CCL3/Macrophage Inflammatory Protein-1α Is Dually Involved in Parasite Persistence and Induction of a TNF- and IFNγ-Enriched Inflammatory Milieu in Trypanosoma cruzi-Induced Chronic Cardiomyopathy.

CCL3, a member of the CC-chemokine family, has been associated with macrophage recruitment to heart tissue and parasite control in the acute infection of mouse with Trypanosoma cruzi, the causative agent of Chagas disease. Here, we approached the participation of CCL3 in chronic chagasic cardiomyopathy (CCC), the main clinical form of Chagas disease. We induced CCC in C57BL/6 (ccl3+/+) and CCL3-deficient (ccl3-/-) mice by infection with the Colombian Type I strain. In ccl3+/+ mice, high levels of CCL3 mRNA and protein were detected in the heart tissue during the acute and chronic infection. Survival was not affected by CCL3 deficiency. In comparison with ccl3+/+, chronically infected ccl3-/- mice presented reduced cardiac parasitism and inflammation due to CD8+ cells and macrophages. Leukocytosis was decreased in infected ccl3-/- mice, paralleling the accumulation of CD8+ T cells devoid of activated CCR5+ LFA-1+ cells in the spleen. Further, T. cruzi-infected ccl3-/-mice presented reduced frequency of interferon-gamma (IFNγ)+ cells and numbers of parasite-specific IFNγ-producing cells, while the T. cruzi antigen-specific cytotoxic activity was increased. Stimulation of CCL3-deficient macrophages with IFNγ improved parasite control, in a milieu with reduced nitric oxide (NOx) and tumor necrosis factor (TNF), but similar interleukin-10 (IL-10), concentrations. In comparison with chronically T. cruzi-infected ccl3+/+ counterparts, ccl3-/- mice did not show enlarged heart, loss of left ventricular ejection fraction, QTc prolongation and elevated CK-MB activity. Compared with ccl3+/+, infected ccl3-/- mice showed reduced concentrations of TNF, while IL-10 levels were not affected, in the heart milieu. In spleen of ccl3+/+ NI controls, most of the CD8+ T-cells expressing the CCL3 receptors CCR1 or CCR5 were IL-10+, while in infected mice these cells were mainly TNF+. Lastly, selective blockage of CCR1/CCR5 (Met-RANTES therapy) in chronically infected ccl3+/+ mice reversed pivotal electrical abnormalities (bradycardia, prolonged PR, and QTc interval), in correlation with reduced TNF and, mainly, CCL3 levels in the heart tissue. Therefore, in the chronic T. cruzi infection CCL3 takes part in parasite persistence and contributes to form a CD8+ T-cell and macrophage-enriched cardiac inflammation. Further, increased levels of CCL3 create a scenario with abundant IFNγ and TNF, associated with cardiomyocyte injury, heart dysfunction and QTc prolongation, biomarkers of severity of Chagas' heart disease.

Interleukin-10 and interferon-gamma modulate surface expression of fractalkine-receptor (CX(3)CR1) via PI3K in monocytes.

The membrane-anchored form of the chemokine fractalkine (CX(3)CL1) has been identified as a novel adhesion molecule that interacts with its specific receptor (CX(3)CR1) expressed in monocytes, T cells and natural killer cells to induce adhesion. In addition, CX(3)CL1 can be cleaved from the cell membrane to induce chemotaxis of CX(3)CR1-expressing leucocytes. Recently, marked variations in CX(3)CR1 monocyte expression have been observed during several pathological conditions. Regulation of CX(3)CR1 in monocytes during basal or inflammatory/anti-inflammatory conditions is poorly understood. The aim of this study was therefore to examine CX(3)CR1 expression during monocyte maturation and the effect of soluble mediators on this process. We found that basal expression of CX(3)CR1 in fresh monocytes was reduced during culture, and that lipopolysacchairde accelerated this effect. In contrast, interleukin-10 and interferon-gamma treatment abrogated CX(3)CR1 down-modulation, through a phosphatidylinositol 3 kinase-dependent pathway. Most importantly, CX(3)CR1 membrane expression correlated with monocyte CX(3)CL1-dependent function. Taken together, our data demonstrate that CX(3)CR1 expression in monocytes can be modulated, and suggest that alterations in their environment are able to influence CX(3)CL1-dependent functions, such as chemotaxis and adhesion, leading to changes in the kinetics, composition and/or functional status of the leucocyte infiltrate.

