Downregulation of CD40 ligand response in monocytes from sepsis patients

It has been suggested that a defective adaptive immune response contributes to septic immunosuppression. Here, the response of monocytes to CD40 ligand (CD40L) for patients with sepsis due to infection with gram-negative organisms has been analyzed. Compared to cells from controls, monocytes from septic patients showed significantly reduced production of tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-12 and were unable to acquire high levels of CD80 and CD86 molecules. These alterations were observed at the onset of sepsis and persisted at day 7. However, the ability of monocytes to respond to CD40L stimulation was partially but significantly restored in cells from patients who recovered from sepsis. In addition, costimulation of autologous CD4⁺ T lymphocytes by CD40L-activated monocytes from septic patients failed to induce cell proliferation and gamma interferon production. Finally, the ability of CD40L to rescue monocytes from apoptosis was severely impaired. We conclude that downregulation of the CD40L response may be an appropriate model for the monocyte alteration observed during septic immunosuppression and may help in the development of novel therapeutic strategies.     

Differential toll-like receptor expression after ex vivo lipopolysaccharide exposure in patients with sepsis and following surgical stress

INTRODUCTION: Monocytes from septic patients have a reduced capacity to respond to lipopolysaccharide (LPS). We examined whether the same response occurred after surgical injury, and whether this reduced activity was associated with differential monocyte toll-like receptor (TLR) expression. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from septic patients, patients undergoing surgery, and healthy volunteers. Cells were stimulated ex vivo with LPS (1 microg/ml) and stained for CD14, CD16, TLR-2, TLR-4, and HLA-DR surface expression. RESULTS: TLR-2 and -4 expressions were significantly increased in monocytes from both septic and surgical patients. While ex vivo LPS-stimulation significantly increased TNFalpha and IL-1beta production in PBMCs from surgical patients, LPS-stimulation decreased IL-1beta production from septic patients as compared to surgical and control patients. Ex vivo LPS-stimulation induced TLR-4 upregulation in monocytes from both surgical and control patients, but not from septic patients. HLA-DR expression in CD14+CD16+ monocytes was reduced only in septic patients. CONCLUSIONS: PBMCs from septic patients, but not following surgical injury, have a reduced capacity to respond to a secondary inflammatory signal, but this defect is not associated with reduced TLR-4 or CD14 expression.     

Aberrant activation profile of cytokines and mitogen-activated protein kinases in type 2 diabetic patients with nephropathy

Cytokine-induced inflammation is involved in the pathogenesis of type 2 diabetes mellitus (DM). We investigated plasma concentrations and ex vivo production of cytokines and chemokines, and intracellular signalling molecules, mitogen-activated protein kinases (MAPK) in T helper (Th) cells and monocytes in 94 type 2 diabetic patients with or without nephropathy and 20 healthy controls. Plasma concentrations of inflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-18 and chemokine CCL2 in patients with diabetic nephropathy (DN) were significantly higher than control subjects, while IL-10, CXCL8, CXCL9, CXCL10 and adiponectin concentrations of DN were significantly higher than patients without diabetic nephropathy (NDN) and control subjects (all P < 0.05). Plasma concentrations of TNF-alpha, IL-6, IL-10, IL-18, CCL2, CXCL8, CXCL9, CXCL10 and adiponectin exhibited significant positive correlation with urine albumin : creatinine ratio in DN patients. The percentage increases of ex vivo production of IL-6, CXCL8, CXCL10, CCL2 and CCL5 upon TNF-alpha activation were significantly higher in both NDN and DN patients than controls (all P < 0.05). The percentage increases in IL-18-induced phosphorylation of extracellular signal-regulated kinase (ERK) in Th cells of NDN and DN were significantly higher than controls (P < 0.05), while the percentage increase in TNF-alpha-induced phosphorylation of p38 MAPK in monocytes and IL-18-induced phosphorylation of p38 MAPK in Th cells and monocytes were significantly higher in NDN patients than controls. These results confirmed that the aberrant production of inflammatory cytokines and chemokines and differential activation of MAPK in different leucocytes are the underlying immunopathological mechanisms of type 2 DM patients with DN.     

