尼古丁通过调节巨噬细胞极化加重实验性脉络膜新生血管的形成

目的:探究巨噬细胞在尼古丁加重脉络膜新生血管(CNV)形成中的作用。方法:饮用尼古丁水溶液(100 mg/L)4周后,使用激光诱导小鼠CNV模型,7 d后用异硫氰酸荧光素标记的葡聚糖心脏灌注,测量CNV面积。分别于激光后1、3、7和14 d采用免疫荧光法检测巨噬细胞的时间和空间分布。采用RT-qPCR检测巨噬细胞相关分子标志物的mRNA表达。同时采用ELISA法检测激光后第1和3天视网膜色素上皮-脉络膜-巩膜组织中血管内皮生长因子(VEGF)、细胞间黏附分子1(ICAM-1)、肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的表达。结果:尼古丁显著增加CNV的渗漏程度及CNV的面积,尼古丁组的重度渗漏率和CNV面积分别为56.25%和(17 569.96±1 444.00)μm²,显著高于对照组的31.25%和(10 158.63±711.00)μm²(P<0.05)。尼古丁促进了M2型巨噬细胞的浸润,增加了M2/M1型巨噬细胞的比率(P<0.05)。尼古丁也促进了M2型巨噬细胞相关分子标志物的mRNA表达,并且促进了VEG...     

CCR2 regulates development of Theiler's murine encephalomyelitis virus-induced demyelinating disease

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a murine model for multiple sclerosis, involves recruitment of T cells and macrophages to the CNS after infection. We hypothesized that CCR2, the only known receptor for CCL2, would be required for TMEV-induced demyelinating disease development because of its role in macrophage recruitment. TMEV-infected SJL CCR2 knockout (KO) mice showed decreased long-term clinical disease severity and less demyelination compared with controls. Flow cytometric data indicated that macrophages (CD45(high) CD11b(+) ) in the CNS of TMEV-infected CCR2 KO mice were decreased compared with control mice throughout disease. CD4(+) and CD8(+) T cell percentages in the CNS of TMEV-infected control and CCR2 KO mice were similar over the course of disease. There were no apparent differences between CCR2 KO and control peripheral immune responses. The frequency of interferon-gamma-producing T cells in response to proteolipid protein 139-151 in the CNS was also similar during the autoimmunity stage of TMEV-induced demyelinating disease. These data suggest that CCR2 is important for development of clinical disease by regulating macrophage accumulation after TMEV infection.     

Leishmania-derived murine monocyte chemoattractant protein 1 enhances the recruitment of a restrictive population of CC chemokine receptor 2-positive macrophages

Transgenic Leishmania parasites that encode the murine chemokine monocyte chemoattractant protein 1 (MCP-1) were generated. These parasites transcribed MCP-1 mRNA and secreted MCP-1 protein. Infection of BALB/c, C57BL/6, or MCP-1 knockout (KO) mice with these parasites resulted in minimal lesion development with fewer parasites in the infected foot, lymph node, and spleen compared to wild-type-infected mice. In contrast, transgenic parasites caused substantial lesions with relatively high numbers of parasites in CC chemokine receptor 2 (CCR2) KO mice, indicating that the parasites are viable and healthy and that the lack of lesion development is CCR2 dependent. Prior infection of mice with transgenic parasites offered no protection to subsequent wild-type L. major challenge, suggesting that the transgenic parasites are controlled by an early innate immune response. Consistent with innate immunity, flow cytometry of cells from the ears of mice infected with transgenic parasites revealed an increase in the number of CCR2-positive macrophages by day 7 postinfection. The enumeration of transgenic parasites in ear lesions demonstrated a significant reduction in parasite numbers, which coincided with the increased CCR2-positive macrophage migration. CCR2-positive macrophages isolated from ears of mice infected with transgenic parasites contained virtually no parasites. In vitro studies revealed that optimal parasite killing required the recruitment of CCR2-positive macrophages, followed by stimulation with a combination of both MCP-1 and gamma interferon (IFN-gamma). This work suggests that the parasite-derived MCP-1 can recruit a restrictive population of CCR2-positive macrophages into lesions that can be optimally stimulated by MCP-1 and IFN-gamma to efficiently kill Leishmania parasites.     