Altered chemokine receptor expression in papillary thyroid cancer.

BACKGROUND: Papillary thyroid cancer (PTC), the most prevalent type of differentiated thyroid carcinoma, displays a strikingly high frequency of lymph node metastasis (LNM). Recent data suggest that chemokines can play an important role in promoting tumor progression and metastatic migration of tumor cells. Here we have evaluated whether PTC tissues express a different pattern of chemokine receptors and if the expression of these receptors correlates with LNM. METHODS: We assessed by immunohistochemistry and flow cytometry the expression of the chemokine receptors CCR3, CCR7, and CXCR4 in tumor and nonmalignant thyroid tissues from patients suffering from PTC. Expression of these receptors in PTC was correlated with the clinical pathological condition of PTC. RESULTS: Our data show a significant enhancement of CCR3 (2.5 times higher, p = 0.038) and CXCR4 (1.7 times higher, p = 0.02) expression in PTC tissues as determined by immunohistochemical staining, and of CCR3 (3.5 times higher, p < 0.002) in the plasma membrane as determined by flow cytometric analyses, compared to controls. In addition, while CCR3 (100%) and CXCR4 (90%) were present in both tumor and control thyroid tissues, expression of CCR7 was scarcely detected in PTC cells (5-10%) and not found in control cells. CXCR4 expression correlated with the classical variant of PTC (p < 0.035) and extranodal extension (p < 0.010) in patients with LNM. CONCLUSIONS: Our data support the notion that CCR3, CCR7, and CXCR4 are increasingly expressed in tumor cells from PTC and that CXCR4 expression in PTC could be a potential marker for enhanced tumor aggressiveness.

Effect of pro-inflammatory cytokine stimulation on human breast cancer: implications of chemokine receptor expression in cancer metastasis.

Interactions between tumour cells and microenvironments may affect their growth and metastasis formation. In search for a better understanding of the role of cellular mediators in the progression of cancer, we investigated the effect of pro-inflammatory cytokines IL-1, IL-6, TNF-alpha and IFN-gamma on the regulation of expression of chemokine receptors CXCR4, CXCR2, CX3CR1, CCR9, and CCR5 in the human breast cancer cell line MCF-7. Our results showed that IL-1 increased CXCR4 expression whereas TNF-alpha increased CX3CR1, CCR9 and CCR5. Interestingly, this regulation was not homogeneous, emphasizing the inherent heterogeneity in cancer that may be responsive to specific inflammatory microenvironments.

Differential expression of cytokines, chemokines and chemokine receptors in patients with coronary artery disease.

Monocytes/macrophages and lymphocytes have a key role in the pathogenesis of atherosclerosis through the production of inflammatory and anti-inflammatory cytokines. We evaluated mRNA expression and protein production of CCL2, CXCL8, CXCL9, CXCL10, IFN-gamma and IL-10 in vitro as well as the expression of the CCR2 and CXCR3 receptors in peripheral blood mononuclear cells (PBMCs) of patients with coronary artery disease (CAD) and healthy controls in the presence or absence of oxidized LDL (oxLDL). Patients with CAD showed higher constitutive expression of CCL2, CXCL8, CXCL9, CXCL10 and IFN-gamma mRNA and, after stimulation with oxLDL, higher expression of CCL2 and CXCL8 mRNA than the control group. We also detected higher levels of CCL2 and CXCL8 in supernatants of oxLDL-stimulated PBMCs from CAD patients than in corresponding supernatants from controls. Patients with CAD had a higher percentage of constitutive CCR2(+) and CXCR3(+) cells after stimulation with oxLDL. Among CAD patients, the main differences between the stable (SA) and unstable angina (UA) groups were lower IL-10 mRNA production in the latter group. Altogether, our data suggest that PBMCs from CAD patients are able to produce higher concentrations of chemokines and cytokines involved in the regulation of monocyte and lymphocyte migration and retention in atherosclerotic lesions.

Flow-cytometric analysis of cytokine production in human paracoccidioidomycosis.

