Cryptococcus neoformans induces macrophage inflammatory protein 1alpha (MIP-1alpha) and MIP-1beta in human microglia: role of specific antibody and soluble capsular polysaccharide

We characterized the expression of the beta-chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES by primary human microglia after exposure to Cryptococcus neoformans. In the absence of specific antibody, C. neoformans failed to elicit a chemokine response, while in the presence of specific antibody, microglia produced MIP-1alpha and MIP-1beta in amounts comparable to those induced by lipopolysaccharide. RANTES was also induced but at much lower levels. In addition to MIP-1alpha and MIP-1beta mRNA, we observed a robust induction of monocyte chemoattractant protein 1 and interleukin-8 mRNA following incubation of microglia with opsonized C. neoformans. In contrast, cryptococcal polysaccharide did not induce a chemokine response even when specific antibody was present and inhibited the MIP-1alpha induction associated with antibody-mediated phagocytosis of C. neoformans. The role of the Fc receptor in the observed chemokine induction was explored in several experiments. Treatment of microglia with cytochalasin D inhibited internalization of C. neoformans but did not affect MIP-1alpha induction. In contrast, treatment with herbimycin A, a tyrosine kinase inhibitor, inhibited MIP-1alpha induction. Microglia stimulated with immobilized murine immunoglobulin also produced MIP-1alpha and RANTES (MIP-1alpha > RANTES). Our results show that microglia produce several chemokines when stimulated by C. neoformans in the presence of specific antibody and that this process is likely to be mediated by Fc receptor activation. This response can be down-regulated by cryptococcal capsular polysaccharide. These findings suggest a mechanism by which C. neoformans infections fail to induce strong inflammatory responses in patients with cryptococcal meningoencephalitis and have important implications for antibody therapy.     

Potential role of the chemokine macrophage inflammatory protein 1alpha in human and experimental schistosomiasis

In human schistosomiasis, the concentrations of the chemokine macrophage inflammatory protein 1alpha (MIP-1alpha/CCL3) is greater in the plasma of patients with clinical hepatosplenic disease. The objective of the present study was to confirm the ability of CCL3 to detect severe disease in patients classified by ultrasonography (US) and to evaluate the potential role of CCL3 in Schistosoma mansoni-infected mice. CCL3 was measured by enzyme-linked immunosorbent assay in the plasma of S. mansoni-infected patients. CCL3-deficient mice were infected with 25 cercariae, and various inflammatory and infectious indices were evaluated. The concentration of CCL3 was higher in the plasma of S. mansoni-infected than noninfected patients. Moreover, CCL3 was greater in those with US-defined hepatosplenic than with the intestinal form of the disease. In CCL3-deficient mice, the size of the granuloma and the liver eosinophil peroxidase activity and collagen content were diminished compared to wild-type mice. In CCL3-deficient mice, the worm burden after 14 weeks of infection, but not after 9 weeks, was consistently smaller. The in vitro response of mesenteric lymph node cells to antigen stimulation was characterized by lower levels of interleukin-4 (IL-4) and IL-10. CCL3 is a marker of disease severity in infected humans, and experimental studies in mice suggest that CCL3 may be a causative factor in the development of severe schistosomiasis.     

Fcgamma receptor signaling in primary human microglia: differential roles of PI-3K and Ras/ERK MAPK pathways in phagocytosis and chemokine induction