Monocyte chemoattractant protein 1 contributes to an adequate immune response in influenza pneumonia

Monocyte chemoattractant protein 1 (MCP-1) and its receptor CCR2 have been shown to play an import role in leukocyte recruitment to sites of infection and inflammation. To investigate the role of MCP-1 during infection with influenza we inoculated wild-type (WT) and MCP-1 knockout (KO) mice with a non-lethal dose of a mouse adapted strain of influenza A. Influenza infection of WT mice resulted in a profound increase in pulmonary MCP-1 levels. MCP-1 KO mice had enhanced weight loss and did not fully regain their body weight during the 14-day observation period. In addition, MCP-1 KO mice demonstrated elevated viral loads 8 days after infection, which was accompanied by reduced leukocyte recruitment into the infected lungs, primarily caused by a diminished influx of macrophages and granulocytes. Moreover, pulmonary levels of IgA were reduced in MCP-1 KO mice. The pulmonary concentrations of tumor necrosis factor-alpha, interleukin-6, macrophage inflammatory protein 2 and interferon-gamma were higher in MCP-1 KO mice. This study shows that MCP-1 contributes to an adequate protective immune response against influenza infection in mice.     

Caveolin-1 knockout mice show an impaired angiogenic response to exogenous stimuli

Recent studies have shown that caveolin-1 (Cav-1) plays an important role as a regulator of angiogenesis in vitro. Here, we use Cav-1 knockout (KO) mice as a model system to examine the in vivo relevance of these findings. A primary mediator of angiogenesis is basic fibroblast growth factor (bFGF). Thus, we studied bFGF-induced angiogenesis in Cav-1 KO mice using a reconstituted basement membrane system, ie, Matrigel plugs, supplemented with bFGF. In Cav-1 KO mice, implanted Matrigel plugs showed a dramatic reduction in both vessel infiltration and density, as compared with identical plugs implanted in wild-type control mice. We also examined the necessity of Cav-1 to support the angiogenic response of an exogenous tumor by subcutaneously injecting Cav-1 KO mice with the melanoma cell line, B16-F10. We show that tumor weight, volume, and vessel density are all reduced in Cav-1 KO mice, consistent with diminished angiogenesis. Ultrastructural analysis of newly formed capillaries within the exogenous tumors reveals a lack of endothelial caveolae and incomplete capillary formation in Cav-1 KO mice. These results provide novel evidence that Cav-1 and caveolae play an important positive role in the process of pathological angiogenesis in vivo.     

Are the protective effects of 17beta-estradiol on splenic macrophages and splenocytes after trauma-hemorrhage mediated via estrogen-receptor (ER)-alpha or ER-beta?

The depression in cell-mediated immune function following trauma-hemorrhage is shown to be restored by 17beta-estradiol (E2) administration. However, it remains unknown which of the two estrogen-receptors, (ER)-alpha or ER-beta, plays the predominant role in mediating the beneficial effects of E2. Female B57BL/J6 ER-beta(-/-) transgenic mice [knockout (KO)] and corresponding ovariectomized wild-type (WT) mice were subjected to laparotomy and hemorrhagic shock (35.0+/-5.0 mmHg for 90 min) and treated with E2 (50 microg/25 g) or ER-alpha agonist propyl pyrazole triol (PPT; 50 microg/25 g) following trauma-hemorrhage. Four hours after resuscitation, systemic cytokine concentrations and cytokine release by splenocytes and splenic macrophages were determined by cytometric bead array. Trauma-hemorrhage resulted in a significant increase in plasma tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, and IL-10. In contrast, the release of these cytokines by splenic macrophages was decreased significantly in WT and KO animals. Administration of E2 or PPT following trauma-hemorrhage produced a significant reduction in systemic TNF-alpha and IL-6 concentrations in WT and KO mice. Although the suppression in the productive capacity of these cytokines following trauma-hemorrhage by macrophages and splenocyte was also prevented in E2- and PPT-treated WT mice, the release of cytokines by macrophages and splenocytes in E2- and PPT-treated KO mice was not restored to the levels observed in sham animals. These findings collectively suggest that both receptors appear to play a significant role in mediating the immunoprotective effects of E2 in different tissue compartments following trauma-hemorrhage.     