Human infection with Paracoccidioides brasiliensis may result in three major outcomes: paracoccidioidomycosis-infection (PI), which is observed in healthy carriers living in endemic areas and the adult form (AF) and juvenile form (JF) of the disease. In this study we proposed to examine the intracellular expression of IFN-gamma, TNF-alpha, IL-2, IL-10, IL-12, CXCL8, CXCL9 and CXCL10 by peripheral blood mononuclear cells (PBMC) of patients with the JF and AF of the disease, as well as of PI individuals stimulated with PMA plus ionomycin, LPS or anti-CD3 plus anti-CD28, by flow cytometry. The results showed that PI individuals present a higher percentage of cells producing IFN-gamma, TNF-alpha, IL-2, CXCL9 and CXCL10 when compared to AF and JF patients. IFN-gamma was predominantly detected in CD3(+)CD8(+) T cells, whereas IL-2 and TNF-alpha were mainly expressed in CD3(+)CD4(+) cells. Monocytes of PI individuals also presented higher expression of CD80 and lower expression of CD86 when compared to JF and AF patients, and higher expression of HLA-DR, only when compared to JF patients. These results indicate that the differential production of cytokines and chemokines, as well as the expression of co-stimulatory molecules involved in antigen presentation, may influence the outcome of PCM infection.

Leishmania infection impairs beta 1-integrin function and chemokine receptor expression in mononuclear phagocytes.

Leishmania spp. are intracellular parasites that cause lesions in the skin, mucosa, and viscera. We have previously shown that Leishmania infection reduces mononuclear phagocyte adhesion to inflamed connective tissue. In this study, we examined the role of adhesion molecules and chemokines in this process. Infection rate (r = -0.826, P = 0.003) and parasite burden (r = -0.917, P = 0.028) negatively correlated to mouse phagocyte adhesion. The decrease (58.7 to 75.0% inhibition, P = 0.005) in phagocyte adhesion to connective tissue, induced by Leishmania, occurred as early as 2 h after infection and was maintained for at least 24 h. Interestingly, impairment of cell adhesion was sustained by phagocyte infection, since it was not observed following phagocytosis of killed parasites (cell adhesion varied from 15.2% below to 24.0% above control levels, P > 0.05). In addition, Leishmania infection diminished cell adhesion to fibronectin (54.1 to 96.2%, P < 0.01), collagen (15.7 to 83.7%, P < 0.05), and laminin (59.1 to 82.2%, P < 0.05). The CD11b(hi) subpopulation was highly infected (49.6 to 97.3%). Calcium and Mg(2+) replacement by Mn(2+), a treatment that is known to induce integrins to a high state of affinity for their receptors, reverted the inhibition in adhesion caused by Leishmania. This reversion was completely blocked by anti-VLA4 antibodies. Furthermore, expression of CCR4 and CCR5, two chemokine receptors implicated in cell adhesion, was found to be downregulated 16 h after infection (2.8 to 4.1 times and 1.9 to 2.8 times, respectively). Together, these results suggest that mechanisms regulating integrin function are implicated in the change of macrophage adhesion in leishmaniasis.

Distinct Leishmania braziliensis isolates induce different paces of chemokine expression patterns.

Inflammatory events during Leishmania braziliensis infection in mice were investigated. Large lesions were directly correlated with the inflammatory reaction but not with parasite burden. Different L. braziliensis strains induce different paces of chemokine expression patterns, leading to diverse cell recruitment and differential inflammatory responses.

Loss of circulating CD27+ memory B cells and CCR4+ T cells occurring in association with elevated EBV loads in XLP patients surviving primary EBV infection.

