Biological Characters Of Macrophages In Severe Combined Immunodeficiency Mice With Endotoxemia

Background and objectives Sepsis, a common complication of patients with severe infection, trauma, burn or surgery, can develop to septic shock and multiple organ dysfunction syndrome which cause death of severe patients. The cases of patients with sepsis are 18,000,000 per year all around the world, 750,000 per year in USA, and 135,000 per year in Europe. The death cases are 14,000 per day all around the world, 215,000 per year in USA. Except death caused by cardiac diseases, sepsis became the main death cause in USA. Although the recognition and treatment for sepsis improved, the death rate of sepsis is still so high that it becomes the difficult problem of modern critical care medicine. The basic reason is that the mechanism of sepsis is unclear, so early and efficient prevention and treatment are lacked.Uncontrolled inflammation leads bad prognosis of sepsis. Innate immune cells are important participants in inflammation, and abnormal activation of innate immune cells can cause uncontrolled inflammation. The pathophysiology of diseases such as infection associated hemophilia syndrome (IAHS), severe acute respiratory syndrome (SARS) and severe H1N1 is characterized by uncontrolled inflammation. Abnormal activation of innate immune cells is one of the important causes for those diseases. However, what causes abnormal activation of innate immune cells needs further study. Recent researches showed the complexity of interaction between innate immune cells and adaptive immune cells. Our previous study showed T cell (-) nude mice had more severe inflammation than wild mice after RSV infection. This result suggested T cells may inhibit inflammation. Kim's research showed lymphocytes inhibited activation of innate immune cells in early inflammation. Thus, we take this into speculation: sepsis, IAHS and SARS may be caused by abnormal activation of innate immune cells triggered by differential deficiencies of lymphocytes. Researches on activation of innate immune cells in severe combined immune deficiency mice with sepsis may support a new way for mechanism of those diseases.Endotoxin plays an important role in Gram negative bacteria infection. Endotoxemia can exist without bacteria tested in blood that it can be independent pathogenic factors of sepsis. Endotoxin has so spread and complex biological affect that uncontrolled inflammation, immunologic disorders, hypermetabolic state, and multiple organ injuries can be triggered by endotoxin directly or indirectly. The pathological immune response of severe combined immune deficiency mice with Gram negative bacteria infection depends on persistent reproduction diffusion of bacteria and immune response of the body. However, this model will take interference to study on immune response singly. To avoid such interference, we set up endotoxemia model by injecting LPS into SCID mice to study biological characters of macrophages. Therefore, effect of lymphocytes defect on inflammation and macrophage activation and the mechanism can be observed.Part one Comparation of inflammation between BALB/c mouse and SCID mouse with endotoxemia induced by lipopolysaccharideObjective To compare the inflammation between wild BALB/c mice and severe combined immunodeficiency (SCID) mice with endotoxemia induced by lipopolysaccharide (LPS).Methods Endotoxemia models of wild and SCID mice were established by injecting LPS intraperitoneally. Serum, liver and lung were taken before and after LPS injection. Alanine transarninase (ALT), aspartate aminotransferase (AST) and blood urea nitrogen (BUN) levels in serum were measured by automatic biochemical analyzer. Liver and lung inflammation injury were observed by H.E staining. Inflammatory and anti-inflammatory cytokines in serum and liver were detected by Cytometric Bead Array (CBA) or ELISA. Results All of SCID mice (8/8) were dead at 12～24 h after LPS injection, and only one BALB/c mouse (1/8) was dead. ALT and AST levels of SCID mice 12 h after LPS injection were higer than those of BALB/c mice, but there was no difference of BUN levels between them. The blind liver and lung pathology scores of SCID mice were higher than those of BALB/c mice. Levels of TNF-α, IFN-γ, IL-10, IL-6, MCP-1 and IL-10 in serum increased significantly after LPS injection, and the cytokine levels of SCID mice were higher than those of BALB/c mice. Levels of TNF-αin liver exacts at 3 h after