a separate study, we have also documented calcium dependent, e tracellular selleck catalog signal reg ulated kinase mediated MC proliferation in response to Hcy. These prior studies suggest that elevated levels of Hcy may contribute to MC proliferation or apoptosis, processes that may mediate kidney injury and contribute to chronic kidney disease. Given the observation that MC are able to secrete chemok ines in response to e tracellular stimuli, it has been pro posed that these chemokines serve an important role of mediating leukocyte infiltration that participate in glomerular response to injury and in the progression of kidney disease. Indeed, in circumstances where MC are e posed to no ious stimuli, they secrete macrophage inflammatory protein 2 that mediate neutrophil infiltration.
MIP 2 is a potent neutrophil chemotactic stimulant that is typically secreted by macrophages in response to inflam mation induced by endoto in. MIP 2 is a member of the C C chemokine sub family of cytokines that includes IL 8 and KC among others. Structur ally, C C chemokines are characterised by possessing one amino acid residue between the first two conserved cysteine residues. This is in contrast to the CC chemokines in which the first two conserved cysteine residues are adjacent. The C C chemokines are capable of regulating all stages of neutrophil recruitment to inflam matory or injury foci. their actions are mediated by C C receptors. MCs are capable of producing and secreting MIP 2 and, MC derived MIP 2 has been demonstrated to mediate glomerulonephritis in a rat model of the aforementioned disorder.
Accordingly, the current study had two major objectives namely a to e amine the role of Hhcy in cytokine production by MC and b to define some of the signalling mechanism that may participate in this proc esses. In particular, given our earlier observation that MC response to e tracellular Hcy involves activation of MAPK, the role of MAPK activation in MIP 2 production by MC was evaluated. Methods Cell Culture Sprague Dawley rat MCs were isolated by the sieving method. The cells were cultured in Dulbeccos Modi fied Eagles Medium supplemented with 10% fetal bovine serum, streptomycin, penicillin and 2 mM glutamine at 37 C in 95% air 5% CO2. Cells from passage 8 15 were used throughout these studies. All other chemicals were obtained from Sigma Aldrich unless oth erwise indicated.
Cytokine Antibody Array A rat cytokine antibody array was employed to assess cytokine production by MC following e posure to Hcy. The protocol was e ecuted according to the manufac turers specifications. Briefly, MCs were initially seeded unto plastic dishes in DMEM supplemented with Batimastat FBS. Brefeldin A 20350-15-6 Subsequently, cultures were serum starved overnight, followed by incubation in medium with L cysteine or Hcy for 24 hours at 37 C. The cells were harvested and cellular protein was prepared from lysates as described below. Protein form lysates was used to determine chemokine production using rat cytokine antibody arra