Modulation Of Human Peripheral Blood Immune Cell Subtypes And Monocyte-endothelial Cell Adhesion By Insulin

BackgroundInflammation is the crucial basis for insulin resistance and atherogenesis in obese and diabetic states, leading to increased risk of cardiovascular diseases. To down-regulate the inflammation level within the human body is an important issue in combating insulin resistance and preventing atherosclerosis.Traditionally recognized as a metabolism regulating hormone, insulin has also been shown recently to exert potent anti-inflammatory and anti-oxidative effects. Recently, cytological and immunological studies revealed various unique subtypes of the immune cells, which play important immune modulating effects, either inducing or suppressing the inflammation level. Of these, special attentions were paid to the regulatory T cells (Treg), inflammatory monocytes (CD16+), and CD146+ T cells. Whether insulin could exert its anti-inflammatory effects through modulation of these cell subtypes remains unclear. Meantime, it has been shown that insulin therapy in the long term could increase the peripheral blood CD34+ stem/progenitor cell level. Whether this effect was a direct effect of insulin or secondary to overall metabolic improvement by insulin still needs to be clarified.Insulin has been shown to be closely related to atherogenesis at various stages and also insulin resistance. Based on our previous data and literature, we raised the hypothesis that insulin may directly or indirectly rectify insulin resistance. (Medical Hypothesis, 2009) Besides, we are also interested in the effect of insulin on the first step of atherosclerosis which is also part of general inflammatory response, i.e. endothelial dysfunction and the adhesion of monocytes to the endothelial cells.Here in the present study we sought to comprehensively explore the effects of insulin on various subtypes of immune cells, and the distribution of these cells in the overall population. The effect of insulin on monocyte-endothelial cells adhesion and the insulin signaling pathways were also studied.MethodsPartⅠFlow cytometry analysis of human peripheral blood immune cell subtypes and optimizationIn order to minimize the effects of interrupting factors, we compared the staining stability of different staining strategies, including the effects of preservation time, the effects of whole blood staining with red blood cell lysis versus mononuclear cell staining. We also did preliminary observation of the expression of TLR2 and TLR4 on different cell subtypes.PartⅡThe distribution of immune cell subtypes within metabolic syndrome and type 2 diabetes patients and evolvement with age Volunteer subjects were recruited according to the inclusion criteria and exclusion criteria and their demographic and baseline physiological data were collected. After overnight fasting, blood samples were collected from the subjects. The glucose and triglycerides levels were measured. The mononuclear cells (MNCs) were collected by Ficoll density gradient centrifugation. The cells were stained with various directly labeled antibody combinations and assessed by flow cytometry. The cell marker combinations included: Treg cells (CD4+/CD25+/FoxP3+); inflammatory monocytes (CD14+/CD16+); CD146+ T cells (CD3+/CD146+); CD34+ stem/progenitor cells. The expression levels of TLR2 and TLR4 were also assessed by their mean fluorescent intensity. The flow cytometry results were compared with the metabolic parameters and demographic data. The results were also analyzed by comparing among different groups (diabetic, obese or lean).PartⅢThe effects of insulin infusion on immune cell subtypes levelsTen non-diabetic obese volunteers were recruited in the study. After overnight fasting, introvenous insulin infusion was administered with concomitant glucose infusion to achieve a state of hyperinsulinemia and normoglycemia. Blood samples were collected at baseline and 2, 4, 8, 14 and 16 hours after the initiation. Mononuclear cells from the 0 (baseline, right before infusion), 4 and 8 hour samples were isolated and stained for flow cytometry observation. The cells were fixed after staining and stored at 4℃overnight. The samples were assessed together the following day. The same patient was given glucose infusion (at the same rate as the insulin infusion procedure) or