Cryptococcus neoformans monoclonal antibody immune complex (IC) induces beta-chemokines and phagocytosis in primary human microglia via activation of Fc receptor for immunoglobulin G (FcgammaR). In this report, we investigated microglial FcgammaR signal-transduction pathways by using adenoviral-mediated gene transfer and specific inhibitors of cell-signaling pathways. We found that Src inhibitor PP2 and Syk inhibitor piceatannol inhibited phagocytosis, macrophage-inflammatory protein-1alpha (MIP-1alpha) release, as well as phosphorylation of extracellular-regulated kinase (ERK) and Akt, consistent with Src/Syk involvement early in FcgammaR signaling. Constitutively active mitogen-activated protein kinase kinase (MEK) induced MIP-1alpha, and Ras dominant-negative (DN) inhibited IC-induced ERK phosphorylation and MIP-1alpha production. These results suggest that the Ras/MEK/ERK pathway is necessary and sufficient in IC-induced MIP-1alpha expression. Neither Ras DN nor the MEK inhibitor U0126 inhibited phagocytosis. In contrast, phosphatidylinositol-3 kinase (PI-3K) inhibitors Wortmannin and LY294002 inhibited phagocytosis without affecting ERK phosphorylation or MIP-1alpha production. Conversely, Ras DN or U0126 did not affect Akt phosphorylation. Together, these results demonstrate distinct roles played by the PI-3K and Ras/MEK/ERK pathways in phagocytosis and MIP-1alpha induction, respectively. Our results demonstrating activation of functionally distinct pathways following microglial FcgammaR engagement may have implications for human central nervous system diseases.     

Role for platelet-endothelial cell adhesion molecule-1 in macrophage Fcgamma receptor function

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) (CD31), a 130-kD transmembrane glycoprotein that functions in adhesion and signaling, is thought to play a role in some forms of leukocyte transmigration. In the lung, PECAM-1 is highly expressed, yet there have been few studies examining its role in pulmonary pathology. We therefore examined the inflammatory response (measured by bronchoalveolar lavage cell counts and protein content) after several types of lung injury in wild-type and PECAM-1 knockout mice. Consistent with studies in other organs, instillation of an endothelial stimulant (interleukin-1) was PECAM-1-dependent. In contrast, we noted that three other forms of acute lung injury (acid aspiration, adenoviral instillation, and tumor necrosis factor instillation) were completely PECAM-1-independent. Interestingly, in situ immune complex deposition injury, another complex lung disease, was also PECAM-1-dependent. This surprising finding was investigated in more detail and found to be due to a defect in macrophage activation, and not to a blockade of leukocyte transmigration. Experiments in bone marrow chimeric mice as well as ex vivo data demonstrated that Fcgamma receptor-dependent phagocytosis and tumor necrosis factor release were significantly reduced in macrophages derived from PECAM-1 knockout mice. Although PECAM-1 may not be required for transmigration of leukocytes into the alveolar space in many forms of complex lung inflammation, it is important in the function of Fcgamma receptors on alveolar macrophages.     

Contrasting roles for RANTES and macrophage inflammatory protein-1 alpha (MIP-1 alpha) in a murine model of allergic peritonitis.

Cell accumulation and CC chemokine production were assessed in the peritoneal cavity of ovalbumin (OVA)-sensitized mice following antigen challenge. Intraperitoneal challenge with OVA induced a significant eosinophil influx from 6 h post-challenge with increased numbers persisting at 24 h. At 6 h there was also a marked presence of neutrophils. Messenger RNA expression and protein levels for the chemokines RANTES and MIP-1 alpha were measured in the cell pellets and supernatants, respectively, from peritoneal washes following OVA challenge. RANTES mRNA was detected from 2 h to 4 h following OVA injection, whereas mRNA for MIP-1 alpha was only detectable at 4 h. RANTES protein was first detected from 4 h after OVA injection and by 24 h the protein levels had increased further. Basal levels of MIP-1 alpha were detected in peritoneal washes. These levels peaked at 2 h after OVA challenge and rapidly declined to basal levels by 6 h. A functional role for the chemokines was assessed using neutralizing polyclonal antibodies. Co-injection of OVA with anti-RANTES antibodies resulted in a significant inhibition of eosinophil infiltration into the cavity at 6 h and 24 h (63% and 52% inhibition, respectively) without significantly influencing the number of neutrophils present. In contrast, injection of anti-MIP-1 alpha antibodies only inhibited neutrophil migration at the 6 h time point by 44% without significantly affecting the accumulation of eosinophils. These results demonstrate an important role for RANTES in mediating eosinophil influx in allergic inflammation and a contrasting role for MIP-1 alpha in mediating neutrophil recruitment.     