Detailed longitudinal studies of patients with X-linked lymphoproliferative disease (XLP) may increase our understanding of the immunologic defects that contribute to the development of lymphoma and hypogammaglobulinemia in XLP. We describe progressive changes observed in immunoglobulin concentrations, lymphocyte subsets, and Epstein-Barr virus (EBV) loads occurring in a 2-year period in a newly infected, but otherwise healthy, carrier (patient 9). We compare these findings with those observed in the patient's brother, who had hypogammaglobulinemia and XLP (patient 4). Immunoglobulin G (IgG), IgM, and IgA concentrations increased in patient 9 during acute EBV infection, but thereafter they decreased steadily to concentrations consistent with hypogammaglobulinemia, reaching a plateau 5 months after infection. In both patients, CD19+ B-lymphocyte rates remained lower than 3%, with a contraction of the B-cell memory compartment (CD27+ CD19+/CD19+) to 20% (normal range, 32%-56%). T-lymphocyte subpopulations showed a reduction in CD4+ T-cell counts and a permanent CD8+ T-cell expansion. Interestingly, CXCR3 memory TH1 cells were expanded and CCR4+ TH2 lymphocytes were reduced, suggesting that abnormal skewing of memory T-cell subsets might contribute to reduced antibody synthesis. Despite an expanded number of CD3+CD8+ lymphocytes, increased EBV loads occurred in both patients without overt clinical symptoms of mononucleosis, lymphoproliferative disease, or lymphoma.

Role of monocyte chemotactic protein-1/CC chemokine ligand 2 on gamma delta T lymphocyte trafficking during inflammation induced by lipopolysaccharide or Mycobacterium bovis bacille Calmette-Guérin.

Gammadelta T lymphocytes are involved in a great variety of inflammatory and infectious responses. However, the mechanisms by which gammadelta T lymphocytes migrate to inflamed sites are poorly understood. In this study we investigate the role of monocyte chemotactic protein (MCP)-1 in regulating gammadelta T cell migration after LPS or Mycobacterium bovis bacille Calmette-Guérin (BCG) challenge. LPS-induced gammadelta T cell influx was significantly inhibited by either pretreatment with dexamethasone or vaccinia virus Lister 35-kDa chemokine binding protein, vCKBP, a CC chemokine neutralizing protein, suggesting a role for CC chemokines in this phenomenon. LPS stimulation increased the expression of MCP-1 mRNA and protein at the inflammation site within 6 h. It is noteworthy that LPS was unable to increase MCP-1 production or gammadelta T cell recruitment in C3H/HeJ, indicative of the involvement of Toll-like receptor 4. Gammadelta T cells express MCP-1 receptor CCR2. Pretreatment with anti-MCP-1 mAb drastically inhibited LPS-induced in vivo gammadelta T cell mobilization. Indeed, MCP-1 knockout mice were unable to recruit gammadelta T cells to the pleural cavity after LPS stimulation, effect that could be restored by coadministration of MCP-1. In addition, BCG-induced gammadelta lymphocyte accumulation was significantly reduced in MCP-1 knockout mice when compared with wild-type mice. In conclusion, our results indicate that LPS-induced gammadelta T lymphocyte migration is dependent on Toll-like receptor 4 and sensitive to both dexamethasone and CC chemokine-binding protein inhibition. Moreover, by using MCP-1 neutralizing Abs and genetically deficient mice we show that LPS- and BCG-induced gammadelta T lymphocyte influx to the pleural cavity of mice is mainly orchestrated by the CC chemokine MCP-1.

Simian immunodeficiency virus dramatically alters expression of homeostatic chemokines and dendritic cell markers during infection in vivo.

Dendritic cells (DCs) are potent antigen-presenting cells that likely play multiple roles in human immunodeficiency virus type 1 (HIV-1) pathogenesis. We used the simian immunodeficiency virus (SIV)/macaque model to study the effects of infection on homeostatic chemokine expression and DC localization directly in secondary lymphoid tissues. SIV infection altered the expression of chemokines (CCL19/MIP-3beta, CCL21/ 6Ckine, and CCL20/MIP-3alpha) and of chemokine receptors (CCR7 and CCR6) that drive DC trafficking. CCL19/MIP-3beta, CCL20/MIP-3alpha, CCR6, and CCR7 expression increased in lymph nodes during the early systemic burst of viral replication (acute infection), whereas CCL21/6Ckine expression progressively decreased throughout disease to AIDS. Parallel with the SIV-induced perturbations in chemokine expression were changes in the expression of the DC-associated markers, DC-SIGN, DC-LAMP, and DECTIN-1. During AIDS, DC-LAMP mRNA expression levels were significantly reduced in lymph nodes and spleen, and DC-SIGN levels were significantly reduced in spleen. These findings suggest that the disruption of homeostatic chemokine expression is responsible, in part, for alterations in the networks of antigen-presenting cells in lymphoid tissues, ultimately contributing to systemic immunodeficiency.