Differential chemokine response of murine macrophages stimulated with cytokines and infected with Listeria monocytogenes

During inflammatory processes the infected macrophage is a rich source of chemokines which induce infiltration of leukocytes to the site of infection. We investigated the regulation of chemokine production by murine macrophages in response to infection with the intracellular bacterial pathogen, Listeria monocytogenes. As a source of quiescent macrophages, murine bone marrow-derived macrophages (BMM) cultured under serum-free conditions were used. With RT-PCR, we detected induction of RNA message for the chemokines macrophage inflammatory protein (MIP)-2, KC, MIP-1alpha, MIP-1beta, IFN-gamma-inducible protein- 10 and RANTES in L. monocytogenes-infected macrophages. Accordingly, ELISA-detectable MIP-1alpha, MIP-2 and KC protein was induced by infection with L. monocytogenes. In contrast, L. monocytogenes infection of BMM alone failed to induce considerable expression of monocyte chemoattractant protein (MCP)-1 at the mRNA or protein level, but co-treatment with IFN-gamma was necessary. Release of infection- triggered MIP-2, MIP-1alpha and KC was negatively regulated by IFN- gamma. Similarly, IL-4 stimulated MCP-1 release by infected macrophages but reduced production of MIP-1alpha, MIP-2 and KC. IL-10 turned out to be a general deactivator in terms of macrophage chemokine production. IL-13 had no effect on MIP-1alpha, MIP-2 and KC production by infected BMM, but slightly reduced MCP-1 release. By using IFN-gamma and IL-4 gene deletion mutant mice, in vivo regulation of these chemokines by IL- 4 and IFN-gamma in listeriosis was studied. In summary, our results show that chemokines are produced by macrophages infected with L. monocytogenes, and that chemokine release is differentially regulated by the macrophage modulators IFN-gamma, IL-4, IL-10 and IL-13.      

Chemokine production and leukocyte recruitment to the lungs of Paracoccidioides brasiliensis-infected mice is modulated by interferon-gamma

Chemokines and chemokine receptors play a role in cell recruitment during granulomatous inflammatory reactions. Here, we evaluated the expression of chemokines and chemokine receptors and their regulation by IFN-gamma in the course of Paracoccidioides brasiliensis (Pb) infection in mice. We found an association between KC and MIP-1alpha (CCL3) production and neutrophil infiltration in the lungs of Pb-infected mice during the early acute phase of infection. High levels of RANTES/CCL5, MCP-1/CCL2, IP-10/CXCL10, and Mig/CXCL9 simultaneously with mononuclear cell infiltration in the lungs was found. In the absence of IFN-gamma (GKO mice) we observed increased production of KC and MIP-1alpha and chronic neutrophilia. Moreover, we found a change in the chemokine receptor profiles expressed by wild-type (WT) versus GKO animals. Increased expression of CXCR3 and CCR5, and low levels of CCR3 and CCR4 were observed in the lungs of Pb-infected WT mice, whereas the opposite effect was observed in the lungs of GKO mice. Consistent with these results, infected cells from WT mice preferentially migrated in response to IP-10 (CXCR3 ligand), while those from GKO mice migrated in response to eotaxin/CCL11 (CCR3 ligand). These results suggest that IFN-gamma modulates the expression of chemokines and chemokine receptors as well as the kind of cells that infiltrate the lungs of Pb-infected mice.     

Mouse Strain-Dependent Chemokine Regulation of the Genital Tract T Helper Cell Type 1 Immune Response