内毒素脂多糖诱导培养的人脐静脉内皮细胞表达巨噬细胞炎性蛋白-1α

目的了解内毒素脂多糖是否诱导人脐静脉内皮细胞(HUVEC)表达巨噬细胞炎性蛋白-1α(MIP-1α)。方法使培养的HUVEC暴露于不同浓度的脂多糖,用地高辛标记的MIP-1αcDNA探针与HUVEC的总。RNA进行斑点杂交,并与HUVEC进行原位杂交,同时用HUVEC的总RNA与MIP-1α引物的混合物进行逆转录．聚合酶链反应(RT-PCR),以检测HUVEC的MIP-1αmRNA的表达;再者,将培养的HUVEC用MIP-1α单克隆抗体进行细胞酶联免疫吸附试验(ELISA),以检测其MIP-1α蛋白表达。结果斑点杂交显示,暴露于浓度为1μg／,ml和10μg／,ml脂多糖时HUVEcmRNA在硝酸纤维素膜上斑点的积分吸光度(A)值分别为1．490和3．310,分别为对照组(0．775)的1．97倍和4．38倍。原位杂交显示,当HUVEC暴露于浓度为1μg／,ml脂多糖后,其MIP-1α mRNA表达与对照组相比有明显增加,方差分析表明,差异有非常显著性(F=142．83,P＜0．01)。但当其暴露于10μg／ml脂多糖后,其MIP-1α mRNA表达则较低。RT-PCR显示,暴露于浓度为1、5和10μg／ml脂多糖时HUVEC的MIP-1α mRNA表达分别为对照组的1．65倍、2．86倍和1．26倍。细胞ELISA显示,各组HUVEC暴露于脂多糖后,其MIP-1α蛋白表达均明显增加,尤以5μg／ml脂多糖组最为显著。方差分析表明,差异有显著性(F=15．36,P＜0．05)。结论内毒素脂多糖可诱导培养的HUVEC表达高水平的MIP-1αmRNA和蛋白,从而在动脉内膜的单核／巨噬细胞的募集起重要作用。     

Levels of macrophage inflammatory protein 1 alpha (MIP-1 alpha) and MIP-1 beta in intervillous blood plasma samples from women with placental malaria and human immunodeficiency virus infection

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta play an important role in modulating immune responses. To understand their importance in immunity to placental malaria (PM) and in human immunodeficiency virus (HIV)-PM coinfection, we investigated levels of these chemokines in the placental intervillous blood plasma (IVB plasma) and cord blood plasma of HIV-negative PM-negative, HIV-negative PM-positive, HIV-positive PM-negative, and HIV-positive PM-positive women. Compared to HIV-negative PM-negative women, the MIP-1 beta concentration in IVB plasma was significantly elevated in HIV-negative PM-positive women and HIV-positive PM-positive women, but it was unaltered in HIV-positive PM-negative women. Also, PM-infected women, irrespective of their HIV status, had significantly higher levels of MIP-1 beta than HIV-positive PM-negative women. The MIP-1 alpha level was not altered in association with either infection. The IVB plasma levels of MIP-1 alpha and MIP-1 beta positively correlated with the cord blood plasma levels of these chemokines. As with IVB plasma, only cord plasma from PM-infected mothers had significantly elevated levels of MIP-1 beta compared to PM-negative mothers, irrespective of their HIV infection status. MIP-1 beta and MIP-1 alpha levels in PM-positive women were positively associated with parasite density and malaria pigment levels. Regardless of HIV serostatus, the IVB MIP-1 beta level was significantly lower in women with PM-associated anemia. In summary, an elevated level of MIP-1 beta was associated with PM. HIV infection did not significantly alter these two chemokine levels in IVB plasma.     

