| Summary: | 碩士 === 國防醫學院 === 生理學研究所 === 96 === Klebsiella pneumoniae (K. pneumoniae) is one of the most common pathogens of nosocomial infection. K. pneumoniae infection of the brain was less reported than in other organs, but an increase in incidence and morbidity has been noted in Taiwan. The incidence of death (> 30%) and long-term neurological sequelae remain high. We have previously demonstrated that glial cells are important cellular sources of proinflammatory cytokines present in CSF in a rat of model of K. pneumoniae meningoencephalitis. The chemokines are classified into CXC, CC, CX3C, and C motif groups by protein structure and function. The CXC chemokines such as growth related oncogene (GRO, CXCL1) and macrophage inflammation protein-2 (MIP-2, CXCL2) are chemoattractant for neutrophils, while monocyte chemotractic protein-1(MCP-1, CCL2) are chemoattractant for monocytes. The chemokine receptors are reported to be involved in neurodegenerative diseases, but whether chemokines/receptors are expressed in brain cells (neurons , astrocytes or microglia) and whether their brain expression are changed during K.p infection remain unclear. In the present study, we further studied the role of chemokines and chemokine receptors in animal models and in primary cultures of rat brain. We found that the increases of WBC counts in CSF and blood were time-dependent. Leukocytosis was observed in CSF at 8 hrs after K.p-infectiont with a predominance of PMNs. The concentrations of neutrophil chemoattractant chemokine macrophage inflammation protein-2 (MIP-2 , CXCL2) in CSF are much higher than those in serum at 4 and 8 hr after K.p infection of the brain, suggesting a likely brain origin. The concentrations of another neutrophil chemoattractant chemokine growth related oncogene (GRO, CXCL1) in CSF also markedly increased as early as 4 hr after K.p infection. After K.p. infection, the mRNA expression of chemokines (GRO, MIP-2, and MCP-1) in brain tissue increased significantly in a time-dependent manner. However, mRNA level of their receptors only showed slight changes with CXCR1 and CXCR2 mRNA level upregulated while the CCR2 mRNA expression rapidly suppressed compared to sham within 8hrs after K.p. inoculation. Intracerebroventricular injection of vMIP-2 (viral macrophage inflammatory protein-2), a broad spectrum peptide antagonist of chemokine receptors, attenuated PMN recruitment into CSF. The highly elevated concentrations of GRO (CXCL1) in CSF induced by K.p. infection was attenuated by vMIP-2. The K.p.-induced upregulation of gene expression of CXCR2, but not CXCR1, in brain tissue at 8 hr after K.p infection was also attenuated by vMIP-2. To further delineated the source of CSF chemokines from brain cells instead of PMNs, we employed primary cultures, prepared from rat cerebral cortex tissue, for in vitro studies. Our primary cortical cultures consisted of neurons, astrocytes and microglia. Exposure of primary cortical cultures to K. pneumoniae caused a moderate decrease in cell viability. Exposure of primary cortical cultures to K. pneumoniae also resulted in elevated levels of nitric oxide / nitrite and reactive oxygen species in the culture media, indicating a functional activation of glia cells. The mRNA levels of GRO and MIP-2, both are PMN chemotracttant, in brain tissue were highly elevated (much higher than MCP) as early as 2 hr after K.p.infection. The K.p.-induced alteration in gene expression for chemokine receptors were to a lesser degree.
Double immunofluorescence staining showed that the CCR2 was expressed on cultured microglia. Taken together, our results suggest a rapid induction of chemokines and chemokine receptors in glial cells, which may play a role in PMN recritment into CSF and and subsequent cell/tissue injury following K. pneumonia infection of CNS.
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