The effects of acute exercise-induced cortisol on CCR2 expression on human monocytes

CC-chemokine receptor 2 (CCR2) and its ligand, monocyte chemotactic protein-1 (MCP-1, also known as CCL2), are crucial for the recruitment of monocytes/macrophages to sites of inflammation. We conducted a series of experiments to investigate the relationship between stress, monocyte CCR2 expression and migration activity. First, we collected peripheral blood mononuclear cells (PBMC) from untrained subjects (n=8) and measured CCR2 expression on CD14(+) monocytes cultured with cortisol, epinephrine and norepinephrine. Second, we collected PBMC from the subjects before and after they cycled for 60 min at 70% peak O(2) uptake (VO2(peak)), and measured alterations in CCR2 expression on monocytes following exercise. Third, we cultured PBMC with serum obtained before and after exercise and the glucocorticoid antagonist RU-486 to determine the effect of cortisol on CCR2 expression in vitro. Last, we measured the ability of PBMC treated with serum or cortisol to migrate through membrane filters in response to CCL2. Cortisol (but not epinephrine or norepinephrine) increased CCR2 expression on monocytes in a dose- and time-dependent manner. Exercise did not influence CCR2 expression on PBMC, whereas incubation of PBMC with post-exercise serum significantly increased CCR2 expression. Both cortisol and post-exercise serum increased the migration of PBMC toward CCL2. The increase in CCR2 expression on PBMC following stimulation with cortisol and serum was blocked by the glucocorticoid receptor antagonist RU-486. In conclusion, cortisol released during exercise increased monocyte CCR2 expression and migration activity in vitro. These alterations may influence inflammation and regeneration of damaged tissue after acute stress.

Human proximal tubule epithelial cells modulate autologous dendritic cell function

Background We have previously demonstrated that human kidney proximal tubule epithelial cells (PTEC) are able to modulate autologous T and B lymphocyte responses. It is well established that dendritic cells (DC) are responsible for the initiation and direction of adaptive immune responses and that these cells occur in the renal interstitium in close apposition to PTEC under inflammatory disease settings. However, there is no information regarding the interaction of PTEC with DC in an autologous human context. Methods Human monocytes were differentiated into monocyte-derived DC (MoDC) in the absence or presence of primary autologous activated PTEC and matured with polyinosinic:polycytidylic acid [poly(I:C)], while purified, pre-formed myeloid blood DC (CD1c+ BDC) were cultured with autologous activated PTEC in the absence or presence of poly(I:C) stimulation. DC responses were monitored by surface antigen expression, cytokine secretion, antigen uptake capacity and allogeneic T-cell-stimulatory ability. Results The presence of autologous activated PTEC inhibited the differentiation of monocytes to MoDC. Furthermore, MoDC differentiated in the presence of PTEC displayed an immature surface phenotype, efficient phagocytic capacity and, upon poly(I:C) stimulation, secreted low levels of pro-inflammatory cytokine interleukin (IL)-12p70, high levels of anti-inflammatory cytokine IL-10 and induced weak Th1 responses. Similarly, pre-formed CD1c+ BDC matured in the presence of PTEC exhibited an immature tolerogenic surface phenotype, strong endocytic and phagocytic ability and stimulated significantly attenuated T-cell proliferative responses. Conclusions Our data suggest that activated PTEC regulate human autologous immunity via complex interactions with DC. The ability of PTEC to modulate autologous DC function has important implications for the dampening of pro-inflammatory immune responses within the tubulointerstitium in renal injuries. Further dissection of the mechanisms of PTEC modulation of autologous immune responses may offer targets for therapeutic intervention in renal medicine.

A rare P2X7 variant Arg307Gln with absent pore formation function protects against neuroinflammation in multiple sclerosis