Enhanced prostaglandin E2 production by monocytes in atopic dermatitis (AD) is not accompanied by enhanced production of IL-6, IL-10 or IL-12

AD is associated with a bias of the T helper cells to show increased IL-4 and reduced interferon-gamma (IFN-γ) production. The production of IFN-γ and IL-4 and the development of Th cells into either high IFN-γ or high IL-4 producers is strongly influenced by factors produced by antigen-presenting cells (APC), like IL-12 and prostaglandin E₂ (PGE₂). IL-12 selectively enhances IFN-γ production and favours the development of IFN-γ-producing Th cells, whereas PGE₂ selectively inhibits IFN-γ production by Th cells. The aim of this study was to test whether the increased IL-4/IFN-γ production ratio by Th cells in AD can be explained by an increased PGE₂/IL-12 production ratio by the APC. Monocytes were used as APC source. PGE₂ and IL-12 production by lipopolysaccharide (LPS)-stimulated monocytes from 12 AD patients and 12 non-atopic controls was determined using two complementary experimental systems, whole blood cultures and purified monocytes. In addition, we determined IL-6 production as a measure of monocyte activation, and IL-10 production because IL-12 production by monocytes is highly influenced by endogenously produced IL-10. The monocytes from AD patients showed normal production levels of IL-6 and IL-10, a two-fold, but non-significant decrease in IL-12 production, and a significantly (three-fold) higher PGE₂ production than those from non-atopic controls. Here we show for the first time that enhanced PGE₂ production by monocytes in AD is not accompanied by a general rise in cytokine production. We conclude that AD is indeed associated with an increased PGE₂/IL-12 production ratio by monocytes.     

Prostaglandin E2 down-regulation of T cell IL-2 production is independent of IL-10 during gram-negative sepsis

The present study ascertained the role of PGE2 in sepsis associated modulation of IL-2 and IL-10 production by T cells. Sepsis was induced in 225-250 g male rats (Sprague Dawley) by implanting fecal pellets containing Escherichia coli (100-150 CFU) and Bacteroides fragilis (10(4) CFU) into the abdominal cavity. Animals implanted with fecal pellets without the bacteria were designated as sterile. For the assessment of PGE2 role in sepsis, a group of septic and sterile rats were pretreated with indomethacin to inhibit endogenous PGE2 synthesis. Splenic T cells were obtained 48 h after septic or sterile implantations, and their IL-2 and IL-10 production was measured. A significant suppression in the levels of IL-2 production and mRNA expression was observed in T cells from septic rats compared with the T cells from sterile and control rats. IL-10 protein and mRNA expression was found to be significantly higher in septic rat T cell compared to sterile and control rat T cells. Although, treatment of animals with indomethacin significantly prevented the sepsis-related suppression of IL-2 production, such treatment of animals was associated with a further upregulation of IL-10 production. These data suggest that although PGE2 released during sepsis can cause T cell IL-2 down-regulation, it may not mediate the T cell IL-10 upregulation. The IL-2 down-regulation may not be an effect of IL-10 upregulation.     

Allergen-specific T cells from birch-pollen-allergic patients and healthy controls differ in T helper 2 cytokine and in interleukin-10 production

BACKGROUND: T helper (Th)2 cells play an important role in the development of IgE-mediated diseases, with local overproduction of Th2 cytokines (IL-4, IL-5 and IL-13) at the site of allergic inflammation. Furthermore, IL-10 has been suggested to play a modulatory role in the induction and maintenance of allergen-specific tolerance in human atopic diseases. AIM: We studied whether circulating allergen-specific Th2 cells persist outside the season of exposure in patients mono-sensitized to birch pollen and whether healthy control individuals also have allergen-specific Th2 cells. We also studied whether IL-10-producing allergen-specific T cells can be found in circulation either in healthy controls or in allergic patients. METHODS: Blood was drawn outside the birch-pollen season from 15 birch-pollen-allergic patients, with seasonal respiratory symptoms and with (n=12) or without (n=3) oral allergy syndrome, and from 10 matched healthy controls. Peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with recombinant Bet v 1 allergen, control antigen tetanus toxoid (TT) and anti-CD3/CD80. In part of the cultures, rIL-4 was added in order to reinforce the allergen-specific Th2 cell responses. RESULTS: In the presence of rBet v 1, T cells from allergic patients, but not from healthy controls, produced IL-4, IL-5 and IL-13. IL-5 production by patients' T cells was further enhanced by adding more IL-4. In contrast, rBet v 1 together with IL-4-induced significant IL-10 production in control subjects but not in patients. Both Th1 and Th2 cytokines were equally induced by polyclonal stimulation in allergic patients and controls, but in the presence of IL-4, polyclonally induced IL-10 production was lower in the patient group. CONCLUSION: rBet v 1-specific Th2 cells circulate outside the season of exposure in the blood of birch-pollen-allergic subjects but not in healthy controls. Allergen-specific T cells were also demonstrated in controls but these cells produce IL-10 when stimulated with rBet v 1 in the presence of IL-4. Our data reveal a different allergen-induced cytokine profile in birch-pollen-allergic patients vs. controls, and suggest that a regulatory mechanism involving IL-4-induced allergen-specific IL-10 production might be defective in allergic subjects.     

