The effect of polymyxin B hemoperfusion on modulation of human leukocyte antigen DR in severe sepsis patients.

BACKGROUND: Recent randomized trials have not found that polymyxin B hemoperfusion (PMX-HP) improves outcomes for patients with sepsis. However, it remains unclear whether the therapy could provide benefit for highly selected patients. Monocyte human leukocyte antigen (mHLA-DR) expression, a critical step in the immune response, is decreased during sepsis and leads to worsening sepsis outcomes. One recent study found that PMX-HP increased mHLA-DR expression while another found that the treatment removed HLA-DR-positive cells. METHODS: We conducted a randomized controlled trial in patients with blood endotoxin activity assay (EAA) level ≥ 0.6. Patients in the PMX-HP group received a 2-h PMX-HP treatment plus standard treatment for 2 consecutive days. Patients in the non-PMX-HP group received only standard treatment. The primary outcome compared the groups on median change in mHLA-DR expression between day 3 and baseline. Secondary outcomes compared the groups on the mean or median change in CD11b expression, neutrophil chemotaxis, presepsin, cardiovascular Sequential Organ Failure Assessment (CVS SOFA) score, vasopressor dose, and EAA level between day 3 and baseline. We further compared the groups on mortality, ICU-free days, ventilator-free days, dialysis dependence status, renal recovery, serum creatinine, vasopressor-free days, and major adverse kidney events (MAKE 28), measured on day 28. RESULTS: Fifty-nine patients were randomized to PMX-HP (n = 29) and non-PMX-HP (n = 30) groups. At baseline, mHLA-DR expression, CD11b, neutrophil chemotaxis, and clinical parameters were comparable between groups. The median change in mHLA-DR expression between day 3 and baseline was higher in PMX-HP patients than in patients receiving standard therapy alone (P = 0.027). The mean change in CD11b between day 3 and baseline was significantly lower in the PMX-HP group than in the non-PMX-HP group (P = 0.002). There were no significant changes from baseline in neutrophil chemotaxis, presepsin, CVS SOFA scores, vasopressor doses, or EAA level between groups. On day 28 after enrollment, mortality, ICU-free days, ventilator-free days, dialysis dependence status, renal recovery, serum creatinine, vasopressor-free days, and MAKE 28 were comparable between groups. CONCLUSION: PMX-HP improved mHLA-DR expression in severe sepsis patients. Future studies should examine the potential benefit of PMX-HP in patients with low mHLA-DR expression. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02413541 . Registered on 3 March 2015.

FGF2 induces RANKL gene expression as well as IL1ß regulated MHC class II in human bone marrow-derived mesenchymal progenitor stromal cells.

OBJECTIVE: Human bone marrow mesenchymal stromal cells (hBM-MSC) are being applied in tissue regeneration and treatment of autoimmune diseases (AD). Their cellular and immunophenotype depend on isolation and culture conditions which may influence their therapeutic application and reflect their in vivo biological functions. We have further characterised the phenotype induced by fibroblast growth factor 2 (FGF2) on healthy donor hBM-MSC focusing on the osteoimmunological markers osteoprotegerin (OPG), receptor activator of nuclear factor kB (RANK), RANK ligand (RANKL) and HLA-DR and their regulation of expression by the inflammatory cytokines IL1ß and IFNγ. METHODS: RANK, RANKL, OPG and HLA-DR expression in hBM-MSC expanded under specific culture conditions, were measured by RT-PCR and flow cytometry. MAPKs induction by FGF2, IL1ß and IFNγ in hBM-MSC was analysed by immunoblotting and RT-PCR. RESULTS: In hBM-MSC, OPG expression is constitutive and FGF2 independent. RANKL expression depends on FGF2 and ERK1/2 activation. IL1ß and IFNγ activate ERK1/2 but fail to induce RANKL. Only IL1ß induces P38MAPK. The previously described HLA-DR induced by FGF2 through ERK1/2 on hBM-MSC, is suppressed by IL1ß through inhibition of CIITA transcription. HLA-DR induced by IFNγ is not affected by IL1ß in hBM-MSC, but is suppressed in articular chondrocytes and lung fibroblasts. CONCLUSIONS: RANKL expression and IL1ß regulated MHC-class II, both induced via activation of the ERK1/2 signalling pathway, are specific for progenitor hBM-MSC expanded in the presence of FGF2. HLA-DR regulated by IL1ß and ERK1/2 is observed on hBM-MSC during early expansion without FGF2 suggesting previous in vivo acquisition. Stromal progenitor cells with this phenotype could have an osteoimmunological role during bone regeneration.

Response of monocyte-derived dendritic cells to rapidly solidified nickel-titanium ribbons with shape memory properties.