Multiple sclerosis (MS) is a chronic relapsing-remitting inflammatory disease of the central nervous system characterized by oligodendrocyte damage, demyelination and neuronal death. Genetic association studies have shown a 2-fold or greater prevalence of the HLA-DRB1*1501 allele in the MS population compared with normal Caucasians. In discovery cohorts of Australasian patients with MS (total 2941 patients and 3008 controls), we examined the associations of 12 functional polymorphisms of P2X7, a microglial/macrophage receptor with proinflammatory effects when activated by extracellular adenosine triphosphate (ATP). In discovery cohorts, rs28360457, coding for Arg307Gln was associated with MS and combined analysis showed a 2-fold lower minor allele frequency compared with controls (1.11% for MS and 2.15% for controls, P = 0.0000071). Replication analysis of four independent European MS case–control cohorts (total 2140 cases and 2634 controls) confirmed this association [odds ratio (OR) = 0.69, P = 0.026]. A meta-analysis of all Australasian and European cohorts indicated that Arg307Gln confers a 1.8-fold protective effect on MS risk (OR = 0.57, P = 0.0000024). Fresh human monocytes heterozygous for Arg307Gln have >85% loss of ‘pore’ function of the P2X7 receptor measured by ATP-induced ethidium uptake. Analysis shows Arg307Gln always occurred with 270His suggesting a single 307Gln–270His haplotype that confers dominant negative effects on P2X7 function and protection against MS. Modeling based on the homologous zP2X4 receptor showed Arg307 is located in a region rich in basic residues located only 12 Å from the ligand binding site. Our data show the protective effect against MS of a rare genetic variant of P2RX7 with heterozygotes showing near absent proinflammatory ‘pore’ function.

Human bladder uroepithelial cells synergize with monocytes to promote IL-10 synthesis and other cytokine responses to Uropathogenic Escherichia coli

Urinary tract infections are a major source of morbidity for women and the elderly, with Uropathogenic Escherichia coli (UPEC) being the most prevalent causative pathogen. Studies in recent years have defined a key anti-inflammatory role for Interleukin-10 (IL-10) in urinary tract infection mediated by UPEC and other uropathogens. We investigated the nature of the IL-10-producing interactions between UPEC and host cells by utilising a novel co-culture model that incorporated lymphocytes, mononuclear and uroepithelial cells in histotypic proportions. This co-culture model demonstrated synergistic IL-10 production effects between monocytes and uroepithelial cells following infection with UPEC. Membrane inserts were used to separate the monocyte and uroepithelial cell types during infection and revealed two synergistic IL-10 production effects based on contact-dependent and soluble interactions. Analysis of a comprehensive set of immunologically relevant biomarkers in monocyte-uroepithelial cell co-cultures highlighted that multiple cytokine, chemokine and signalling factors were also produced in a synergistic or antagonistic fashion. These results demonstrate that IL-10 responses to UPEC occur via multiple interactions between several cells types, implying a complex role for infection-related IL-10 during UTI. Development and application of the co-culture model described in this study is thus useful to define the degree of contact dependency of biomarker production to UPEC, and highlights the relevance of histotypic co-cultures in studying complex host-pathogen interactions.

Bovine colostrum modulates cytokine production in human peripheral blood mononuclear cells stimulated with lipopolysaccharide and phytohemagglutinin

Bovine colostrum has been shown to influence the cytokine production of bovine leukocytes. However, it remains unknown whether processed bovine colostrum, a supplement popular among athletes to enhance immune function, is able to modulate cytokine secretion of human lymphocytes and monocytes. The aim of this investigation was to determine the influence of a commercially available bovine colostrum protein concentrate (CPC) to stimulate cytokine production by human peripheral blood mononuclear cells (PBMCs). Blood was sampled from four healthy male endurance athletes who had abstained from exercise for 48 h. PBMCs were separated and cultured with bovine CPC concentrations of 0 (control), 1.25, 2.5, and 5% with and without lipopolysaccharide (LPS) (3 microg/mL) and phytohemagglutinin (PHA) (2.5 microg/mL). Cell supernatants were collected at 6 and 24 h of culture for the determination of tumor necrosis factor (TNF), interferon (IFN)-gamma, interleukin (IL)-10, IL-6, IL-4, and IL-2 concentrations. Bovine CPC significantly stimulated the release of IFN-gamma, IL-10, and IL-2 (p < 0.03). The addition of LPS to PBMCs cocultured with bovine CPC significantly stimulated the release of IL-2 and inhibited the early release of TNF, IL-6, and IL-4 (p < 0.02). Phytohemagglutinin stimulation in combination with bovine CPC significantly increased the secretion of IL-10 and IL-2 at 6 h of culture and inhibited IFN-gamma and TNF (p < 0.05). This data show that a commercial bovine CPC is able to modulate in vitro cytokine production of human PBMCs. Alterations in cytokine secretion may be a potential mechanism for reported benefits associated with supplementation.

The Expanded Human Kallikrein (KLK) gene family : genomic organisation, tissue specific expression and potential functions

The tissue kallikreins are serine proteases encoded by highly conserved multigene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13 26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5 or 3 untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25–44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.