TNF-alpha and IL-12 production in response to stimulation with LPS. In the present study we expand these prior studies in three areas. First, the potentiating effect of IL-4 pretreatment is both stimulus and gene selective. Pretreatment of mouse macrophages with IL-4 for a minimum of 6 h produces a 2- to 4-fold enhancement of LPS-induced expression of several cytokines and chemokines, including TNF-alpha, IL-1alpha, macrophage-inflammatory protein-2, and KC, but inhibits the production of IL-12p40. In addition, the production of TNF-alpha by macrophages stimulated with IFN-gamma and IL-2 is inhibited by IL-4 pretreatment, while responses to both LPS and dsRNA are enhanced. Second, the ability of IL-4 to potentiate LPS-stimulated cytokine production appears to require new IL-4-stimulated gene expression, because it is time dependent, requires the activation of STAT6, and is blocked by the reversible protein synthesis inhibitor cycloheximide during the IL-4 pretreatment period. Finally, IL-4-mediated potentiation of TNF-alpha production involves specific enhancement of mRNA translation. Although TNF-alpha protein is increased in IL-4-pretreated cells, the level of mRNA remains unchanged. Furthermore, LPS-stimulated TNF-alpha mRNA is selectively enriched in actively translating large polyribosomes in IL-4-pretreated cells compared with cells stimulated with LPS alone.     

IFN-gamma-independent autocrine cytokine regulatory mechanism in reprogramming of macrophage responses to bacterial lipopolysaccharide.

Macrophages are now well recognized to have a critical role in both innate and acquired immunity. The sentinel macrophage function is highly regulated and serves to allow for intrinsic plasticity of the innate immune responses to potential environmental signals. However, the mechanisms underlying the dynamic properties of the cellular arm of innate immunity are poorly understood. Therefore, we have conducted a series of in vitro studies to evaluate the contribution of immunoregulatory cytokines, such as IFN-gamma, IL-10, and IL-12, in modulation of macrophage responses. We found that macrophages from IFN-gamma knockout (IFN-gamma(-/-)) mice exhibit only marginal LPS-induced TNF-alpha, IL-12, and NO responses, all of which can be fully restored in the presence of rIFN-gamma. Pretreatment with substimulatory LPS concentrations led to reprogramming of IFN-gamma(-/-) macrophage responses in a dose-dependent manner that manifested by an increased TNF-alpha and IL-12, but not NO, production upon the subsequent LPS challenge. These reprogramming effects were substantially attenuated and profoundly enhanced in macrophages from IL-12(-/-) and IL-10(-/-) mice, respectively, as compared with those modulated in macrophages from the congenic wild-type mice. LPS-dependent reprogramming was also fully reproduced in macrophages isolated from SCID mice after immunodepletion of NK cells. Our data strongly imply that cytokine (TNF-alpha and IL-12), but not NO, responses in macrophages may, at least in part, be governed by an autocrine IFN-gamma-independent regulatory mechanism reciprocally controlled by IL-10 and IL-12. This mechanism may serve as an alternative/coherent pathway to the canonical IFN-gamma-dependent induction of antimicrobial and tumoricidal activity in macrophages.     

A pathway through interferon-gamma is the main pathway for induction of nitric oxide upon stimulation with bacterial lipopolysaccharide in mouse peritoneal cells.

Production of nitric oxide (NO) in response to bacterial lipopolysaccharide (LPS) was investigated using cultures of mouse peritoneal exudate cells (PEC) and the macrophage cell line RAW264.7. In the presence of anti-(interferon-gamma) (IFN-gamma), NO production was markedly suppressed in the PEC culture but not in the RAW264.7 culture. In the PEC culture, LPS induced both IFN-gamma production and activation of IFN response factor-1, which leads to the gene expression of inducible NO synthase, but neither was induced in the culture of RAW264.7 cells. In addition to anti-(IFN-gamma), antibodies against interleukin (IL)-12 and IL-18 showed a suppressive effect on LPS-induced NO production in the PEC culture, and these antibodies in synergy showed strong suppression. Stimulation of the PEC culture with IL-12 or IL-18 induced production of IFN-gamma and NO, and these cytokines, in combination, exhibited marked synergism. Stimulation of the culture with IFN-gamma induced production of NO, but not IL-12. The macrophage population in the PEC, prepared as adherent cells, responded well to LPS for IL-12 production, but weakly for production of IFN-gamma and NO. The macrophages also responded well to IFN-gamma for NO production. For production of IFN-gamma by stimulation with LPS or IL-12 + IL-18, nonadherent cells were required in the PEC culture. Considering these results overall, the indirect pathway, through the production of intermediates (such as IFN-gamma-inducing cytokines and IFN-gamma) by the cooperation of macrophages with nonadherent cells, was revealed to play the main role in the LPS-induced NO production pathway, as opposed to the direct pathway requiring only a macrophage population.     

The production of IFN-gamma by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4

Macrophages release IFN-gamma on combined stimulation with IL-12 and IL-18, but the signaling requirements of this process and its regulation by other cytokines are unknown. Here, we demonstrate that STAT4 is indispensable for IL-12/IL-18-induced production of IFN-gamma by mouse peritoneal macrophages. Type 2 NO synthase (NOS2), which we previously found to be a prerequisite for IL-12-induced IFN-gamma production in NK cells, was not required for IFN-gamma production by these macrophages. IL-12 alone already induced the expression of IFN-gamma mRNA, but nuclear translocation of STAT4, the release of IFN-gamma protein, and the subsequent production of NO was strictly dependent on the simultaneous presence of IL-18. NF-kappa B, which mediates IL-18 effects in T cells, was only weakly activated by IL-12 and/or IL-18 in macrophages. Known inhibitors of macrophage functions (e.g., IL-4 and TGF-beta) also suppressed macrophage IFN-gamma production and the subsequent production of NOS2-derived NO. The inhibitory effect of IL-4 was paralleled by nuclear translocation of STAT6, which in EMSAs was able to bind to the same DNA oligonucleotide as STAT4. These results further define the production of IFN-gamma by macrophages and point to a diversity in the signals required for IFN-gamma production by various cell types.     