Wound healing is impaired in MyD88-deficient mice: a role for MyD88 in the regulation of wound healing by adenosine A2A receptors

Synergy between Toll-like receptor (TLR) and adenosine A2A receptor (A2AR) signaling switches macrophages from production of inflammatory cytokines such as tumor necrosis factor-alpha to production of the angiogenic growth factor vascular endothelial growth factor (VEGF). We show in this study that this switch critically requires signaling through MyD88, IRAK4, and TRAF6. Macrophages from mice lacking MyD88 (MyD88(-/-)) or IRAK4 (IRAK4(-/-)) lacked responsiveness to TLR agonists and did not respond to A2AR agonists by expressing VEGF. Suppression of TRAF6 expression with siRNA in RAW264.7 macrophages also blocked their response to TLR and A2AR agonists. Excisional skin wounds in MyD88(-/-) mice healed at a markedly slower rate than wounds in wild-type MyD88(+/+) mice, showing delayed contraction, decreased and delayed granulation tissue formation, and reduced new blood vessel density. Although macrophages accumulated to higher levels in MyD88(-/-) wounds than in controls, expression of VEGF and HIF1-alpha mRNAs was elevated in MyD88(+/+) wounds. CGS21680, an A2AR agonist, promoted repair in MyD88(+/+) wounds and stimulated angiogenesis but had no significant effect on healing of MyD88(-/-) wounds. These results suggest that the synergistic interaction between TLR and A(2A)R signaling observed in vitro that switches macrophages from an inflammatory to an angiogenic phenotype also plays a role in wound healing in vivo.     

Role of B7 costimulatory molecules in mediating systemic and mucosal antibody responses to attenuated Salmonella enterica serovar Typhimurium and its cloned antigen

The purpose of the present study was to evaluate the ability of an attenuated Salmonella enterica serovar Typhimurium vaccine strain to up-regulate B7-1 and B7-2 on antigen-presenting cells and to examine the functional roles these costimulatory molecules play in mediating immune responses to Salmonella and to an expressed cloned antigen, the saliva-binding region (SBR) of antigen I/II. In vitro stimulation of B cells (B220+), macrophages (CD11b+), and dendritic cells (CD11c+) with S. enterica serovar Typhimurium induced an up-regulation of B7-2 and, especially, B7-1 expression. The in vivo functional roles of B7-1, B7-2, and B7-1/2 were evaluated in BALB/c wild-type and B7-1, B7-2, and B7-1/2 knockout (KO) mice following intranasal immunization with the Salmonella expressing the cloned SBR. Differential requirements for B7-1 and B7-2 were observed upon primary and secondary immunizations. Compared to wild-type controls, B7-1 and B7-2 KO mice had reduced mucosal and systemic anti-Salmonella antibody responses after a single immunization, while only B7-1 KO mice exhibited suppressed anti-Salmonella antibody responses following the second immunization. Mucosal and systemic antibody responses to SBR were reduced following the primary immunization, whereas a compensatory role for either B7-1 or B7-2 was observed after the second immunization. B7-1/2 double KO mice failed to induce detectable levels of mucosal or systemic immunoglobulin A (IgA) or IgG antibody responses to either Salmonella or SBR. These findings demonstrate that B7-1 and B7-2 can play distinct as well as redundant roles for mediating mucosal and systemic antibody responses, which are likely dependent upon the nature of the antigen.     

Macrophage Inflammatory Protein 1α/CCL3 Is Required for Clearance of an Acute Klebsiella pneumoniae Pulmonary Infection

