Effects Of Post-hemorrhagic Shock Mesenteric Lymph On The Immune Function Of Spleen Dendritic Cells

Background: Immunologic dysfunction is one of major contributors of systemic inflammatory response syndrome(SIRS), the ensuing multiple organ dysfunction syndrome(MODS) and even death in the episode of severe hemorrhagic shock. A series of immune cells in the body release a large amount of cytokines against the invasion of foreign antigens none-specifically. Among these cells, antigen presenting cells such as dendritic cells(DCs) play the effects of anti-infectious by the means of presenting invasion signal to T lymphocytes and initiating the process of permanent specific immunity. It has been reported that the first affected innate immunity in the process of hemorrhagic shock challenge results in the immune depression and inflammation diffusion sequentially, which is considered to be one of important immunological basis of SIRS, MODS and even sepsis. The return of mesenteric lymph into systemic circulation is the pivotal cause of tissue injury, and organ dysfunction during the development of hemorrhagic shock. Previous studies indicated that the interruption of shock lymph return into systemic circulation can attenuate architecture damage of spleen and thymus and cellular immunity. However, the relationship of shock lymph return and DCs, which initiates cellular immunity through antigen presentation, is remain to be determined.Objective: Based on a mouse model of hemorrhagic shock, DCs were harvested using immunomagnetic separation. In order to reveal the moderating effects of shock mesenteric lymph on the immune function of spleen DCs tentatively, the present study was observed the influence of mesenteric lymph drainage or mesenteric lymph incubation on cytokines producing ability of DCs stimulated by LPS.Methods: Male BALB/C mice were randomized into 3 groups: sham group, shock group, shock plus lymph drainage(shock+drainage) group. The mice in shock and shock+drainage groups were performed hemorrhagic shock(maintaining 40±2 mm Hg for 60 min and then treated by fluid resuscitation). In shock+drainage group mesenteric lymph was drained after completion of fluid resuscitation immediately. At 3h after completion of fluid resuscitation(at the corresponding time point in sham group) in shock and shock+drainage groups the spleens were harvested from mice and DCs were isolated using immunomagnetic separation(anti-CD11c) and then cultured in vitro. The release of TNF-α, IL-10, IL-12 of DCs were observed with or without stimulated by LPS. Part of male BALB/C mice conducted into hemorrhagic shock conditions were drained post-hemorrhagic shock mesenteric lymph during hypotension(PHSML-H), part of those mice were drained post-hemorrhagic shock mesenteric lymph after resuscitation(PHSML-R). The normal mice were drained normal mesenteric lymph(NML). The normal spleens were harvested from normal mice and DCs were isolated using immunomagnetic separation and then cultured with PHSML-H, PHSML-R and NML in vitro. The LPS served as a positive control. The release of TNF-α, IL-10, IL-12 of DCs was observed with or without stimulated by LPS at 3, 6, 12 and 24 h time point.Results: The purity of CD11 c positive cells obtained from spleen using immuno- magnetic separation accounts for upwards of 90 percent. There was none of significant difference in content of TNF-α, IL-10 and IL-12 in supernatant of cultured DCs for 24 h among sham, shock, shock+drainage group(P>0.05). After incubated with LPS for 24 h, the content of TNF-α, IL-10 and IL-12 in supernatant of sham and shock+drainage groups and the content of TNF-α and IL-10 in supernatant of shock group increased significantly compared with that of untreated conditions(P<0.05). More importantly than all of that, the results indicated that the content of TNF-α and IL-12 of shock group, the content of IL-12 of shock+drainage group decreased significantly compared with that of sham group(P<0.05); the content of TNF-α of shock+drainage group increased significantly compared with that of shock group(P<0.05). The results from acute isolated DCs treated with mesenteric lymph indicated that the content of TNF-α and IL-10 in supernatant increased at several time points after treated with PHSML-H or PHSML-R, the content of IL-12 has no significant change, but the content of TNF-α and IL-10 decreased significantly compared with that of positive control. DCs incubated with different factors stimulated by LPS again, the ability of TNF-α, IL-10 and IL-12 production increased after treated by PHSML-H or PHSML-R at some extent, but still decreased compared with positive control of LPS, especially treated by PHSML-R.Conclusion: Hemorrhagic shock leads to the dysfunction of cytokines secretion of spleen DCs, which is one of major contributors of immune suppression during the episode of hemorrhagic shock. Lymph drainage to reduce the return of shock mesenteric lymph into systemic circulation has a certain advantage to restore DCs function, which may be one of mechanisms of blocking the return of shock mesenteric lymph into systemic circulation to improve immune function.