LPS injection and levels of IL-10 in liver exacts at 6 h after LPS injection were higher in SCID mice than in BALB/c mice.Conclusions After LPS injection, compared with BALB/c mice, SCID mice secreted more inflammatory and anti-inflammatory cytokines which led SCID mice into uncontrolled inflammation. Such inflammation caused severe organ injuries and death of SCID mice. Abnormal augmentation of innate immune response under condition of lymphocyte defect maybe the important reason for severe systemic inflammatory response syndrome which puts the body in danger.Part two Comparation of macrophage activation between BALB/c mice and SCID mice with endotoxemia induced by LPSObjective To compare activation of peritoneal macrophages between wild BALB/c mice and SCID mice with endotoxemia induced by LPS. And to observe the effect of lymphocytes defect on macrophage activation.Methods Endotoxemia models of wild mice and severe combined immunodeficiency (SCID) mice were established by injecting LPS intraperitoneally. Peritoneal fluids, peritoneal macrophages and spleen NK cells were taken. Peritoneal macrophages from both mice were stimulated by LPS in vitro. Supertants were taken. Cytokines from peritoneal fluids and supertants were tested by ELISA; TNF-α, IL-10 mRNA expression of peritoneal macrophages were tested by real-time PCR. Phagocytosis of macrophages was tested by phagocytic test of chicken red cells. Surface markers such as TLR4, MHC-II, CD80, CD86 and CD40 of macrophages and intracellular cytokine IFN-γof NK cells were tested by flow cytometry.Results Levels of peritoneal TNF-αwere higher at 1 h and levels of peritoneal IL-10 were lower at 3 h after LPS injected from SCID mice than those from BALB/c mice. TNF-αmRNA expression of peritoneal macrophages from SCID mice was higher and IL-10 mRNA expression was lower than peritoneal macrophages from BALB/c mice after and even before LPS injection. Phagocytosis of macrophages from BALB/c mice was stronger than those from SCID mice. Expression of surface markers such as TLR4, MHC-II, CD80 and CD86 of peritoneal macrophages increased in BALB/c mice after LPS injected, and there were no obvious difference in expression of such markers in SCID mice before and after LPS injection. And expression of CD40 of peritoneal macrophages from both mice decreased after LPS injection.Conclusions Compared with wild mice, co-stimulatory molecules CD80 and CD86 of peritoneal macrophages increased spontaneously and phagocytosis of peritoneal macrophages decreased in SCID mice. After LPS injection, surface markers of peritoneal macrophages such as TLR4, MHC-II, CD80 and CD86 had no change in SCID mice. Inflammatory cytokines secreted by peritoneal macrophages and intracellular IFN-γof NK cells were more in SCID mice. Those were the main characters of uncontrolled inflammation of SCID mice with endotoxemia.Part three Role of Mitogen-activated protein kinase1 in adaptive suppression of macrophages activationObjective To discuss the role of SOCS1,SOCS3 and MKP-1 in adaptive control of macrophages activation.Methods Endotoxemia models of BALB/c mice and severe combined immunodeficiency (SCID) mice were established by injecting LPS intraperitoneally. Peritoneal macrophages were taken for measuring SOCS1, SOCS3 and MKP-1 mRNA expression. Thioglycollate-stimulated peritoneal macrophages were taken for LPS stimulation in vitro separately or mixed with pan-T cells. Supertants were taken for measuring TNF-α, IL-10 and IL-10. Peritoneal macrophages were tested for MKP-1 mRNA and protein expression.Results SOCS1 and SOCS3 mRNA expression of peritoneal macrophages increased after LPS injection. And SOCS1 mRNA expression was higher in SCID mice than in BALB/c mice at 3 h,6 h after LPS injection, SOCS3 mRNA expression was higher in BALB/c mice than in SCID mice at 1 h,12 h after LPS injection. At any time point, MKP-1 mRNA expression of peritoneal macrophages from BALB/c mice was higher than those from SCID mice. After LPS stimulation in vitro, separately macrophages secreted more TNF-αand IL-6 than mixed cell culture. And MKP-1 mRNA and protein expression of macrophages were higher in mixed cell culture.Conclusions Adaptive control of macrophages activation in mice with endotoxemia didn't depend on SOCS1 and SOCS3. Pan-T cells can inhibit activation of macrophages. After LPS stimulation, lymphocytes may promote MKP-1 expression of peritoneal macrophages, thus suppress inflammatory cytokines secretion of macrophages.