Vaginal infection with the mouse pneumonitis agent of Chlamydia trachomatis (MoPn) produces shorter courses of infection in C57BL/6 and BALB/c mice than in C3H/HeN mice, while C57BL/6 mice are more resistant to oviduct pathology. A robust Th1 response is extremely important in host defense against chlamydia. In this study we examined gamma interferon (IFN-gamma), interleukin 10 (IL-10), and the T-cell-regulatory chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemoattractant protein-1 (MCP-1) to determine if differences in these responses were associated with the differential courses of infection seen in these three strains of mice. Increased and prolonged IFN-gamma responses and lower IL-10 responses were observed in the C57BL/6 strain compared to BALB/c and C3H. Examination of genital tract chemokines revealed a marked predominance of MIP-1alpha over MCP-1 only in the C57 strain. Thus, a pattern of high MIP-1alpha and low MCP-1 levels during the first week of infection is associated with an increased Th1 response and a shorter, more benign chlamydial infection. Inhibition of the MCP-1 response in C3H mice increased their later T-cell production of IFN-gamma but decreased their early IFN-gamma response and had no effect on the course or outcome of infection. Inhibition of MCP-1 is not beneficial in chlamydial infection because of its pleiotropic effects.     

Interleukin-4 and IFN-gamma differentially stimulate macrophage chemoattractant protein-1 (MCP-1) and eotaxin production by intestinal epithelial cells.

When the intestine becomes infected by pathogenic organisms, intestinal epithelial cells (IEC) respond with the production of chemokines, which then attract and activate specific subsets of leukocytes. During chronic inflammation, the panel of IEC chemokines produced likely represents the net effect of a plethora of mediators present in the milieu, including cytokines from activated T lymphocytes. To explore the influence of T lymphocyte cytokines, we treated IEC-18 cells with interferon-y (IFN-gamma) and interleukin-4 (IL-4) and measured the effect on production of the CC chemokines, monocyte chemoattractant protein-1 (MCP-1) and eotaxin, and the CXC chemokine, macrophage inflammatory protein-2 (MIP-2). Both IFN-gamma and IL-4 enhanced MCP-1 mRNA levels but with different kinetics. IFN-gamma stimulated a transient increase in MCP-1 mRNA levels, which peaked at 2 h, whereas IL-4-stimulated MCP-1 mRNA levels were markedly increased at 1 h and remained elevated at all time points studied. With each stimulus, the increase in MCP-1 mRNA levels was accompanied by a steady time-dependent increase in MCP-1 secretion. In addition, treatment with IFN-gamma or IL-4 enhanced IL-1beta-stimulated MCP-1 mRNA production and protein secretion. Eotaxin mRNA was detectable in unstimulated IEC-18 cells, and IL-4 but not IFN-gamma caused a rapid enhancement in levels, which remained elevated for 24 h after treatment. Finally, IL-1beta but not IFN-gamma or IL-4 enhanced MIP-2 mRNA levels. Knowledge gained from studying the outcome of T lymphocyte-derived stimuli will help understand the complex sequence of events during chronic intestinal inflammation.     

Novel chemokine responsiveness and mobilization of neutrophils during sepsis

Blood neutrophils (PMN) are usually unresponsive to CC chemokines such as monacyte chemotactic protein-1 and macrophage inflammatory protein-1 alpha. In rodents, the lung buildup of PMN as determined by myeloperoxidase (MPO) activity after airway instillation of bacterial lipopolysaccharide (LPS) was independent of MCP-1 and MIP-1 alpha. In striking contrast, during sepsis following cecal ligation and puncture (CLP), blood PMN demonstrated mRNA for CC chemokine receptors. Furthermore, PMN from CLP, but not from sham rodents, bound MCP-1 and MIP-1 alpha and responded chemotactically in vitro to both MCP-1 and MIP-1 alpha. In CCR2(-/-) mice or WT mice treated in vivo with antibodies to either MCP-1 or MIP-1 alpha, MPO activity was greatly attenuated in CLP animals. In CLP mice, increased serum IL-6 levels were found to be dependent on CCR2, MCP-1, and MIP-1 alpha. When PMN from CLP rodents were incubated in vitro with either MCP-1 or MIP-1 alpha, release of IL-6 was also shown. These findings suggest that sepsis fundamentally alters the trafficking of PMN into the lung in a manner that now engages functional responses to CC chemokines.     

Augmented production of chemokines (monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta) in patients with systemic sclerosis: MCP-1 and MIP-1alpha may be involved in the development of pulmonary fibrosis.