The objective of these studies was to determine the role of macrophage inflammatory protein 1alpha/CCL3 in pulmonary host defense during Klebsiella pneumoniae infection. Following intratracheal inoculation, 7-day survival of CCL3(-/-) mice was less than 10%, compared to 60% for CCL3(+/+) mice. Survival of CCR5(-/-) mice was equivalent to that of controls, indicating that the enhanced susceptibility of CCL3(-/-) mice to K. pneumoniae is mediated via another CCL3 receptor, presumably CCR1. At day 3, CFU burden in the lungs of CCL3(-/-) mice was 800-fold higher than in CCL3(+/+) mice, demonstrating that CCL3 is critical for control of bacterial growth in the lung. Surprisingly, CCL3(-/-) mice had no differences in the recruitment of monocytes/macrophages and even showed enhanced neutrophil recruitment at days 1, 2, and 3 postinfection, compared to CCL3(+/+) mice. Therefore, the defect in clearance was not due to insufficient recruitment of leukocytes. No significant differences in cytokine levels of monocyte chemoattractant protein 1 (MCP-1), interleukin 12, gamma interferon, or tumor necrosis factor alpha in lung lavages were found between CCL3(+/+) and CCL3(-/-) mice. CCL3(-/-) alveolar macrophages were found to have significantly lower phagocytic activity toward K. pneumoniae than CCL3(+/+) alveolar macrophages. These findings demonstrate that CCL3 production is critical for activation of alveolar macrophages to control the pulmonary growth of the gram-negative bacterium K. pneumoniae.     

Loss of tumor necrosis factor alpha potentiates transforming growth factor beta-mediated pathogenic tissue response during wound healing

Animal cornea is an avascular transparent tissue that is suitable for research on wound healing-related scarring and neovascularization. Here we show that loss of tumor necrosis factor alpha (TNFalpha) potentiates the undesirable, pathogenic response of wound healing in an alkali-burned cornea in mice. Excessive invasion of macrophages and subsequent formation of a vascularized scar tissue were much more marked in TNFalpha-null knockout (KO) mice than in wild-type mice. Such an unfavorable outcome in KO mice was abolished by Smad7 gene introduction, indicating the involvement of transforming growth factor beta or activin/Smad signaling. Bone marrow transplantation from wild-type mice normalized healing of the KO mice, suggesting the involvement of bone marrow-derived inflammatory cells in this phenomenon. Co-culture experiments showed that loss of TNFalpha in macrophages, but not in fibroblasts, augmented the fibroblast activation as determined by detection of alpha-smooth muscle actin, the hallmark of myofibroblast generation, mRNA expression of collagen Ialpha2 and connective tissue growth factor, and detection of collagen protein. TNFalpha in macrophages may be required to suppress undesirable excessive inflammation and scarring, both of which are promoted by transforming growth factor beta, and for restoration of tissue architecture in a healing alkali-burned cornea in mice.     

Chemokine regulation of the inflammatory response to a low-dose influenza infection in CCR2-/- mice

Influenza virus infections induce chemokines and cytokines, which regulate the immune response. The chemokine receptor CCR2 plays an important role in macrophage recruitment and in the development of T1 immunity. In the present study, we addressed the role of CCR2 in influenza A virus infection. CCR2 knockout (-/-) mice are protected against influenza A virus infection, despite delayed recruitment of macrophages. We show that low-dose influenza infection of CCR2-/- mice leads to increased neutrophilia between Days 5 and 10 after infection and decreased monocyte/macrophage and CD4(+) T cell recruitment to the lungs between Days 5 and 7 after infection. These changes in leukocyte recruitment did not result from or cause increased viral titers or delayed viral clearance. Neutrophilia in the lungs correlated with increased keratinocyte-derived chemokine (KC) and/or MIP-2 expression in CCR2-/- mice between Days 5 to 10 after infection, although the kinetics of neutrophil recruitment was not altered. MIP-2 mRNA and protein expression was increased three- to fivefold, and KC protein levels were increased two- to threefold in CCR2-/- compared with CCR2 wild-type mice at Day 5 after infection. This preceded the peak neutrophil influx, which occurred 7 days after infection. In vitro studies confirmed that MIP-2 and KC accounted for neutrophil chemotactic activity in the bronchoalveolar lavage. CCR2 deficiency also resulted in increased MIP-1alpha, MIP-1beta, MCP-1, and IFN-inducible protein 10 and decreased RANTES mRNA expression. Furthermore, IL-6 and TNF-alpha cytokine production were elevated after infection. These studies suggest that CCR2 plays a multifactorial role in the development of the immune response to influenza.     