Intrapulmonary expression of macrophage inflammatory protein 1alpha (CCL3) induces neutrophil and NK cell accumulation and stimulates innate immunity in murine bacterial pneumonia

Macrophage inflammatory protein 1alpha (MIP-1alpha) (CCL3) is an important mediator of leukocyte recruitment and activation in a variety of inflammatory states, including infection. A recombinant human type 5 adenovirus containing the murine MIP-1alpha cDNA (AdMIP-1alpha) was constructed to determine the effect of transient intrapulmonary expression of MIP-1alpha on leukocyte recruitment, activation, and bacterial clearance in a murine model of Klebsiella pneumoniae pneumonia. The intratracheal administration of AdMIP-1alpha resulted in both time- and dose-dependent expression of MIP-1alpha mRNA and protein within the lung. Importantly, the intrapulmonary overexpression of MIP-1alpha resulted in a maximal 35- and 100-fold reduction in lung and blood bacterial burden, respectively, in animals cochallenged with K. pneumoniae, which was associated with a significant increase in neutrophil and activated NK cell accumulation. Furthermore, the transgenic expression of MIP-1alpha during bacterial pneumonia resulted in enhanced expression of gamma interferon mRNA, compared to that observed in Klebsiella-challenged animals pretreated with control vector. These findings indicate an important role for MIP-1alpha in the recruitment and activation of selected leukocyte populations in vivo and identify this cytokine as a potential immunoadjuvant to be employed in the setting of localized bacterial infection.     

同型半胱氨酸促进内皮细胞表达巨噬细胞炎性蛋白1α

单核细胞迁入动脉内皮下问隙是动脉粥样硬化(AS)发生的早期事件。巨噬细胞炎性蛋白1α(MIP-1α)对单核细胞有趋化作用。高同型半胱氨酸血症是AS的一个独立危险因子。我们观察同型半胱氨酸(HCY)是否诱导培养的人脐静脉内皮细胞(HUVEC)表达MIP-1α,探讨HCY在AS形成中的作用。     

Monocyte chemoattractant protein 1, macrophage inflammatory protein 1 alpha, and RANTES recruit macrophages to the kidney in a mouse model of hemolytic-uremic syndrome

The macrophage has previously been implicated in contributing to the renal inflammation associated with hemolytic-uremic syndrome (HUS). However, there is currently no in vivo model detailing the contribution of the renal macrophage to the kidney disease associated with HUS. Therefore, renal macrophage recruitment and inhibition of infiltrating renal macrophages were evaluated in an established HUS mouse model. Macrophage recruitment to the kidney was evident by immunohistochemistry 2 h after administration of purified Stx2 and peaked at 48 h postinjection. Mice administered a combination of Stx2 and lipopolysaccharide (LPS) showed increased macrophage recruitment to the kidney compared to mice treated with Stx2 or LPS alone. Monocyte chemoattractants were induced in the kidney, including monocyte chemoattractant protein 1 (MCP-1/CCL2), macrophage inflammatory protein 1alpha (MIP-1alpha/CCL3), and RANTES (CCL5), in a pattern that was coincident with macrophage infiltration as indicated by immunohistochemistry, protein, and RNA analyses. MCP-1 was the most abundant chemokine, MIP-1alpha was the least abundant, and RANTES levels were intermediate. Mice treated with MCP-1, MIP-1alpha, and RANTES neutralizing antibodies had a significant decrease in Stx2 plus LPS-induced macrophage accumulation in the kidney, indicating that these chemokines are required for macrophage recruitment. Furthermore, mice exposed to these three neutralizing antibodies had decreased fibrin deposition in their kidneys, implying that macrophages contribute to the renal damage associated with HUS.     