To determine the role of chemokines in the pathogenesis of systemic sclerosis (SSc), we examined serum levels, spontaneous production by peripheral blood mononuclear cells (PBMC), and histological distribution in the affected skin, of MCP-1, MIP-1alpha and MIP-1beta in SSc patients. Serum levels of these chemokines were examined by ELISA in 58 patients with SSc and 20 normal controls. The levels of these chemokines in culture supernatants from PBMC were also measured by ELISA. Serum levels and spontaneous production levels by PBMC of MCP-1, MIP-1alpha, and MIP-1beta were significantly elevated in patients with SSc compared with normal controls. Elevated serum levels of MCP-1 and MIP-1alpha significantly correlated with the presence of pulmonary fibrosis. MCP-1 expression in the skin of SSc was immunohistochemically examined using anti-MCP-1 MoAb. MCP-1 was strongly expressed in the epidermis, inflammatory mononuclear cells, and vascular endothelial cells in the sclerotic skin of SSc patients, but not expressed in any control skin. Furthermore, the MCP-1 expression in inflammatory mononuclear cells and endothelial cells significantly correlated with earlier onset of SSc. Thus, MCP-1, MIP-1alpha and MIP-1beta may be involved in the disease process, possibly by augmenting leucocyte migration into the affected tissues in SSc. Furthermore, MCP-1 and MIP-1alpha may play an important role in the development of pulmonary fibrosis in SSc.     

C-C chemokines differentially alter interleukin-4 production from lymphocytes.

The expression of cytokines can dictate the intensity, chronicity, and type of immune/inflammatory response that is produced. These events may be regulated by accumulation of particular cell populations at a site of immune response that can be regulated by the expression of specific chemokines. Recent data have indicated that chemokines also have direct effects on cellular activation. In particular, T lymphocyte responses have been divided into two distinct phenotypes, designated by TH1- and TH2-type cytokine expression. Although it is recognized that divergent T-lymphocyte-derived cytokine phenotypes exist, the mechanisms that dictate the expression of these cytokines and ultimately the division of these immune responses is not entirely clear. In the present study, we present data that the C-C chemokine family members may be a factor influencing the direction of T-cell-derived lymphokine production. To elucidate the role of C-C chemokines, MIP-1 alpha and MCP-1, we have used both antigen-specific (schistosomal egg antigen (SEA)) and nonspecific (conconavalin (Con) A) stimuli. Using TH2-type lymphocyte populations from SEA-sensitized mice, a significant increase in IL-4 mRNA expression and protein production was observed when MCP-1 was added to the culture. Conversely, MIP-1 alpha treatment appeared to decrease interleukin (IL)-4 production. Interestingly, the proliferative response in the TH2-type (SEA-specific) response was up-regulated by MIP-1 alpha whereas MCP-1 down-regulated the response, inversely correlating with IL-4 production. Primary stimulation of naive lymphocytes with Con A induces a predominant interferon (IFN)-gamma response, whereas the second stimulation of the same lymphocytes with Con A induces both IFN-gamma and IL-4. When the two C-C chemokines were individually co-incubated with Con-A-stimulated lymphocytes, both up-regulated IFN-gamma production and proliferation during the primary stimulation. Similarly, in the secondary response, both chemokines further upregulated IFN-gamma production; however, only MCP-1 co-stimulation increased IL-4 production, whereas MIP-1 alpha significantly decreased IL-4 production in these same cell populations. These results were also reflected in steady-state levels of mRNA expression. These results suggest that the production of C-C chemokines (MCP-1 or MIP-1 alpha) during an immune response may aid in determining the type of cytokines produced and the level of lymphocyte activation during a particular response.     