CCR8 signaling influences Toll-like receptor 4 responses in human macrophages in inflammatory diseases

CCR8 immunity is generally associated with Th2 responses in allergic diseases. In this study, we demonstrate for the first time a pronounced attenuated influx of macrophages in ovalbumin (OVA)-challenged CCR8 knockout mice. To explore whether macrophages in human inflamed lung tissue also were CCR8 positive, human lung tissue from patients with chronic obstructive pulmonary disease (COPD) was evaluated. Indeed, CCR8 expression was pronounced in invading monocytes/macrophages from lungs of patients with Global Initiative for Obstructive Lung Disease (GOLD) stage IV COPD. Given this expression pattern, the functional role of CCR8 on human macrophages was evaluated in vitro. Human peripheral blood monocytes expressed low levels of CCR8, while macrophage colony-stimulating factor (M-CSF)-derived human macrophages expressed significantly elevated surface levels of CCR8. Importantly, CCL1 directly regulated the expression of CD18 and CD49b and hence influenced the adhesion capacity of human macrophages. CCL1 drives chemotaxis in M-CSF-derived macrophages, and this could be completely inhibited by lipopolysaccharide (LPS). Whereas both CCL1 and LPS monotreatment inhibited spontaneous superoxide release in macrophages, CCL1 significantly induced superoxide release in the presence of LPS in a dose-dependent manner. Finally, CCL1 induced production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) and could inhibit LPS-induced cytokine production in a dose-dependent manner. Our data demonstrate, for the first time, the presence of CCR8 on inflammatory macrophages in human COPD lung tissue. Importantly, the functional data from human macrophages suggest a potential cross talk between the CCR8 and the Toll-like receptor 4 (TLR4) pathways, both of which are present in COPD patients.     

Distinct and overlapping roles of CXCR5 and CCR7 in B-1 cell homing and early immunity against bacterial pathogens

CXC chemokine receptor (CXCR)5 and CC chemokine receptor (CCR)7 are the major chemokine receptors required for B cell homing and microenvironmental localization during antigen-independent and -dependent B cell differentiation. Here, we show markedly decreased B-1 B cell numbers in the peritoneal cavity of CXCR5-/- and CXCR5-/-CCR7-/- double-deficient mice paralleled by reduced antigen-induced phosphorylcholine-specific immunoglobulin (Ig)M responses after intraperitoneal (i.p.) administration of streptococcal antigen. CCR7-/- mice also revealed a partial reduction in peritoneal B-1 cell numbers combined with a reduced humoral response to i.p. injected bacterial antigen. However, opposite roles of CXCR5 and CCR7 were observed when the frequency of peritoneal B-2 cells was analyzed. CXCR5-/- mice almost completely lacked B-2 cells, whereas CCR7 deficiency engendered an increase in peritoneal B-2 cells. In addition, CCR7-/- mice had enhanced, splenic IgM+ plasma cell responses, whereas the extrafollicular B cell response of the CXCR5-/- mice was not significantly altered compared with wild-type controls. Thus, the two chemokine receptors exert divergent forces at multiple levels of the innate immune response. CXCR5 plays a dominant role in peritoneal B-1 B cell homing and body cavity immunity, but both chemokine receptors are needed for a proportional peritoneal B-2 cell homing and balanced development of an early splenic B cell response.     

(Pro)renin receptor promotes choroidal neovascularization by activating its signal transduction and tissue renin-angiotensin system

The receptor-associated prorenin system (RAPS) refers to pathogenic mechanisms whereby prorenin binding to its receptor activates both the tissue renin-angiotensin system (RAS) and RAS-independent intracellular signaling pathways. Although we found significant involvement of angiotensin II type 1 receptor (AT1-R)-mediated inflammation in choroidal neovascularization (CNV), a central abnormality of vision-threatening age-related macular degeneration, the association of receptor-associated prorenin system with CNV has not been defined. Here, (pro)renin receptor blockade in a murine model of laser-induced CNV led to the significant suppression of CNV together with macrophage infiltration and the up-regulation of intercellular adhesion molecule-1, (ICAM-1) monocyte chemotactic protein-1, (MCP-1) vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1, and VEGFR-2. To clarify the role of signal transduction via the (pro)renin receptor in CNV, we used mice in which renin-angiotensin system was deactivated by either the pharmacological blockade of AT1-R with losartan or the genetic ablation of AT1-R or angiotensinogen. Compared with wild-type controls, these mice exhibited significant reduction of CNV and macrophage infiltration, both of which were further suppressed by (pro)renin receptor blockade. The (pro)renin receptor and phosphorylated extracellular signal-regulated kinases (ERK) were co-localized in vascular endothelial cells and macrophages in CNV. (Pro)renin receptor blockade suppressed ERK activation and the production of MCP-1 and VEGF, but not ICAM-1, VEGFR-1, or VEGFR-2, in AT1-R-deficient mice with CNV and in losartan-treated microvascular endothelial cells and macrophages. These results indicate the significant contribution of RAPS to CNV pathogenesis.     

