The Potential Importance Of Apolipoprotein M On The Accumulation Of CD4~+ T-lymphocytes In The Murine Spleen

Part I Accumulation of CD4~+ T-lymphocytes in the spleen of Apo M+/+ miceObjective: Taking advantage of wild type(Apo M+/+) and Apo M deficient(Apo M-/-) mice, to investigate whether the genetic knock-out Apo M affects the T-lymphocytes and their subgroups in the spleen with or without challenge of lipopolysaccharide(LPS).Methods: Male Apo M+/+ and Apo M-/- mice at 6-8 weeks were randomly assigned to receive intraperitoneally(i.p.) injection of either vehicle(0.9% saline; control group), 5mg/kg LPS, or 10mg/kg LPS for three times with a 24-hrs interval. All mice were sacrificed at 96 hrs after the first i.p. injection and the spleens were collected for preparing the single-cell suspension. The prepared splenic single-cells suspension(1.0×107/ml, 100μl) were incubated with FITC-conjugated anti-CD3, PE-Cy5 conjugated anti-CD4, PE-Cy7 conjugated anti-CD8 for 30 minutes at 4℃ protected from light, and washed twice with PBS. Then the stained cells were analyzed by flow cytometry immediately.Results:The splenic CD3+ T-lymphocytes were dramatically decreased in both Apo M-/- mice and Apo M+/+ mice(P<0.001, P<0.01, respectively), although there were no statistical differences occurred in the Apo M gene deficient mice compared to the wild type. The basal proportion of CD4~+ T-lymphocytes were significantly lower in Apo M-/- mice than in Apo M+/+ mice(P<0.01). After administration of LPS at both concentrations of 5 mg/kg and 10 mg/kg, the proportion of CD4~+ T-lymphocytes were significantly decreased in the Apo M-/- and Apo M+/+ mice(both P<0.001). Furthermore, the decrease of CD4~+ T-lymphocytes in Apo M-/- mice was much more than those in Apo M+/+ mice(P<0.05, P<0.01, respectively). There was no significant difference for the splenic CD8+ T-lymphocytes between the Apo M+/+ and Apo M-/- mice in the control group and LPS-challenged mice. The similar phenomena with CD4~+ T-lymphocytes were found in the ratio of CD4~+/CD8+ T-lymphocytes, although there was only slightly decrease of basal CD4~+/CD8+ ratio in Apo M-/- mice compared to the Apo M+/+ mice(P=0.0556).Conclusion: Apo M may facilitate the maintenance of CD4~+ T-lymphocytes in the spleen, even it can accumulate the splenic CD4~+ T-lymphocytes and increase the splenic CD4~+/CD8+ T-lymphocytes ratio after LPS administration. Part II Apo M facilitating normal immune response may be associated with the accumulation of CD4~+ T-lymphocytes in murine spleenObjective: Taking advantage of wild type(Apo M+/+) and Apo M deficient(Apo M-/-) mice, to determinate whether the genetic knock-out Apo M affects the m RNA expressions of TNF-α and MCP-1 in liver tissue, and the protein levels in the serum with or without the lipopolysaccharide(LPS) administration.Methods: Male Apo M+/+ and Apo M-/- mice at 6-8 weeks were randomly assigned to receive intraperitoneally(i.p.) injection of either vehicle(0.9% saline; control group), 5mg/kg LPS, or 10mg/kg LPS for three times with a 24-hrs interval. All mice were sacrificed at 96 hrs after the first i.p. injection. The heptic m RNA levels of TNF-α and MCP-1 were quantified by real-time PCR, and the serum protein levels TNF-α and MCP-1 were determined by Luminex x MAP technology.Results:The hepatic m RNA levels of TNF-α were significantly increased after LPS administration in Apo M+/+ and Apo M-/- mice(P<0.001, P<0.001, respectively). The hepatic m RNA levels of MCP-1 were also significantly increased after LPS administration in Apo M+/+ and Apo M-/- mice(P<0.01, P<0.001, respectively). However, there were no statistical differences in hepatic TNF-α and MCP-1 m RNA levels between the Apo M+/+ and Apo M-/- mice before and after LPS administration. The serum levels of TNF-α and MCP-1 in Apo M+/+ mice significantly increased after LPS administration(P<0.001, P<0.01, respectively). Similarly, the serum levels of TNF-α and MCP-1 in Apo M-/- mice significantly increased after LPS administration(both P<0.05). However, the serum levels of TNF-α had no statistical difference between the Apo M+/+ and Apo M-/- mice in the control group and 5mg/kg LPS-treated mice. The serum levels of TNF-α in Apo M-/- mice were extremely lower after 10mg/kg LPS administration compared to Apo M+/+ mice(P<0.01). The serum levels of MCP-1 in Apo M-/- mice was moderately higher than those in the Apo M-/- mice(P<0.05), while no statistical differences were observed between Apo M+/+ and Apo M-/- mice after LPS administration.Conclusion: Apo M can facilitate the normal immune response in vivo, which may be associated with the accumulation of CD4~+ T-lymphocytes in the murine spleen.Part Ⅲ Apo M may accumulate the CD4~+ T-lymphocytes in the murine spleen by increasing the expression of VE-cadherin in the spleenObjective: Taking advantage of wild type(Apo M+/+) and Apo M deficient(Apo M-/-) mice, to determinate whether the genetic knock-out Apo M affects the splenic m RNA levels of E-cadherin, VE-cadherin, S1PR1~5, CXCR3, CXCR4 before and after LPS treament.Methods: Male Apo M+/+ and Apo M-/- mice at 6-8 weeks were randomly assigned to receive intraperitoneally(i.p.) injection of(0.9% saline as the control group) 10mg/kg LPS. The mice were sacrificed at 1, 3, 6, 12 and 24 hrs after the i.p. injection and the splenic m RNA levels of E-cadherin, VE-cadherin, S1PR1~5, CXCR3, CXCR4 were determined by Real-time PCR before and after LPS treament.Restults: After the LPS treatment, the splenic m RNA levels of the E-cadherin and VE-cadherin were significantly decreased over time in Apo M+/+ and Apo M-/- mice(both P<0.01), but there were no significant difference in the m RNA levels of E-cadherin between Apo M+/+ and Apo M-/- mice at each time point(P>0.05). Interestingly, the splenic m RNA levels of VE-cadherin in Apo M-/- mice were significantly lower than Apo M+/+ mice at 0h, 1h, 3h, 6h and 12h(P<0.05). The splenic m RNA levels of S1PR1, S1PR3 and S1PR4 in Apo M+/+ and Apo M-/- mice were decreased over time(both P<0.01); At each time point, there were no significant difference in the splenic m RNA levels of S1PR1,S1PR3 and S1PR4 between the Apo M+/+ and Apo M-/- mice(P>0.05). The splenic m RNA levels of the CXCR3 and CXCR4 were significantly decreased over time in Apo M+/+ and Apo M-/- mice(both P<0.01), but there were no significant difference between the Apo M+/+ and Apo M-/-mice at each time point(P>0.05).Conclusion: Apo M may protect the integrity of the vascular endothelial cells by increasing the expression of VE-cadherin in murine spleen, thereby accumulating the CD4~+ T-lymphocytes in the spleen; CXCR3 and CXCR4 may not participate in the accumulation of CD4~+ T-lymphocytes in the spleen.