Neutrophil depletion exacerbates experimental Chagas' disease in BALB/c, but protects C57BL/6 mice through modulating the Th1/Th2 dichotomy in different directions

To elucidate the roles of neutrophils in experimental Chagas' disease, we depleted the peripheral neutrophils in BALB/c and C57BL/6 mice with a monoclonal antibody 1 day before Trypanosoma cruzi infection. Neutrophil depletion in BALB/ c mice resulted in exacerbation of the disease and decreased expression of mRNA for Th1 cytokines, including IL-2 and IFN-gamma, IL-12p40 and TNF-alpha in their spleens after the infection, while a Th2 cytokine, IL-10, increased especially 1 day after infection. Neutrophils from infected BALB / c mice expressed mRNA for IL-12p40, IFN-gamma, TNF-alpha and Th1 chemoattractive chemokines, monokine induced by IFN-gamma (MIG) and macrophage inflammatory protein-1alpha (MIP-1alpha ). In contrast, in C57BL/6 mice neutrophil depletion induced resistance to the disease and enhanced the expression of the above Th1 cytokines, although IL-10 mRNA in neutrophil-depleted C57BL/6 mice was also higher than in control mice. Neutrophils from C57BL/6 mice did not express IL-12p40, IFN-gamma and MIG but expressed TNF-alpha, MIP-1alpha and IL-10. Therefore, neutrophils may play opposite roles in these two strains of mice with respect to protection versus exacerbation of T. cruzi infection, possibly through modulating the Th1/Th2 dichotomy in different directions.     

Acellular components of Chlamydia pneumoniae stimulate cytokine production in human blood mononuclear cells

Accumulating evidence suggest that infection with Chlamydia pneumoniae is associated with atherosclerosis, but the mechanisms involved remain unclear. Inflammation is important in the initial phase of atherogenesis, and cytokines are important in the initiation and progression of inflammation. The aim of this study was to assess the capacity of acellular components of C. pneumoniae to stimulate the production of pro-inflammatory cytokines and chemokines. Peripheral blood mononuclear cells were stimulated in vitro with sonicated C. pneumoniae. Significant amounts of TNF-alpha, IL-1, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) were produced. Inhibition of endotoxin using polymyxin B revealed that chlamydial endotoxin plays a minor role in the cytokine induction. Neutralization of TNF by TNF-binding protein and blockade of IL-1 receptors by IL-1 receptor antagonist revealed that TNF, IL-1 and IL-6 production was independent from each other, whereas IL-8 synthesis was strongly dependent on endogenous TNF and IL-1. In contrast, synthesis of MCP-1 and MIP-1alpha was dependent on endogenous TNF, but not IL-1. In conclusion, acellular components of C. pneumoniae are a potent stimulus for cytokine production, and this mechanism may have an important role in the inflammatory aspects of atherogenesis.     

CXC chemokines Gro(alpha)/IL-8 and IP-10/MIG in Helicobacter pylori gastritis

Infection with Helicobacter pylori causes chronic active gastritis, which is characterized by neutrophils infiltrating the gastric epithelial layer and the underlying lamina propria as well as by T, B lymphocytes and macrophages accumulating in the lamina propria. In this study, the chemokine profile responsible for the recruitment of these inflammatory cells is investigated. Using both RNA/RNA in situ hybridization and immunohistochemistry, the expression of the neutrophil and/or lymphocyte-attractant CXC chemokines growth-related oncogene alpha (Gro(alpha)), IL-8, interferon-gamma (IFN-gamma)-inducible protein-10 (IP-10), monokine induced by IFN-gamma (MIG) and the CC chemokines macrophage inflammatory protein-1alpha (MIP-1alpha), -1beta, regulated on activation normal T cell expressed and secreted (RANTES) and monocyte chemoattractant protein-1 (MCP-1) is studied and microanatomically localized in the gastric mucosa. Macrophages in the lamina propria at sites with neutrophil infiltration and gastric epithelium infiltrated by neutrophils highly expressed the neutrophil-attractant chemokines Gro(alpha) and IL-8. Additionally, Gro(alpha) and IL-8 were expressed by neutrophils themselves localized within gastric epithelium, in the foveolar lumen and in the cellular debris overlying mucosal erosion. IP-10 and to a lower extent MIG, both selectively chemotactic for inflammatory T cells, were expressed by endothelial cells of gastric mucosal vessels and by mononuclear cells at sites with T cell infiltration. Expression of all other CC chemokines tested was significantly lower. These in vivo data indicate that a set of predominantly CXC chemokines modulates the inflammation in H. pylori gastritis. Gro(alpha) and IL-8 may play an important role in neutrophil trafficking from the mucosal vessel into the gastric epithelium, whereas IP-10 and MIG contribute to the recruitment of inflammatory T cells into the mucosa.     