Neither Dectin-2 nor the Mannose Receptor Is Required for Resistance to Coccidioides immitis in Mice

We investigated the roles of the mannose receptor (MR) and Dectin-2 in resistance to pulmonary coccidioidomycosis in C57BL/6 (B6) mice and in the interaction of myeloid cells with spherules, using B6 mice with targeted mutations in Mrc1 and Clec4n. Spherules are the tissue form of Coccidioides, and we determined that the MR on bone marrow-derived dendritic cells (BMDC) was important for recognition of spherules (formalin-killed spherules [FKS]) and for secretion of interleukin 10 (IL-10) and proinflammatory cytokines in response to FKS by both elicited macrophages and BMDC. Infected MR knockout (KO) mice produced more IL-10 in their lungs than did B6 mice, and MR KO mice also made more protective Th-17 cytokines. In contrast to the MR, Dectin-2 was not required for recognition of FKS by BMDC or for the production of cytokines by BMDC in response to FKS. However, Dectin-2 KO was required for stimulation of elicited peritoneal macrophages. Despite that, lung cytokine levels were not significantly different in Dectin-2 KO mice and B6 mice 14 days after infection, except for IL-1β, which was higher in Dectin-2 KO lungs. Although both Dectin-2^−/− and MR^−/− myeloid cells had reduced proinflammatory cytokine responses to FKS in vitro, neither MR nor Dectin-2 deficiency reduced the resistance of B6 mice to pulmonary coccidioidomycosis.     

Mycobacterium tuberculosis in chemokine receptor 2-deficient mice: influence of dose on disease progression

Within a Mycobacterium tuberculosis-induced granuloma, lymphocytes and macrophages work together to control bacterial growth and limit the spread of infection. Chemokines and chemokine receptors are involved in cell migration and are logical candidates for a role in granuloma formation. In the present study we addressed the role of CC chemokine receptor 2 (CCR2) in M. tuberculosis infection. In previous studies (W. Peters et al., Proc. Natl. Acad. Sci. USA 98:7958-7963, 2001), CCR2(-/-) mice were found to be highly susceptible to a moderate or high dose of H37Rv administered intravenously (i.v.). We have expanded those studies to demonstrate that the susceptibility of CCR2(-/-) mice is dose dependent. After low-dose aerosol or i.v. infection of CCR2(-/-) mice with M. tuberculosis, there was a substantial delay in cell migration to the lungs and delayed expression of gamma interferon and inducible nitric oxide synthase. The CCR2(-/-) mice had a severe and prolonged deficiency in the number of macrophages in the lungs and an early increase in the number of neutrophils. Despite these deficiencies in cell migration, the CCR2(-/-) mice did not have increased bacterial loads in the lungs compared to wild-type (C57BL/6) mice and successfully formed granulomas. This finding is in contrast to CCR2(-/-) mice infected with a high dose of M. tuberculosis administered i.v. These results indicate that with low-dose infection, a delay in immune response in the lungs does not necessarily have detrimental long-term effects on the progression of the disease. The fact that CCR2(-/-) mice survive with substantially fewer macrophages in the low-dose models implies that the immune response to low-dose M. tuberculosis infection in mice is more robust than necessary to control the infection. Finally, these data demonstrate that, in cases of infectious disease in knockout models, clear phenotypes may not be evident when one is solely evaluating bacterial numbers and survival. Functional assays may be necessary to reveal roles for components of the multifactorial immune system.