A macrophage inflammatory protein homolog encoded by guinea pig cytomegalovirus signals via CC chemokine receptor 1

Cytomegaloviruses encode homologs of cellular immune effector proteins, including chemokines (CKs) and CK receptor-like G protein-coupled receptors (GPCRs). Sequence of the guinea pig cytomegalovirus (GPCMV) genome identified an open reading frame (ORF) which predicted a 101 amino acid (aa) protein with homology to the macrophage inflammatory protein (MIP) subfamily of CC (β) CKs, designated GPCMV-MIP. To assess functionality of this CK, recombinant GPCMV-MIP was expressed in HEK293 cells and assayed for its ability to bind to and functionally interact with a variety of GPCRs. Specific signaling was observed with the hCCR1 receptor, which could be blocked with hMIP −1α in competition experiments. Migration assays revealed that GPCMV-MIP was able to induce chemotaxis in hCCR1-L1.2 cells. Antisera raised against a GST-MIP fusion protein immunoprecipitated species of ∼12 and 10 kDa from GPCMV-inoculated tissue culture lysates, and convalescent antiserum from GPCMV-infected animals was immunoreactive with GST-MIP by ELISA assay. These results represent the first substantive in vitro characterization of a functional CC CK encoded by a cytomegalovirus.     

同型半胱氨酸对单核细胞NF-κB活化及巨噬细胞炎性蛋白-1α表达的影响

目的　探讨同型半胱氨酸对体外培养的人单核细胞株THP -1核因子-κB(NF -κB)、抑制因子(IκB- α)活性的影响及其与巨噬细胞炎性蛋白-1α(MIP- 1α)表达上调的关系。方法　THP -1单核细胞分别用同型半胱氨酸和NF- κB抑制剂PDTC预处理后,应用Northernblot和流式细胞术分别检测MIP- 1αmRNA和蛋白的表达,并应用Western蛋白印迹进一步检测核蛋白NF -κB蛋白含量和胞质IκB -α含量。结果　与未加任何处理因素的对照组比较,MIP- 1αmRNA和蛋白在0. 1mmol/L同型半胱氨酸处理后明显增加,分别为对照组的3 .69倍和1 .16倍(P<0 .01),同时NF κBP65亚基核转位亦增加。而加入100μmol/LNF κB抑制剂PDTC预处理30min后,再用同样浓度的同型半胱氨酸刺激,则MIP -1αmRNA和蛋白表达受到明显的抑制,NF- κBP65核转位增加。单独加入PDTC对MIP -1αmRNA、蛋白表达及NF -κBP65核转位无明显影响。此外,同型半胱氨酸( 0 .1mmol/L)处理THP- 1可引起IκB -α蛋白水平显著降低, 120min后有所回升。结论　同型半胱氨酸在病理浓度可促进NF -κB活化,发生核转位,进而促进THP -1细胞表达MIP 1αmRNA和蛋白,这种作用与IκB -α蛋白磷酸化降解有关。     

Endotoxin induces the expression of macrophage inflammatory protein 1 alpha mRNA by rat alveolar and bone marrow-derived macrophages.

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is a newly described cytokine that is present in large amounts in the culture supernatant of an endotoxin-stimulated murine macrophage-like cell line (RAW 264.7). There is increasing information that suggests that this cytokine mediates acute neutrophilic inflammation, although the mechanism of mediation is unknown. Data examining the production and regulation of MIP-1 alpha by primary rat macrophages are lacking, and MIP-1 alpha has not been studied previously in an animal model of endotoxin-induced neutrophilic alveolitis. In this study, we performed Northern analysis of steady-state rat MIP-1 alpha mRNA using an oligonucleotide probe complementary to amino acids 4-13 of murine MIP-1 alpha. Our data demonstrate that rat alveolar and bone marrow-derived macrophages can be induced by in vitro endotoxin treatment to express a 1.1-kb MIP-1 alpha mRNA. Expression of the mRNA could be elicited by treatment with 0.1 to 10.0 micrograms/ml of endotoxin in vitro with peak steady-state levels detectable up to 9 h after adding endotoxin to the media. Alveolar macrophages recovered by whole lung lavage from endotoxin-treated rats expressed increased amounts of the mRNA homologous to MIP-1 alpha mRNA when treated in vitro with endotoxin. We also found that rat neutrophils could be induced by endotoxin in vitro to express the MIP-1 alpha mRNA. We were able to identify MIP-1 alpha in culture supernatant from endotoxin-stimulated rat alveolar and bone marrow-derived macrophages by immunoprecipitation with a specific goat anti-murine MIP-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rabies viruses infect primary cultures of murine, feline, and human microglia and astrocytes