Expression patterns of cytokines and chemokines genes in human hepatoma cells

Various cytokines and chemokines play a role in carcinogenesis. However, no study has previously been undertaken to investigate comprehensively the expressions of cytokines and chemokines in hepatoma cells. In this study, we determined which cytokines and chemokines are expressed in hepatoma cells. Recently, it was reported that the expressions of several chemokines could be increased by Fas stimulus in many normal and cancer cells. Therefore, we also investigated whether chemokines expression is regulated by Fas ligation. To address this issue, we performed RNase protection assays upon 13 cytokines and 8 chemokines genes in 10 human hepatoma cell lines, comprising 8 hepatitis B virus (HBV)-associated hepatoma cell lines. Transforming growth factor-beta2 (TGF-beta2) was found to be expressed in 8 HBV-associated hepatoma cell lines, and to be potently expressed in 5 cell lines; however, the mRNA expressions of interleukin-10 (IL-10), IL-12, interferon-gamma(IFN-gamma) and tumor necrosis factor-alpha(TNF-alpha) were not detected in any cell lines examined. Among the chemokines investigated in this study, IL-8 was expressed by 8 HBV- associated hepatoma cell lines, and monocyte chemoattractant protein-1 (MCP-1) by 7 HBV-associated hepatoma cell lines. However, the mRNA expressions of macrophage inflammatory protein-1alpha(MIP-1alpha), MIP-1beta, interferon-inducible protein-10 (IP-10), RANTES, lymphotactin and I-309 were either very weak or undetectable. Fas ligation did not increase chemokines expression in hepatoma cells. Conclusively, TGF-beta2, IL-8 and MCP-1 were overexpressed in HBV-associated hepatoma cells, and the expressions of chemokines were not increased by Fas ligation in human hepatoma cells.     

Induction of host protective Th1 immune response by chemokines in Leishmania donovani-infected BALB/c mice

The resolution from leishmanial infection is dependent on the coordinated interactions between the components of the cell mediated immune system and the activation of T-cell population into appropriate cytokine production and the activation of macrophages. Earlier reports established that C-C chemokines particularly macrophage inflammatory protein (MIP)-1alpha and macrophage chemoattractant protein (MCP)-1 restrict the parasitic burden via the regulation of impaired protein kinase C (PKC) signalling and induction of free-radical generation in murine leishmaniasis. This study explored the role of MIP-1alpha and MCP-1 in the induction of T helper 1 (Th1) immune response and suppression of T helper 2 (Th2) response in Leishmania donovani-infected BALB/c mice. These chemokines induced the known pro-inflammatory cytokine interleukin (IL)-12 secretion and inhibited the secretion of anti-inflammatory cytokines IL-10 and transforming growth factor-beta in infected macrophages. Impaired antigen presentation capability of infected macrophages was also restored by the chemokine treatment. C-C chemokine treatment resulted in reduced levels of mRNA expression of IL-10, but increased levels of mRNA expression of IL-12p40, interferon (IFN)-gamma, tumour necrosis factor-alpha and inducible nitric oxide synthase in both liver mononuclear cells as well as in splenocytes, reflecting a switch of CD4+ differentiation from Th2 to Th1. Flow cytometric analysis of infected spleen cells suggested that C-C chemokine treatment enhances the CD4+ T cells to produce increased levels of IFN-gamma. These studies hypothesize a promising immuno-prophylactic effect of chemokines against leishmaniasis by induction of Th1 cytokine release imparting a long-term resistance.     