saline infusion after 2 weeks. The cell levels as well as theTLR2 and TLR4 abundance were compared among different time points. NF-κB nuclear translocation was assessed by imaging flow cytometry, and plasma MCP-1 and MMP-9 levels were determined by ELISA.PartⅣEffects of insulin on THP-1 monocyte- HAEC adhesion and mechanismThe effects of insulin and/or LPS on THP-1– HAEC adhesion was assessed. First, HAECs were plated into 96 well plates and allowed to reach confluence. THP-1 cells were labeled with CFDA-SE dye and added to each well and respective treatments were added and incubated for 4 hours. The unattached cells were washed away and the attached cells were trypinized and the fluorescent intensity were determined to reflect the amount of attached THP-1 cells.In a separate larger scale adhesion experiment, the attached cells were sorted by flow cytometer to obtain the THP-1 cells and the HAECs. mRNA was collected and relative expression of ICAM-1 and TNF-αin HAECs as well as TNF-αand SOCS-3 in THP-1 cells were assessed by real time PCR.ResultsPartⅠThe cell staining quality was best preserved by immediate staining, fixation and 4℃storage within 3 days. Staining of Ficoll isolated mononuclear cells was more stable and better preserved the CD16+ expression than staining whole blood. We detected similar levels of immune cell subtypes as reported. TLR2 and TLR4 were relatively abundant on monocytes, but not detectable on lymphocytes or CD34+ stem/progenitor cells.PartⅡCD146+ T cells level was higher in the type 2 diabetes mellitus (T2DM) group compared with non-diabetic obese group and the healthy control group. While the CD34+ stem/progenitor cells had a slight trend toward decrease and TLR2, TLR4 tended to increase in the T2DM group, the differences were not significant. There were no obvious differences of Treg cells and CD16+ monocytes among T2DM, non-diabetic obese and healthy subjects.Further analysis revealed that CD34+ cells level was inversely correlated with age, while CD146+ T cells level was correlated with age. CD14+/CD16+ cells level was inversely correlated with serum triglycerides level, and Treg was inversely correlated with blood glucose level.PartⅢCompared with saline or glucose infusion, insulin infusion significantly upregulated the Treg level and downregulated the CD16+ inflammatory monocytes level. Insulin infusion for a duration of 8 hours didn't produce significant changes on the CD34+ stem/progenitor cells level or the TLR2, TLR4 expression on monocyte surface.Meantime, insulin infusion suppressed the NF-κB nuclear translocation of monocytes and the plama levels of MMP-9 and MCP-1.PartⅣLPS significantly promoted the adhesion of THP-1 monocytes to the human aortic endothelial cells over a duration of 4 hours, while insulin dose-dependently suppressed the LPS effect.Insulin downregulated the ICAM-1 and TNF-αexpression of HAEC and suppressed the up-regulatory effect of LPS after 4 hours of treatment. Insulin downregulated the expression of TNF-αand SOCS-3 by THP-1 cells and suppressed the up-regulatory effect of LPS.Conclusions1. For comparison of human peripheral blood immune cell subtypes at different time points, to stain the Ficoll isolated mononuclear cells immediately and fix them for detection within 3 days can minimize the effect of confounding factors.2. Human peripheral blood immune cell subtypes were altered at states of metabolic syndrome, type 2 diabetes and aging. CD146+ T cells are upregulated in type 2 diabetes state and increase with age, Tregs decrease with increasing hyperglycemia, and CD34+ stem/progenitor cells decrease with aging. These altogether indicate pro-inflammatory states in metabolic syndrome, type 2 diabetes and aging, as well as the decreased anti-inflammatory and regenerating capacity that accompany aging.3. Insulin infusion could upregulate the peripheral blood Treg cells level and downregulate the peripheral blood CD16+ mononuclear cells level, indicating a new mechanism of insulin's anti-inflammatory effect. And insulin's inhibition of NF-κB translocation and MMP-9, MCP-1 secretion was once again confirmed.4. Insulin inhibits monocyte-endothelial cell adhesion, and suppresses ICAM-1 expression, meanwhile insulin downregulates the TNF-αand SOCS-3 exppression, indicating a potential way that insulin improves insulin resistance.