CD14+CD16+ monocytes in the course of sepsis in neonates and small children: monitoring and functional studies

The phenotype and function of peripheral blood monocytes change after trauma and during sepsis. The aim of the study was to evaluate monocyte expression of human leucocyte antigen (HLA)-DR and Fc receptor III (FcR III) (CD16) in neonates and small children with high risk of sepsis (hospitalized at the intensive care unit). The reduced proportion of CD14+HLA-DR+ monocytes was observed in all patients at the intensive care unit, while the increase of CD16 expression on monocytes was observed in the course of sepsis. The measurement of CD16 expression on monocytes also proved to be more useful for monitoring patient. The proportion of both CD14dimCD16+ and CD14highCD16+ monocytes increased during sepsis; however, monocytes showed reduced ability to phagocytose Escherichia coli, compromised ability to cooperate with T cells and reduced CD86 expression in parallel to HLA-DR depression. The reduced interleukin (IL)-1 but rather increased IL-10 production was associated with sepsis. The differences between CD14+CD16+ monocytes of healthy donors and patients with sepsis are discussed.     

Dysregulation of B7.2 (CD86) expression on monocytes of HIV-infected individuals is associated with altered production of IL-2.

T helper (Th) responses are mediated in part by immunoregulatory cytokines and the signals delivered by the costimulatory CD28-B7 pathway. In this study, we have investigated the relationship between the regulation of B7 isoform expression on antigen-presenting cells from HIV+ individuals and the production of Th cytokines. The level of expression of both B7.1 and B7.2 isoforms as measured by mean channel fluorescence was significantly decreased on freshly isolated monocytes from HIV+ individuals compared with HIV- controls. However, the levels of expression of B7.1 and B7.2 on both B cells and monocytes increased significantly following culture in HIV+ individuals compared with HIV- controls. B7 expression is subject to regulation by immunoregulatory cytokines. Therefore, we analysed the regulation of B7 expression by cytokines, namely IL-10 and tumour necrosis factor-alpha (TNF-alpha), the production of which is enhanced in HIV infection and have similar inhibitory effects on B7 expression. Two groups of HIV+ individuals were distinguished on the basis of the inhibitory effect of IL-10 and TNF-alpha on monocyte B7.2 expression. IL-10 inhibited B7.2 expression on monocytes from some HIV+ individuals (termed responders) like the HIV- controls. However, in a subset of HIV+ individuals (non-responders) this inhibitory effect was lost. Loss of inhibition of B7.2 expression by IL-10 was associated with significantly reduced IL-2 production by phytohaemagglutinin (PHA)- stimulated peripheral blood mononuclear cells (PBMC). These observations showing an association of B7 dysregulation on monocytes and B cells with altered production of IL-2 may have implications in HIV immunopathogenesis.     

B-cell activating factor of the tumour necrosis factor family expression in blood monocytes and T cells from patients with primary Sjögren's syndrome

We investigated B-cell activating factor of the tumour necrosis factor family (BAFF) level in peripheral blood mononuclear cells (PBMCs), monocytes and T cells from patients with primary Sjögren's syndrome (pSS) and controls both ex vivo and in vitro after cytokine stimulation. PBMCs, monocytes and T cells were isolated from 15 patients with pSS and 17 controls. Cells were cultured alone or with interferon (IFN)α, IFNγ and interleukin 10 (IL-10). T cells were stimulated with phytohaemagglutin and anti-CD3. BAFF protein was assessed by enzyme-linked immunosorbent assay. Ex vivo , no difference was observed in BAFF mRNA level in PBMCs and monocytes from patients and controls. Blood monocytes were the main cell type secreting BAFF both in patients and controls. In vitro , after IFNα stimulation, BAFF mRNA level was significantly higher in cells from patients than from controls (63.8 versus 20.7, P  = 0.03). T cells from patients secreted a higher level of BAFF protein than those from healthy donor cells (17.4 versus 2.9 pg/ml, respectively, P  = 0.04) but at a lower level than that from monocytes. Stimulation of T cells did not change BAFF secretion level. The induction of Th17 cells showed no increased BAFF expression. In conclusion, similar to epithelial cells, blood monocytes in patients with pSS show increased production of BAFF under IFNα, which confirms the involvement of IFNα in pSS. BAFF expression is also increased in blood T cells of such patients, independently of T-cell stimulation.     

