Effect Of L-arginine On Phaenotype And Function Of Dendritic Cells

Background and objective Sepsis is a systemic inflammatory response syndrome(SIRS) caused by infection, and sepsis is the common complication caused by serioustrauma, burn and infection．Septic shock and multiorgan dysfunction are the mostcommon causes of death in the patients with sepsis. Recent studies had found that theoccurrence and development of sepsis were related to the immune dysfunction closely.The restoring immune homeostasis and environmental balance were very important intreatment and improvement of their prognosis. Dendritic cell (DC) is the mostpowerful antigen-presenting cell (APC), so it is considered to be the initiator of theimmune response. As the main regulator of the immune response, DC can activate theinnate immune system to start a longer-lasting adaptive immune, induce thymus toremove autoreactive T cells, differentiation regulatory T cells, and maintain their ownimmune tolerance. In sepsis pathogenesis, dendritic cell plays an important role inimmune regulation, and it will gradually become possible to change the prognosis ofpatients with sepsis by changing the DC number and function. In this study, peripheralblood monocyte-derived dendritic cell was researched. In order to clear the regulativemechanism of L-arginine to dendritic cell and the application of L-arginine (L-arg) asimmunomodulatory drugs in the course of treatment of sepsis, the regulative functionof L-arginine to dendritic cells was observed from the cell phenotype and antigen-presenting ability.Methods DC were separated from human monocytes by culturing in presence ofcytokines (GM-CSF, IL-4) after twelve hours, and divided into group A, B, C, D, Eand F randomly. In the6th day, Group A was treated with cytokines (GM-CSF, IL-4)only as control group, and group B, C, D, E, F were treated with TNF-α10ng/ml,L-arg20μmol/ml, TNF-α10ng/ml+L-arg20μmol/ml, TNF-α10ng/ml+L-arg50μmol/ml, TNF-α10ng/ml+L-arg100μmol/ml respectively. The change in morphology wasobserved by inverted microscope everyday. On the first day and the8th day thecultivating DC in the six groups was taken to adjust the cell concentration of5×106/ml after washing in PBS respectively.100μl suspension was put into the testtube for cytometry,20μl antibody of FITC-CD1a, FITC-CD80, FITC-HLA-DR andPE-CD83was added. Flow cytometry was used to detect cell phenotype after washingwith PBS again. PBMC was separated from20ml peripheral blood of healthy adultsby the above method, and non-adherent cells was taken to co-culture with six groups’DC on the8th day for4days,10μl MTT solution was added to each hole, and100μldimethyl sulfoxide (DMSO) was added after1hour, optical density (OD) value wasmeasured in a microplate reader at490nm.Results The cell of group B (TNF-α10ng/ml), C (L-arg20μmol/ml), D(TNF-α10ng/ml+L-arg20μmol/ml), E (TNF-α10ng/ml+L-arg50μmol/ml), F(TNF-α10ng/ml+L-arg100μmol/ml) developed characteristic dendritic cellsmorphology except control group. Compared with group A, CD80, CD83, CD1a andHLA-DR were increased in group B, C, D, E and F obviously (all P＜0.05). However,there was no significant difference between group B and C (P＞0.05). The samesituation was between D and C (P＞0.05). Compared with group B, CD80, CD83,CD1a and HLA-DR were increased in group E and F obviously (all P＜0.05).Compared with group D, CD80, CD83, CD1a and HLA-DR were also increased ingroups E and F (all P＜0.05). But four kinds of phenotypic above-mentionedexpression were no significant difference between group E and F. In mixedlymphocyte reactions, the OD value of group B, C, D, E and F was0.36±0.04,0.32±0.04,0.43±0.05,0.88±0.06,0.86±0.07respectively, while the OD value ofgroup A was0.09±0.03(all P＜0.05). Compared with group B, the OD valueincreased in group D, E and F obviously(all P＜0.05). The OD value is no significantdifference between group E and F (P＞0.05). Compared with group D, the OD valueincreased in group E and F obviously(all P＜0.05). The OD value was no significantdifference between group B and C (all P＞0.05).Conclusion L-arginine can promote the differentiation and maturation of DC. DC treated with L-arginine presented higher lever of phaenotype and antigen-presentingability than DC without L-arginine. It is expected to play an important role in thepyemic immunomodulatory therapy.