The class A macrophage scavenger receptor type I (SR-AI) recognizes complement iC3b and mediates NF-κB activation

The macrophage scavenger receptor SR-AI binds to host tissue debris to perform clearance and it binds to bacteria for phagocytosis. In addition, SR-AI modulates macrophage activation through cell signaling. However, investigation of SR-AI signaling on macrophages is complicated due to its promiscuous ligand specificity that overlaps with other macrophage receptors. Therefore, we expressed SR-AI on HEK 293T cells to investigate its ligand binding and signaling. On 293T cells, SR-AI could respond to E. coli DH5α, leading to NF-κB activation and IL-8 production. However, this requires E. coli DH5α to be sensitized by fresh serum that is treated with heat-inactivation or complement C3 depletion. Anti-C3 antibody inhibits the binding of SR-AI to serum-sensitized DH5α and blocks DH5α stimulation of SR-AI signaling. Further analysis showed that SR-AI can directly bind to purified iC3b but not C3 or C3b. By mutagenesis, The SRCR domain of SR-AI was found to be essential in SR-AI binding to serum-sensitized DH5α. These results revealed a novel property of SR-AI as a complement receptor for iC3b-opsonized bacteria that can elicit cell signaling.     

IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells

Murine IL-10 (cytokine synthesis inhibitory factor) inhibits cytokine production by Th1 cell clones when they are activated under conditions requiring the presence of APC. By preincubating APC with IL-10, we demonstrate that IL-10 acts principally on APC to inhibit IFN-gamma production by Th1 clones. Moreover, IL-10 is not active when Th1 cells are stimulated with glutaraldehyde-fixed APC, which also indicates that its action involves regulation of APC function. Furthermore, IL-10 inhibits cytokine synthesis by Th1 cells stimulated with the super-antigen Staphylococcus enterotoxin B, which does not appear to require processing. Flow microfluorimetry purified splenic or peritoneal B cells and macrophages, and B cell and macrophage cell lines can present Ag to Th1 clones. However, IL-10 acts only on sorted macrophages and the macrophage cell line to suppress IFN-gamma production by Th1 clones. IL-10 does not show this effect when B cells are used as APC. In contrast, IL-10 does not impair the ability of APC to stimulate cytokine production by Th2 cells. IL-10 does not decrease IFN-gamma-induced I-Ad levels on a macrophage cell line. Inasmuch as IL-10 also inhibits IL-2-induced IFN-gamma production by Th1 cells in an Ag-free system requiring only the presence of accessory cells, these data suggest that IL-10 may inhibit macrophage accessory cell function which is independent of TCR-class II MHC interactions.     

Mechanisms of enhanced macrophage-mediated prostaglandin E2 production and its suppressive role in Th1 activation in Th2-dominant BALB/c mice

PGE(2) has been known to suppress Th1 responses. We studied the difference in strains of mice in PGE(2) production by macrophages and its relation to Th1 activation. Macrophages from BALB/c mice produced greater amounts of PGE(2) than those from any other strains of mice, including C57BL/6, after LPS stimulation. In accordance with the amount of PGE(2) produced, macrophage-derived IL-12 and T cell-derived IFN-gamma production were more strongly suppressed in BALB/c macrophages than in C57BL/6 macrophages. When macrophages were treated with indomethacin or EP4 antagonist, Th1 cytokines were more markedly increased in cells from BALB/c mice than in those from C57BL/6 mice. Although cyclooxygenase-2 was expressed similarly after LPS stimulation in these mouse strains, the release of arachidonic acid and the expression of type V secretory phospholipase A(2) mRNA were greater in BALB/c macrophages. However, exogenous addition of arachidonic acid did not reverse the lower production of PGE(2) by C57BL/6 macrophages. The expression of microsomal PGE synthase, a final enzyme of PGE(2) synthesis, was also greater in BALB/c macrophages. These results indicate that the greater production of PGE(2) by macrophages, which is regulated by secretory phospholipase A(2) and microsomal PGE synthase but not by cyclooxygenase-2, is related to the suppression of Th1 cytokine production in BALB/c mice.     

Anti-class a scavenger receptor autoantibodies from systemic lupus erythematosus patients impair phagocytic clearance of apoptotic cells by macrophages <Emphasis Type="Italic">in vitro</Emphasis>

Introduction  

Inadequate clearance of apoptotic cells by macrophages is one of the reasons for the breakdown of self-tolerance. Class A scavenger receptors, macrophage receptor with collagenous structure (MARCO) and scavenger receptor A (SR-A), which are expressed on macrophages, play important roles in the uptake of apoptotic cells. A previous study reported the presence of the anti-MARCO antibody in lupus-prone mice and systemic lupus erythematosus (SLE) patients. The purpose of this study was to investigate the prevalence of anti-class A scavenger receptor antibodies in patients with various autoimmune diseases, in particular SLE, and the functional implication of those autoantibodies in the phagocytic clearance of apoptotic cells by macrophages.