T-cell cytokine responses in human infection with Mycobacterium tuberculosis.

Compared with healthy tuberculin reactors, Mycobacterium tuberculosis-stimulated peripheral blood mononuclear cells from tuberculosis patients had diminished production and mRNA expression of the Th1 cytokines gamma interferon and interleukin 2 (IL-2), with no change in production and mRNA expression for the Th2 cytokines IL-4, IL-10, and IL-13. These results were confirmed by evaluation of T cells and CD4+ cells. At the level of systemic T cells, development of tuberculosis is associated with diminished Th1 but not enhanced Th2 responses.     

Depressed interleukin-12 (IL-12), but not IL-18, production in response to a 30- or 32-kilodalton mycobacterial antigen in patients with active pulmonary tuberculosis

The secreted 30-kDa antigen (Ag) of Mycobacterium tuberculosis directly stimulates Th1-type protective cytokine responses in healthy tuberculin reactors but not in patients with active tuberculosis (TB). To examine the cytokine profiles attributable to Th1 suppression associated with active TB, interleukin-12 (IL-12), IL-18, and IL-10 production in response to a 30- or 32-kDa Ag in 16 patients with active pulmonary TB and 24 healthy controls was investigated by enzyme-linked immunosorbent assay. In TB patients, production of IL-12 p40, as well as gamma interferon (IFN-gamma), by 30- or 32-kDa Ag-stimulated peripheral blood mononuclear cells (PBMC) was significantly decreased compared with that in healthy tuberculin reactors. There were no significant differences in IL-18 production between patients and controls early during stimulation (16 h). However, PBMC from patients showed significantly enhanced IL-18 proteins after 96 h of stimulation. Similarly, higher IL-10 production was observed in the TB patients than in healthy tuberculin reactors. After 2 months of anti-TB therapy, the mean IFN-gamma and IL-12 p40 production and the mean blastogenic responses were significantly increased in PBMC in the 10 TB patients who were followed up. Our findings provide evidence that depressed IL-12 in response to the 30- or 32-kDa Ag is involved in the immunopathogenesis of human active pulmonary TB.     

肺癌患者外周血单个核细胞Th1/Th2反应状态及黄芪的调节作用

目的:探讨肺癌患者外周血单个核细胞（PBMC）Th1/Th2免疫反应状态,观察中药黄芪（AG）对其作用。方法:采集并分离肺癌患者和健康志愿者的PBMCs,各分为4组:其中1组即刻冻存待测,其余3组分别为不加药对照组、加 10％AG、20％AG组,培养48h,收集细胞。以IL-2、IFN-γ代表Th1型细胞因子,IL-4、IL-6、IL-10代表Th2型细胞因子,用RT-PCR方法检测肺癌患者PBMC中Th1/Th2型细胞因子mRNA的表达。结果:肺癌患者PBMC中,IL-4、IL-6和IL-10表达阳性率显著高于正常对照（P<0.05）,而IL-2无1例表达（0/23）,IFN-γ仅1例表达;经与黄芪培养后（2种浓度）,肺癌患者Th2型因子与未加AG培养组相比,IL-4、IL-6和IL-10的表达率下降（P<0.05）;而IL-2的表达率显著提高（P<0.05）。结论:肺癌患者外周血的免疫细胞呈现Th2型免疫反应优势状态;黄芪对于肺癌宿主PBMCs Th1/Th2的状态有良好的调节作用,可使肺癌患者PBMCs的Th2型优势免疫反应向Th1方向逆转。     

Cytokine production in peripheral blood cells during and outside the pollen season in birch-allergic patients and non-allergic controls

BACKGROUND: The naturally occurring pollen season permits observation of the kinetic changes in the process of allergic inflammation. We examined cytokine production in peripheral blood (PB) T cells and monocytes obtained from birch-allergic patients both during and outside the pollen season. METHODS: PB from 16 patients and six healthy controls was obtained during the alder pollen season, at the beginning and the peak of the birch pollen season and outside the pollen season. Mononuclear cells (MNC) were stimulated with allergen and polyclonal activators. For flow cytometric analysis, MNC were stained with monoclonal antibodies (MoAbs) against the cell surface markers CD3, CD8, CD14 and the intracellular cytokines IL-4, IL-5, IL-10, IL-12, IL-13, granulocyte macrophage-colony stimulating factor (GM-CSF) and IFN-gamma. RESULTS: In allergic patients, significant increases in clinical symptoms, use of medication, eosinophil numbers and birch-specific IgE were found during the pollen season. In vitro allergen stimulation increased the number of GM-CSF+ monocytes (P<0.01) and this increase was dependent on allergen exposure. The IL-4/IFN-gamma ratio rose (P<0.001) at the peak of birch pollen season and the ratio correlated with symptom scores during the birch season. In the CD4+ cell population, the numbers of GM-CSF+ cells were higher throughout the alder and birch seasons compared with outside the pollen season (P<0.05). No such changes were seen in the healthy controls. CONCLUSIONS: The main finding of our study was the increased percentage of GM-CSF+ monocytes in atopic subjects compared with healthy controls. In allergic patients, natural seasonal pollen exposure resulted in increased numbers of GM-CSF+ cells among both monocytes and CD4+ T cells. We have also shown that a seasonal change in Th2/Th1 cytokine ratio requires an adequate and prolonged allergen stimulation that is seen late in the pollen season.     

The functional role of natural killer cells early in clinical sepsis

Although much information is available for the function of circulating monocytes when signs of sepsis are apparent, little is known for natural killer (NK) cells. NK cells were isolated from 10 healthy controls and from 103 patients with sepsis within the first 24 h from diagnosis. NK cells were stimulated with lipopolysaccharide for cytokine production. Release of tumor necrosis factor‐alpha and of interleukin (IL)‐6 was below the limit of detection. Release of IL‐23 and of interferon‐gamma (IFNγ) was significantly greater among patients than among healthy volunteers. Release of IFNγ was pronounced in septic shock. Patients were divided into two subgroups based on the ratio of IFNγ to IL‐23 released by the NK cells after stimulation: those with ratio ≤5 and 28‐day survival 13.5%, and those with ratio >5 and 28‐day survival 29.4% (p: 0.048). It is concluded that early after clinical development of sepsis, NK cells remain active for the production of IFNγ. Their activity is associated with the final outcome.     

In vitro kinetics of allergen- and microbe-induced IL-4 and IFN-gamma mRNA expression in PBMC of pollen-allergic patients

BACKGROUND: According to a hypothesis allergens induce Th2 responses in allergic patients, and microbes induce Th1 responses. We studied the kinetics of in vitro allergen-, tuberculin (PPD)- and tetanus toxin (TT)-induced IFN-gamma and IL-4 mRNA expression in peripheral blood mononuclear cell (PBMC) cultures of pollen-allergic patients and healthy controls. METHODS: PBMC of 10 birch or timothy pollen-allergic patients and of 13 healthy controls were stimulated in vitro with allergen (birch or timothy), PPD or TT. Pellets and supernatants were collected at 24, 48, 72 and 96 h after stimulation. IFN-gamma and IL-4 production was measured by enzyme linked immunosorbent assay and mRNA expression using RT-PCR and time-resolved fluorometry. RESULTS: Allergen induced IFN-gamma production and mRNA expression in PBMC more in allergic patients than in healthy controls. Also allergen induced IL-4 mRNA expression more in allergic patients than in healthy controls. PPD induced IFN-gamma mRNA expression both in allergic patients and healthy controls, whereas IFN-gamma production was induced only in healthy controls and IL-4 was not induced at all. TT induced IFN-gamma mRNA expression in both groups, IFN-gamma production in allergic patients, and IL-4 mRNA expression in both allergic patients and healthy controls. CONCLUSIONS: In vitro stimulation with allergen induced both IFN-gamma and IL-4 mRNA expression of PBMC in allergic patients. These observations challenge the clearcut division of microbe-specific Th1 and allergen-specific Th2 responses in peripheral blood.