Ni-Ti Shape Memory Alloys (SMAs) have attracted considerable attention as biomaterials for medical devices. However, the biocompatibility of Ni-Ti SMAs is often unsatisfactory due to their poor surface structure. Here we prepared Rapidly Solidified (RS) Ni-Ti SMA ribbons by melt-spinning and their surface was characterised by Auger-electron spectroscopy, X-ray photoelectron spectrometry and scanning electron microscopy. The biocompatibility of the produced ribbons and their immunomodulatory properties were studied on human monocyte-derived dendritic cells (MoDCs). We showed that melt-spinning of Ni-Ti SMAs can form a thin homogenous oxide layer, which improves their corrosion resistance and subsequent toxicity to MoDCs. Ni-Ti RS ribbons stimulated the maturation of MoDCs, as detected by changes in the cells' morphology and increased expression of HLA-DR, CD86, CD40 and CD83 molecules. However, Ni-Ti RS ribbons enhanced the tolerogenic properties of immature MoDCs, which produced higher levels of IL-10 and IL-27, driving the differentiation of IL-10- and TGF-ß-producing CD4+T cells. On the other hand, in the presence of lipopolysaccharide, an important pro-inflammatory biomolecule, Ni-Ti RS ribbons enhanced the allostimulatory and Th1 polarising capacity of MoDCs, whereas the production of Th2 and Th17 cytokines was down-regulated. In conclusion, Ni-Ti RS ribbons possess substantial immunomodulatory properties on MoDCs. These findings might be clinically relevant, because implanted Ni-Ti SMA devices can induce both desired and adverse effects on the immune system, depending on the microenvironmental stimuli.

Association of HLA-DR14 with the treatment response in Japanese patients with autoimmune hepatitis.

BACKGROUND: Influence of human lymphocyte antigen (HLA) on the therapeutic response in autoimmune hepatitis (AIH) is not known. AIMS: To evaluate if HLA-DR types influence biological and histological responses to corticosteroids in patients with AIH. METHODS: During 28 years from 1979 through 2007, 48 patients with definite diagnosis of AIH received long-term corticosteroid therapy (median 9 years [range: 5-28 years]) in a single Japanese center. They were followed for transaminase levels and received liver biopsy before and after the treatment. RESULTS: DR4 was detected in 32 and DR14 in 11 patients; seven possessed both DR4 and DR14. DR4 was more frequent in AIH patients than in the general population (67% vs. 22%), while DR14 was comparably frequent between them (23% vs. 17%). Overall, biochemical response was achieved in 43 (90%) of the 48 patients. The sustained biochemical response to a maintenance prednisolone dose < 10 mg was gained more frequently in the patients with than without DR14 (10/11 [91%] vs. 10/37 [27%], P < 0.001). Marked histological improvement with a decrease in histology activity index (HAI) score by > 2 points was achieved in 31 of the 32 (97%) biochemical responders. Histological aggravation with an increase in HAI score occurred in 4 of the 16 (25%) patients without biochemical response (non-responders and relapsers combined), but in none of the 32 responders. CONCLUSION: Long-term immunosuppressive treatment can improve the outcome of Japanese patients with AIH, and DR14 is associated with excellent biochemical response.

Granulocyte-macrophage colony-stimulating factor to reverse sepsis-associated immunosuppression: a double-blind, randomized, placebo-controlled multicenter trial.

RATIONALE: Sustained sepsis-associated immunosuppression is associated with uncontrolled infection, multiple organ dysfunction, and death. OBJECTIVES: In the first controlled biomarker-guided immunostimulatory trial in sepsis, we tested whether granulocyte-macrophage colony-stimulating factor (GM-CSF) reverses monocyte deactivation, a hallmark of sepsis-associated immunosuppression (primary endpoint), and improves the immunological and clinical course of patients with sepsis. METHODS: In a prospective, randomized, double-blind, placebo-controlled, multicenter trial, 38 patients (19/group) with severe sepsis or septic shock and sepsis-associated immunosuppression (monocytic HLA-DR [mHLA-DR] <8,000 monoclonal antibodies (mAb) per cell for 2 d) were treated with GM-CSF (4 microg/kg/d) or placebo for 8 days. The patients' clinical and immunological course was followed up for 28 days. MEASUREMENTS AND MAIN RESULTS: Both groups showed comparable baseline mHLA-DR levels (5,609 +/- 3,628 vs. 5,659 +/- 3,332 mAb per cell), which significantly increased within 24 hours in the GM-CSF group. After GM-CSF treatment, mHLA-DR was normalized in 19/19 treated patients, whereas this occurred in 3/19 control subjects only (P < 0.001). GM-CSF also restored ex-vivo Toll-like receptor 2/4-induced proinflammatory monocytic cytokine production. In patients receiving GM-CSF, a shorter time of mechanical ventilation (148 +/- 103 vs. 207 +/- 58 h, P = 0.04), an improved Acute Physiology and Chronic Health Evaluation-II score (P = 0.02), and a shorter length of both intrahospital and intensive care unit stay was observed (59 +/- 33 vs. 69 +/- 46 and 41 +/- 26 vs. 52 +/- 39 d, respectively, both not significant). Side effects related to the intervention were not noted. CONCLUSIONS: Biomarker-guided GM-CSF therapy in sepsis is safe and effective for restoring monocytic immunocompetence. Use of GM-CSF may shorten the time of mechanical ventilation and hospital/intensive care unit stay. A multicenter trial powered for the improvement of clinical parameters and mortality as primary endpoints seems indicated. