CSF-1-dependent red pulp macrophages regulate CD4 T cell responses

The balance between immune activation and suppression must be regulated to maintain immune homeostasis. Tissue macrophages (MΦs) constitute the major cellular subsets of APCs within the body; however, how and what types of resident MΦs are involved in the regulation of immune homeostasis in the peripheral lymphoid tissues are poorly understood. Splenic red pulp MΦ (RPMs) remove self-Ags, such as blood-borne particulates and aged erythrocytes, from the blood. Although many scattered T cells exist in the red pulp of the spleen, little attention has been given to how RPMs prevent harmful T cell immune responses against self-Ags. In this study, we found that murine splenic F4/80(hi)Mac-1(low) MΦs residing in the red pulp showed different expression patterns of surface markers compared with F4/80(+)Mac-1(hi) monocytes/MΦs. Studies with purified cell populations demonstrated that F4/80(hi)Mac-1(low) MΦs regulated CD4(+) T cell responses by producing soluble suppressive factors, including TGF-β and IL-10. Moreover, F4/80(hi)Mac-1(low) MΦs induced the differentiation of naive CD4(+) T cells into functional Foxp3(+) regulatory T cells. Additionally, we found that the differentiation of F4/80(hi)Mac-1(low) MΦs was critically regulated by CSF-1, and in vitro-generated bone marrow-derived MΦs induced by CSF-1 suppressed CD4(+) T cell responses and induced the generation of Foxp3(+) regulatory T cells in vivo. These results suggested that splenic CSF-1-dependent F4/80(hi)Mac-1(low) MΦs are a subpopulation of RPMs and regulate peripheral immune homeostasis.     

Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: methodology and new insights.

BACKGROUND: The need to accurately discriminate dendritic cells (DCs) and macrophages (Mphs) in mouse lungs is critical given important biological differences. However, a validated flow cytometry-based method is still lacking, resulting in much confusion between both cell types. METHODS: Single-cell suspensions freshly obtained from collagenase-digested lung tissue were stained with a CD11c-specific monoclonal antibody, detected using a PE-Cy5 or APC-conjugated secondary reagent. Cellular immunophenotype was simultaneously explored using a panel of PE-conjugated markers. The FL1 or FITC-detection channel was reserved for the assessment of autofluorescence. RESULTS: CD11c-bright cells were heterogeneous and displayed a bimodal distribution with regard to autofluorescence (AF). CD11c+/low-AF cells were lineage-negative and showed features compatible with myeloid DCs. This was confirmed by morphology, potent T-cell stimulatory function in a mixed-leukocyte reaction, surface expression of MHCII and costimulatory molecules, and further immunophenotypical criteria, including the expression of Mac-1 and absence of CD8alpha. In contrast, CD11c+/high-AF cells displayed the features of pulmonary Mphs, including typical Mph morphology, very weak induction of T-cell proliferation, low to absent expression of MHCII and costimulatory molecules, and very low levels of Mac-1 as well as F4/80. We also show that only CD11c+/high-AF cells strongly expressed the macrophage marker MOMA-2, while interestingly Mac-3 was expressed at high levels by CD11c+/high-AF and low-AF alike. CONCLUSIONS: This study shows that the combination of CD11c-expression and autofluorescence is necessary and sufficient to accurately separate DCs from macrophage subpopulations in mouse lungs.     

Genetic segregation of inflammatory lung disease and autoimmune disease severity in SHIP-1-/- mice

Alternatively activated M2 macrophages are implicated as both regulators and agents of lung disease, but their control is poorly understood. SHIP-1 is a 5' inositol phosphatase that negatively regulates the PI3K signaling pathway implicated in inflammation. SHIP-1-deficient mice have defects in hematopoiesis and B cell development, and die prematurely due to consolidation of lungs with M2-skewed macrophages. SHIP-1 is thought to restrain M2 macrophage polarization, with deregulated M2 skewing coinciding with severe lung disease in SHIP-1-deficient mice. To determine the influence of genetic background on the lung phenotype in SHIP-1(-/-) mice, we backcrossed the SHIP-1 null mutation onto C57BL/6 (Th2-resistant) and BALB/c (Th2-prone) backgrounds. Remarkably, we found that inflammatory lung disease was severe in C57.SHIP-1(-/-) mice, but absent in BALB.SHIP-1(-/-) mice. C57.SHIP-1(-/-), but not BALB.SHIP-1(-/-) mice had greatly increased myeloid progenitors, myeloid hyperplasia, markedly enhanced numbers of activated alveolar macrophages, and elevated amounts of Th2 and proinflammatory cytokines in bronchoalveolar lavage fluid and serum, suggesting that deregulated cytokine production induced disease. C57.SHIP-1(-/-) mice also developed severe B cell-dependent autoimmune disease, which was markedly attenuated on the BALB/c background. These data demonstrate that, contrary to current concepts, loss of SHIP-1 alone is not sufficient to cause lung inflammation, with disease only manifest on a permissive genetic background. This finding questions the nature of the lung disease in SHIP-1(-/-) mice, suggesting that its M2 classification is not strictly correct. Future identification of disease-promoting loci might reveal determinants of comorbid lung disease and autoimmunity and uncover potentially useful therapeutic targets.     

Modulation of macrophage subtypes by IRF5 determines osteoclastogenic potential

Bone-resorbing osteoclasts are differentiated from macrophages (MΦ) by M-CSF and RANKL. MΦ can be mainly classified into M1 and M2 MΦ, which are proinflammatory and anti-inflammatory, respectively, but little is known about their osteoclastogenic potential. Here, we investigated the osteoclastogenic potential of MΦ subtypes. When the two MΦ subtypes were differentiated into osteoclasts using M-CSF and RANKL, M2 MΦ more potently differentiated into osteoclasts than M1 MΦ. M2 MΦ generated with IL-4 or IL-10 also showed enhanced osteoclast differentiation compared with M1 MΦ induced by IFN-γ and lipopolysaccharide. In addition, robust bone-resorptive capacity and giant actin rings, which are features of mature osteoclasts, were observed in M2, but not M1 MΦ, under the osteoclast differentiation condition. Osteoclast differentiation was significantly increased in CD206⁺ M2 MΦ but not in CD86⁺ M1 MΦ. Compared with M1 MΦ, c-Fms and RANK were highly expressed in M2 MΦ. Enhanced osteoclastogenesis of M2 MΦ was mediated through sustained ERK activation, followed by efficient c-Fos and NFATc1 induction. Notably, the osteoclastogenic potential of M1 MΦ converted into M2 MΦ by exposure to M-CSF was higher than that of M2 MΦ converted into M1 MΦ by exposure to GM-CSF. Silencing IRF5, which is responsible for M1 MΦ polarization, increased osteoclast differentiation by enhancing c-Fms expression and activation of ERK, c-Fos, CREB, and NFATc1, which was inhibited by overexpression of IRF5. Collectively, M2 MΦ are suggested to be more efficient osteoclast precursors than M1 MΦ because of the attenuated expression of IRF5.     

Suppression of TNF-α and IL-1 signaling identifies a mechanism of homeostatic regulation of macrophages by IL-27

IL-27 is a pleiotropic cytokine with both activating and inhibitory functions on innate and acquired immunity. IL-27 is expressed at sites of inflammation in cytokine-driven autoimmune/inflammatory diseases, such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, and sarcoidosis. However, its role in modulating disease pathogenesis is still unknown. In this study, we found that IL-27 production is induced by TNF-α in human macrophages (MΦ) and investigated the effects of IL-27 on the responses of primary human MΦ to the endogenous inflammatory cytokines TNF-α and IL-1. In striking contrast to IL-27-mediated augmentation of TLR-induced cytokine production, we found that IL-27 suppressed MΦ responses to TNF-α and IL-1β, thus identifying an anti-inflammatory function of IL-27. IL-27 blocked the proximal steps of TNF-α signaling by downregulating cell-surface expression of the signaling receptors p55 and p75. The mechanism of inhibition of IL-1 signaling was downregulation of the ligand-binding IL-1RI concomitant with increased expression of the receptor antagonist IL-1Ra and the decoy receptor IL-1RII. These findings provide a mechanism for suppressive effects of IL-27 on innate immune cells and suggest that IL-27 regulates inflammation by limiting activation of MΦ by inflammatory cytokines while preserving initial steps in host defense by augmenting responses to microbial products.     

HIV-1 proteins preferentially activate anti-inflammatory M2-type macrophages

HIV-1 proteins, including Tat, gp120, and Nef, activate macrophages (MΦ), which is consistent with the fact that HIV-1 infection is characterized by sustained immune activation. Meanwhile, MΦ are functionally classified into two types: proinflammatory M1-MΦ and anti-inflammatory M2-MΦ. We show that HIV-1 proteins, particularly Nef, preferentially activate M2-MΦ. Extracellular Tat, gp120, and Nef activated MAPK and NF-κB pathways in human peripheral blood monocyte-derived MΦ. However, the activation was marked in M-CSF-derived M2-MΦ but not GM-CSF-derived M1-MΦ. Nef was the most potent activator, and its signaling activation was comparable to that by TNF-α. Indeed, Nef was internalized more rapidly by M2-MΦ than by M1-MΦ. The myristoylation and proline-rich motif of Nef were responsible for the observed signaling activation. Consistent with the activation of MAPK/NF-κB pathways, Nef stimulated the production of a number of proinflammatory cytokines/chemokines by M2-MΦ. However, Nef reduced the expression of CD163 and phagocytosis, the characteristic markers of M2-MΦ, indicating that Nef drives an M2-like to M1-like phenotypic shift. Because the differentiation of most tissue MΦ depends on M-CSF and its receptor, which is the essential axis for the anti-inflammatory M2-MΦ phenotype, the current study reveals an efficient mechanism by which HIV-1 proteins, such as Nef, induce the proinflammatory MΦ.     

Production of LPS-induced inflammatory mediators in murine peritoneal macrophages: neocuproine as a broad inhibitor and ATP7A as a selective regulator

Copper chelation regulates the production of inflammatory mediators in vivo during vascular inflammation and atherogenesis. Little is known about how the copper egress pump ATP7A regulates the production of these mediators. In this study, we isolated ATP7A deficient macrophages (MΦ) from the peritoneal cavity of blotchy mice and identified the lipopolysaccharide (LPS)-induced inflammatory mediators that were altered by ATP7A deficiency. These results were compared with the effect of neocuproine (a copper chelator) treatment on both ATP7A deficient and control MΦ. Seven of the 24 inflammatory mediators examined in this study had significant changes in expression in the ATP7A deficient MΦ compared to controls; 16 of these mediators were significantly reduced in MΦ treated with neocuproine compared to controls. Both neocuproine treatment and ATP7A deficiency reduced IFN-γ, MCP-1, MCP-3, and VEGF-A levels. Interestingly, the production of KC/GRO was upregulated by ATP7A deficiency but downregulated by neocuproine treatment. Neocuproine, but not ATP7A deficiency, reduced the production of FGF-9, IL-1α, IL-12p70, IL-2, IL-3, IL-4, IL-6, MIP-1β, MIP-2, RANTES, and TNFα. ATP7A deficiency but not neocuproine treatment reduced IP-10 and MCP-5 levels. In addition, both ATP7A deficiency and neocuproine treatment had no effect on GM-CSF, IL-10, IL-11, IL-7, OSM, and SCF. Together, these findings provide evidence that MΦ ATP7A selectively regulates LPS-induced inflammatory mediators, in part, via modulation of cellular copper availability, whereas neocuproine generally inhibits the production of inflammatory mediators. These results also imply that although copper chelation and ATP7A downregulation may result in different copper concentrations, gradients, and/or distribution in the cells, they may not lead to opposite biological effects on inflammatory mediator production.     

A macrophage subpopulation recruited by CC chemokine ligand-2 clears apoptotic cells in noninfectious lung injury

CC chemokine ligand-2 (CCL2)/monocyte chemoattractant protein (MCP)-1 expression is upregulated during pulmonary inflammation, and the CCL2-CCR2 axis plays a critical role in leukocyte recruitment and promotion of host defense against infection. The role of CCL2 in mediating macrophage subpopulations in the pathobiology of noninfectious lung injury is unknown. The goal of this study was to examine the role of CCL2 in noninfectious acute lung injury. Our results show that lung-specific overexpression of CCL2 protected mice from bleomycin-induced lung injury, characterized by significantly reduced mortality, reduced neutrophil accumulation, and decreased accumulation of the inflammatory mediators IL-6, CXCL2 (macrophage inflammatory protein-2), and CXCL1 (keratinocyte-derived chemokine). There were dramatic increases in the recruitment of myosin heavy chain (MHC) II IA/IE(int)CD11c(int) cells, exudative macrophages, and dendritic cells in Ccl2 transgenic mouse lungs both at baseline and after bleomycin treatment compared with levels in wild-type mice. We further demonstrated that MHCII IA/IE(int)CD11c(int) cells engulfed apoptotic cells during acute lung injury. Our data suggested a previously undiscovered role for MHCII IA/IE(int)CD11c(int) cells in apoptotic cell clearance and inflammation resolution.     

Identification of a urokinase receptor-integrin interaction site. Promiscuous regulator of integrin function

Adhesion and signaling by integrins require their dynamic association with nonintegrin membrane proteins. One such protein, the glycolipid-anchored urokinase receptor (uPAR), associates with and modifies the function of the beta(2)-integrin Mac-1 (CD11b/CD18). In this study, a critical non-I-domain binding site for uPAR on CD11b (M25; residues 424-440) is identified by homology with a phage display peptide known to bind uPAR. Recombinant soluble uPAR and cells expressing uPAR bound to immobilized M25, binding being promoted by urokinase and blocked by soluble M25, but not a scrambled control or homologous peptides from other beta(2)-associated alpha-chains. Mac-1, but not a mutated Mac-1 in which M25 was replaced with the homologous sequence of CD11c, co-precipitated with uPAR. In the beta-propeller model of alpha-chain folding, M25 spans an exposed loop on the ligand-binding, upper surface of alphaM, identifying uPAR as an atypical alphaM ligand. Although not blocking ligand binding to Mac-1, M25 (25-100 microM) inhibited leukocyte adhesion to fibrinogen, vitronectin, and cytokine-stimulated endothelial cells. M25 also blocked the association of uPAR with beta(1)-integrins and impaired beta(1)-integrin-dependent spreading and migration of human vascular smooth muscle cells on fibronectin and collagen. These observations indicate that uPAR associates with integrins directly and that disruption of this association broadly impairs integrin function, suggesting a novel strategy for regulation of integrins in the settings of inflammation and tumor progression.     

Systemic inflammation in chronic obstructive pulmonary disease: a population-based study

Background

Investigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue.

Methods

Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay.

Results

IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6.

Conclusion

AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.     

Colonic eosinophilic inflammation in experimental colitis is mediated by Ly6C(high) CCR2(+) inflammatory monocyte/macrophage-derived CCL11

Recent genome-wide association studies of pediatric inflammatory bowel disease have implicated the 17q12 loci, which contains the eosinophil-specific chemokine gene CCL11, with early-onset inflammatory bowel disease susceptibility. In the current study, we employed a murine model of experimental colitis to define the molecular pathways that regulate CCL11 expression in the chronic intestinal inflammation and pathophysiology of experimental colitis. Bone marrow chimera experiments showed that hematopoietic cell-derived CCL11 is sufficient for CCL11-mediated colonic eosinophilic inflammation. We show that dextran sodium sulfate (DSS) treatment promotes the recruitment of F4/80(+)CD11b(+)CCR2(+)Ly6C(high) inflammatory monocytes into the colon. F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocytes express CCL11, and their recruitment positively correlated with colonic eosinophilic inflammation. Phenotypic analysis of purified Ly6C(high) intestinal inflammatory macrophages revealed that these cells express both M1- and M2-associated genes, including Il6, Ccl4, Cxcl2, Arg1, Chi3l3, Ccl11, and Il10, respectively. Attenuation of DSS-induced F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocyte recruitment to the colon in CCR2(-/-) mice was associated with decreased colonic CCL11 expression, eosinophilic inflammation, and DSS-induced histopathology. These studies identify a mechanism for DSS-induced colonic eosinophilia mediated by Ly6C(high)CCR2(+) inflammatory monocyte/macrophage-derived CCL11.     

Random phage-epitope library based identification of a peptide antagonist of Mac-1 β2 integrin ligand binding

The leukocyte β2 integrin Mac-1 (CD11b/CD18) plays a pivotal role in inflammation and host defense. To develop peptide antagonists selectively inhibiting the function of Mac-1, we used a random constrained 6-mer (cys-6aa-cys) peptide library to map the structural features of CD11b, by determining the epitope of neutralizing monoclonal antibody mAb 44a (anti-CD11b). We have used a stringent phage display strategy, which resulted in the identification of one disulfide C-RLKEKH-C constrained peptide by direct biopanning of library on decreasing amounts of purified mAb 44a. The selected peptide mimics a discontinuous epitope, a peculiar shape on the CD11b-I-domain surface. Competitive ELISA experiments with different Mac-1 ligands showed that C-RLKEKH-C is able to bind to fibrinogen, iC3b, and C1q. Furthermore, the monomeric circular peptide C-RLKEKH-C, was effective in blocking the interaction between (125)I-fibrinogen and Mac-1 (IC(50)=3.35±0.1×10(-6)M), and inhibited the adhesion of human neutrophils to fibrinogen and iC3b. These data provide information about the relative location of amino acids on the I-domain surface using mAb 44a imprint of the CD11b protein. The derived mimotope may help in the design of future anti-inflammatory therapeutic agents that can act as specific therapeutic agents targeting PMNs mediated inflammation.     

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion

Background

Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated proline–glycine–proline (N-ac-PGP). In the current study, we investigate whether N-ac-PGP influences β₂-integrin activation and function in neutrophilic firm adhesion to endothelium.

Methods

Human polymorphonuclear leukocytes (PMNs) were isolated from fresh human blood. Subsequently, a transmigration assay was performed to evaluate the active migration of PMNs towards N-ac-PGP. Furthermore, the effect of the tripeptide on β₂-integrin activation was assessed by performing the adhesion assay using fibrinogen as a ligand. To determine whether this effect was due to conformational change of β₂-integrins, antibodies against CD11b and CD18 were used in the adhesion assay and the expression pattern of CD11b was determined.

Results

Human neutrophils transmigrated through an endothelial cell layer in response to basolateral N-ac-PGP. N-ac-PGP induced also a neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that CD11b/CD18 (Mac-1) was responsible for the N-ac-PGP-induced firm adhesion of neutrophils to fibrinogen. Pertussis toxin decreased the Mac-1 activation indicating the involvement of G-proteins. N-ac-PGP most likely activated Mac-1 by initiating a conformational change, since the expression pattern of Mac-1 on the cell surface did not change significantly.

Conclusions

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion.

General significance

This is the first study to describe that the chemo-attractant N-ac-PGP also activates Mac-1 on the surface of neutrophils, which can additionally contribute to neutrophilic transmigration into the lung tissue during lung inflammation.     

Hemolysin induces Toll-like receptor (TLR)-independent apoptosis and multiple TLR-associated parallel activation of macrophages

Vibrio cholerae hemolysin (HlyA) displays bipartite property while supervising macrophages (MΦ). The pore-forming toxin causes profound apoptosis within 3 h of exposure and in parallel supports activation of the defying MΦ. HlyA-induced apoptosis of MΦ remains steady for 24 h, is Toll-like receptor (TLR)-independent, and is driven by caspase-9 and caspase-7, thus involving the mitochondrial or intrinsic pathway. Cell activation is carried forward by time dependent up-regulation of varying TLRs. The promiscuous TLR association of HlyA prompted investigation, which revealed the β-prism lectin domain of HlyA simulated TLR4 up-regulation by jacalin, a plant lectin homologue besides expressing CD86 and type I cytokines TNF-α and IL-12. However, HlyA cytolytic protein domain up-regulated TLR2, which controlled CD40 for continuity of cell activation. Expression of TOLLIP before TLR2 and TLR6 abrogated TLR4, CD40, and CD86. We show that the transient expression of TOLLIP leading to curbing of activation-associated capabilities is a plausible feedback mechanism of MΦ to deploy TLR2 and prolong activation involving CD40 to encounter the HlyA cytolysin domain.     

Role of intercellular adhesion molecule 1 in indomethacin-induced ileitis

Adhesion molecules have been implicated in the pathogenesis of inflammatory bowel diseases. We investigated their expression and contribution to leukocyte recruitment in experimental intestinal inflammation. Ileitis was induced in Sprague-Dawley rats by two injections of indomethacin (7.5 mg/kg), given 24 h apart. Endothelial intercellular adhesion molecule-1 (ICAM-1) expression was quantified using the dual radiolabeled monoclonal antibody technique and Mac-1 (CD11b/CD18) expression on leukocytes by flow cytometry. Leukocyte infiltration was monitored by tissue myeloperoxidase (MPO) activity. The first indomethacin injection induced a time- and site-dependent increase of ICAM-1 expression in ileal mucosa and muscularis. The second injection resulted in a reduction of ICAM-1 expression below constitutive levels whereas Mac-1 was upregulated. MPO changes paralleled lesion development over 48 h. ICAM-1 and MPO values were correlated for the first 24 h. Immunoneutralization of either ICAM-1 or Mac-1 attenuated mucosal injury. We conclude that (i) indomethacin-induced ileitis is associated with a temporally disassociated upregulation of ICAM-1 and (ii) despite a reduction in ICAM-1 after 24 h, ICAM-1, in concert with Mac-1, contributes to mucosal injury and leukocyte infiltration elicited by indomethacin.     

Role of SHIP-1 in the adaptive immune responses to aeroallergen in the airway

Background

Th2-dominated inflammatory response in the airway is an integral component in the pathogenesis of allergic asthma. Accumulating evidence supports the notion that the phosphoinositide 3-kinase (PI3K) pathway is involved in the process. We previously reported that SHIP-1, a negative regulator of the PI3K pathway, is essential in maintaining lung immunohomeostasis, potentially through regulation of innate immune cells. However, the function of SHIP-1 in adaptive immune response in the lung has not been defined. We sought to determine the role of SHIP-1 in adaptive immunity in response to aeroallergen stimulation in the airway.

Methodology/Principal Findings

SHIP-1 knockout (SHIP-1^−/−) mice on BALB/c background were immunized with ovalbumin (OVA) plus aluminum hydroxide, a strong Th2-inducing immunization, and challenged with OVA. Airway and lung inflammation, immunoglobulin response, Th2 cytokine production and lymphocyte response were analyzed and compared with wild type mice. Even though there was mild spontaneous inflammation in the lung at baseline, SHIP-1^−/− mice showed altered responses, including less cell infiltration around the airways but more in the parenchyma, less mucus production, decreased Th2 cytokine production, and diminished serum OVA-specific IgE, IgG1, but not IgG2a. Naïve and OVA sensitized SHIP-1^−/− T cells produced a lower amount of IL-4. In vitro differentiated SHIP-1^−/− Th2 cells produced less IL-4 compared to wild type Th2 cells upon T cell receptor stimulation.

Conclusions/Significance

These findings indicate that, in contrast to its role as a negative regulator in the innate immune cells, SHIP-1 acts as a positive regulator in Th2 cells in the adaptive immune response to aeroallergen. Thus any potential manipulation of SHIP-1 activity should be adjusted according to the specific immune response.     

Shifts in macrophage (M phi) surface phenotypes during tumor growth: association of Mac-2+ and Mac-3+ M phi with immunosuppressive activity

Rat anti-mouse monoclonal antibodies (mAb), anti-Mac-1, -2, and -3, directed against macrophage (M phi) glycoprotein surface antigens, were used to demonstrate a tumor-induced shift in peritoneal M phi subpopulations. This study of the tumor-induced shift was approached in two steps. First, to show that separate phenotypic M phi subpopulations existed and second, to show that a shift in these populations was involved in immunosuppression of the host during tumor growth. Endogenous peroxidase activity was examined among normal and tumor-bearing host (TBH) M phi. A significant increase in the number of peroxidase-positive M phi occurred during tumor growth. Indirect immunofluorescence showed a decrease in Mac-2+ cells and an increase in Mac-3+ cells in TBH M phi populations. When the mAb, anti-Mac-1,-2, and -3 were used in the presence of complement (C), they were cytotoxic for M phi and showed differential depletion of normal and TBH M phi. Peroxidase-positive TBH M phi were susceptible to C-mediated lysis by anti-Mac-1 and -3 but not by anti-Mac-2, whereas no direct relationship was observed among normal host M phi. To demonstrate differences between normal and TBH M phi subpopulations, soluble inhibitory factors were examined from mAb plus C-modified M phi populations. Anti-Mac plus C-treated normal and TBH M phi produced supernatants with different regulatory capabilities as assessed in the mixed-lymphocyte reaction (MLR). Anti-Mac-2 plus C treatment significantly reduced the ability of TBH M phi to produce a soluble suppressor(s) but did not alter normal host M phi-derived suppressor production. In contrast, anti-Mac-1 and -3 plus C treatment of normal host M phi significantly reduced suppressor production. In the TBH, however, anti-Mac-1 plus C had no effect, while anti-Mac-3 plus C had only a limited reduction as compared to the normal host. Determination of levels of prostaglandin E2 (PGE2) in M phi supernatants showed that normal host Mac-1+ M phi were involved in down regulation of PGE2 production. This control was missing in the TBH M phi. Mac-2+ M phi were the apparent producers of PGE2 which accounts for the factor-mediated MLR suppression attributed to TBH Mac-2+ M phi. Collectively, these data suggest that tumor-induced aberrations in immunoregulation can in part be attributed to differences in anti-Mac mAb-defined M phi subpopulations.     