Summary.  Recent studies have reported the detection of rabies viral antigens and virions in astrocytes and microglia of rabies-infected animals. As a first step toward understanding whether these glial cells may be involved in rabies virus replication, persistence, and/or pathogenesis, we explored their potential to be infected in vitro. Primary cultures of murine, feline, and human microglia and astrocytes were infected with several different rabies viruses: two unpassaged street virus isolates, a cell culture-adapted strain, and a mouse brain-passaged strain. Infection, as determined by immunofluorescence, was detected in 15 of the 16 (94%) virus-glial cell combinations. Replication of infectious virus, determined by infectivity assay, was detected in 7 of the 8 (88%) virus-cell combinations. These results show that astrocytes and microglia can be infected by rabies viruses, suggesting that they may have a potential role in disease, perhaps contributing to viral spread, persistence and/or neuronal dysfunction. Accepted November 19, 1996 Received October 21, 1996     

Macrophage chemotactic protein-1 and macrophage inflammatory protein-1 alpha induce nitric oxide release and enhance parasite killing in Leishmania infantum-infected human macrophages

Chemokines are a group of structurally defined small proteins that act as chemoattractants for leukocytes and are involved in many different biological activities, including leukocyte activation for antimicrobial mechanisms. We studied the effect of the chemokines monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α on nitric oxide release and parasitocidal ability of peripheral blood-derived human macrophages in vitro infected with Leishmania infantum, zymodeme MON1. In infected human macrophages, treatment with MCP-1 or MIP-1α significantly enhanced nitric oxide production and leishmanicidal ability, compared with untreated cells, to the same levels induced by interferon-γ. Both nitric oxide release and parasitocidal ability of macrophages were significantly reduced by addition of L-N ^Gmonomethylarginine (L-NMMA), which is a competitive inhibitor of the L-arginine nitric oxide pathway. These data suggest that MCP-1 and MIP-1α mediate macrophage activation for nitric oxide release and subsequent parasite clearance, and thus may play a role in the containment of Leishmania infection. Received: 9 November 2001 / Accepted: 16 September 2002 Correspondence to O. Brandoniso     

Elevated levels of macrophage inflammatory protein 2 in severe murine peritonitis increase neutrophil recruitment and mortality.

We hypothesized that chemokines may play important roles in a cecal ligation and puncture (CLP) model of septic peritonitis in CD-1 mice. Concentrations of C-X-C (macrophage inflammatory protein 2 [MIP-2] and ENA-78) and C-C (MIP-1alpha and JE) chemokines were measured (by enzyme-linked immunosorbent assay) in serum, peritoneal lavage fluid, lung, and liver at 4, 8, 24, 48, and 96 h after CLP. Significant elevations in all measured chemokines occurred in peritoneal fluid after CLP (P < 0.05). MIP-2, in particular, increased dramatically (>400-fold, P < 0.001) in peritoneal fluid, serum, and to a lesser extent lung and liver (P < 0.05). Increased MIP-2 was correlated with severity of sepsis (P < 0.001). To determine the significance of this finding, mice were passively immunized prior to CLP with polyclonal antibody to MIP-2, which decreased mortality from 85 to 38% at 96 h (P < 0.01). To further understand the mechanism of the effect of MIP-2, additional measurements demonstrated that anti-MIP-2 prior to CLP decreased the percent neutrophils in peritoneal fluid (55% +/- 12%, compared with 82% +/- 10% in controls), but no significant changes in tumor necrosis factor alpha, interleukin-6, or interleukin-10 occurred. MIP-2 contributes to the inflammatory response and overall mortality in this model of severe septic peritonitis, possibly by increasing recruitment of neutrophils, which clear bacteria but may also injure the host.