IFN-gamma and IL-12 differentially regulate CC-chemokine secretion and CCR5 expression in human T lymphocytes

Interleukin (IL)-12, especially in the presence of neutralizing anti-IL-4 monoclonal antibodies, primed CD45RO(-) T clones for high CCL3/macrophage-inflammatory protein-1alpha (MIP-1alpha) and CCL4/MIP-1beta levels. In CD4(+) and CD8(+) clones from two patients deficient for IL-12Rbeta1 (IL-12Rbeta1(-/-)), production of CCL3/MIP-1alpha and CCL4/MIP-1beta was defective. CD4(+) clones from two patients deficient for interferon-gamma (IFN-gamma) R1 (IFN-gammaR1(-/-)) produced somewhat decreased CCL4/MIP-1beta levels. IL-12 failed to prime CD4(+) or CD8(+) healthy clones for high CCL5/regulated on activation, normal T expressed and secreted (RANTES) production, although its secretion was impaired in CD4(+) clones from IL-12Rbeta1(-/-) and IFN-gammaR1(-/-) patients. CCR5 surface expression was up-regulated in resting peripheral blood mononuclear cells and CD4(+) clones from both kinds of patients, rendering them more susceptible to CCR5-dependent (R5) HIV-1 infection. Neutralization of IFN-gamma increased CCR5 expression and decreased CC-chemokine secretion by CD4(+) clones from healthy and IL-12Rbeta1(-/-) individuals, suggesting an IFN-gamma-dependent control of CCR5 expression. These data provide the first documented analysis of chemokine secretion and chemokine receptor expression on T cells from IL-12 and IFN-gamma receptor-deficient patients and dissect the role of IL-12 and IFN-gamma on inducing inflammatory chemokine secretion and down-regulating CCR5 expression in human T cells.     

Monocyte chemotactic protein-1 in the follicle of the menstrual and IVF cycle

Ovulation constitutes an inflammatory-like process, with macrophages migrating into the follicle. This study evaluates the production of two macrophage-specific chemokines, monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha), in the human follicle at ovulation. Blood samples, follicular fluids and follicular cells were collected during menstrual and IVF cycles. Levels of MCP-1 and MIP-1alpha were measured in follicular fluid, blood plasma and cultured media (granulosa, theca and granulosa-lutein cells [GLCs]). Cells were cultured with or without LH, FSH, interleukin (IL)-1alpha, IL-1beta, tumour necrosis factor (TNF) alpha, progesterone or estradiol. The levels of MCP-1 were markedly higher in follicular fluid as compared with blood plasma in both menstrual and IVF cycles. The difference in MCP-1 levels between follicular fluid and plasma in menstrual cycles increased from the follicular phase (three-fold difference) to the late ovulatory phase (25-fold). Levels of MIP-1alpha were low in plasma and follicular fluid of both menstrual and IVF cycles. Theca cells from follicles of menstrual cycles secreted both MCP-1 and MIP-1alpha under basal conditions, and the secretion was increased by addition of IL-1beta (MCP-1 and MIP-1alpha) and IL-1alpha (MCP-1). GLCs secreted MCP-1 under basal conditions and also MIP-1alpha after IL-1beta stimulation. The macrophage-specific chemokine MCP-1 is highly expressed and is induced by IL-1 in the theca layer of the human follicle at ovulation.     

Human peripheral blood T cells, monocytes, and macrophages secrete macrophage inflammatory proteins 1alpha and 1beta following stimulation with heat-inactivated Brucella abortus

Heat-killed Brucella abortus (HBa) has been proposed as a carrier for therapeutic vaccines for individuals with immunodeficiency, due to its abilities to induce interleukin-2 (IL-2) and gamma interferon (IFN-gamma) in both CD4(+) and CD8(+) T cells and to upregulate antigen-presenting cell functions (including IL-12 production). In the current study, we investigated the ability of HBa or lipopolysaccharide isolated from HBa (LPS-Ba) to elicit beta-chemokines, known to bind to the human immunodeficiency virus type 1 (HIV-1) coreceptor CCR5 and to block viral cell entry. It was found that human peripheral blood mononuclear cells secreted beta-chemokines following stimulation with HBa, and this effect could not be blocked by anti-IFN-gamma neutralizing antibodies. Among purified T cells, macrophage inflammatory protein 1alpha and 1beta (MIP-1alpha and MIP-1beta, respectively) secretion was observed primarily in human CD8(+) T cells. The kinetics of beta-chemokine induction in T cells were slow (3 to 4 days). The majority of beta-chemokine-producing CD8(+) T cells also produced IFN-gamma following HBa stimulation, as determined by triple-color intracellular staining. A significant number of CD8(+) T cells contained stored MIP-1beta that was released after HBa stimulation. Both HBa and LPS-Ba stimulated high levels of MIP-1alpha and MIP-1beta production in elutriated monocytes and even higher levels in macrophages. In these cells, beta-chemokine mRNA was upregulated within 30 min and proteins were secreted within 4 h of stimulation. The monocyte- and macrophage-derived beta-chemokines were sufficient to block CCR5-dependent HIV-1 envelope-mediated cell fusion. These data suggest that, in addition to the ability of HBa to elicit antigen-specific humoral and cellular immune responses, HBa-conjugated HIV-1 proteins or peptides would also generate innate chemokines with antiviral activity that could limit local viral spread during vaccination in vivo.     