Regulation of macrophage activation markers by IL-4 and IFN-gamma is subpopulation-specific

We have addressed the differential regulatory properties that IFN-gamma and IL-4 exert on macrophage (M phi) subpopulations. For this purpose, Thyoglicolate-, Peptone-, and Con A-elicited M phi, as well as bone marrow-derived M phi and P388D1 cells, were cultured in the presence of either IFN-gamma or IL-4. The expression of LFA-1, Mac-1, and Mac-2 after this treatment was studied by FACS analysis. We have found that these surface molecules are differentially modulated by the two lymphokines, depending on the M phi subpopulation studied. Mac-1 is upregulated only in Thyoglicolate-elicited cells after treatment with IFN-gamma, while no change in the expression of Mac-2 was observed in any of the groups. LFA-1 is upregulated by IFN-gamma in Thyoglicolate- and bone marrow-derived M phi and P388D1 cells, while IL-4 does not induce LFA-1 on these cells. Interestingly, however, we have observed the reverse situation on Con A-elicited M phi, where a strong induction of LFA-1 is achieved by treatment of the cells with IL-4, while IFN-gamma does not modify the expression of this antigen. Our results obtained with the lymphokine-stimulated M phi are interpreted in the context of functionally induced M phi subpopulations, which might be regulated by either Th1 or Th2 CD4+ T cells. Thyoglicolate-elicited M phi may represent the in vitro equivalent of a M phi subpopulation regulated in vivo by Th1 cells while Con A-elicited M phi could be the equivalent of a subpopulation regulated by Th2 cells.     

Enhanced macrophage activity in granulomatous lesions of immune mice challenged with Mycobacterium tuberculosis

In this study, we evaluated the cellular influx and cytokine environment in the lungs of mice made immune by prior vaccination with Mycobacterium bovis bacillus Calmette-Guérin compared with control mice after infection with Mycobacterium tuberculosis to characterize composition of protective lesions in the lungs. Immune mice controlled the growth of the M. tuberculosis challenge more efficiently than control mice. In immune animals, granulomatous lesions were smaller and had a more lymphocytic core, less foamy cells, less parenchymal inflammation, and slower progression of lung pathology than in lungs of control mice. During the chronic stage of the infection, the bacterial load in the lungs of immune mice remained at a level 10 times lower than control mice, and this was associated with reduced numbers of CD4P(+P) and CD8P(+P) T cells, and the lower expression of protective (IL-12, IFN-gamma), inflammatory (TNF-alpha), immunoregulatory (GM-CSF), and immunosuppressive (IL-10) cytokines. The immune mice had higher numbers of CD11b- CD11c(high) DEC-205(low) alveolar macrophages, but lower numbers of CD11b+ CD11c(high) DEC-205(high) dendritic cells, with the latter expressing significantly lower levels of the antiapoptotic marker TNFR-associated factor-1. Moreover, during the early stage of chronic infection, lung dendritic cells from immune mice expressed higher levels of MHC class II and CD40 molecules than similar cells from control mice. These results indicate that while a chronic disease state is the eventual outcome in both control and immune mice infected with M. tuberculosis by aerosol exposure, immune mice develop a protective granulomatous lesion by increasing macrophage numbers and reduced expression of protective and inflammatory cytokines.