Interleukin-25-induced chemokines and interleukin-6 release from eosinophils is mediated by p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and nuclear factor-kappaB

Interleukin (IL)-25, a novel Th2 cytokine, is capable of amplifying allergic inflammation. We investigated the modulation of nuclear factor (NF)-kappaB and mitogen-activated protein kinases (MAPK) pathways in IL-25-activated eosinophils, the principal effector cells of allergic inflammation, for the in vitro release of chemokines including monocyte chemoattractant protein-1 (MCP-1), IL-8, and macrophage inflammatory protein (MIP)-1alpha, and inflammatory cytokine IL-6. Gene expression of chemokines and IL-6 was evaluated by RT-PCR, and concentrations of chemokines and cytokine were measured by cytokine protein array, cytometric bead array, and enzyme-linked immunosorbent assay. NF-kappaB, c-Jun amino-terminal kinase (JNK), and p38 MAPK activities in eosinophils were assessed by electrophoretic mobility shift assay and Western blot. IL-25 was found to upregulate the gene expression of chemokines MCP-1, MIP-1alpha, and IL-8, and cytokine IL-6, in eosinophils, and to significantly increase the release of the above chemokines and IL-6 from eosinophils. IL-25 could also activate the JNK, p38 MAPK, and NF-kappaB activities of eosinophils, while inhibitor of IkappaB-alpha phosphorylation (BAY11-7082), JNK (SP600125), and p38 MAPK (SB203580) could suppress the release of IL-8, MIP-1alpha, MCP-1, and IL-6. Together, the above results showed that the induction of MCP-1, MIP-1alpha, IL-8, and IL-6 in IL-25-activated eosinophils are regulated by JNK, p38 MAPK, and NF-kappaB pathways.     

MCP-1 and MIP-2 response in Trichinella spiralis infected mice treated with 4-deoxypyridoxine (4-DPD)

Chemokines are involved in a number of pathophysiological conditions, such as inflammatory processes and are divided in two major subfamilies, C-X-C and C-C chemokines. The C-C chemokines are monocyte chemotactic protein 1-2-3-4-5, while C-X-C chemokines include MIP-2, IL-8, etc. We studied the levels of MCP-1 and MIP-2 in diaphragmatic and intercostal muscle tissue and serum in Trichinella spiralis infected mice treated and not treated with 4-deoxypyridoxine, a potent Vit. B6 antagonist which inhibits humoral and cellular immune response. MCP-1 and MIP-2 were measured in homogenized tissue and serum and determined by a specific ELISA. Here we found the levels of MCP-1 and MIP-2 in diaphragmatic and intercostal muscle tissue of T. spiralis infected mice were significantly increased after 10 days and peaked on day 20 post-infection; however, the levels of MIP-2 in mice treated with 4-DPD was lower than that of untreated mice at day 20. MCP-1 also peaked at days 20 and 40. Animals treated with 4-DPD also inhibited the production of MCP-1, compared with untreated animals. The maximum inhibition was at day 40. These inhibitory effects on MIP-2 and MCP-1 were also repeated in the serum determinations, but were not significant. This study demonstrates that MIP-2 and MCP-1 are stimulated in serum and tissue of T. spiralis infected mice and 4-DPD-treated animals significantly inhibited them.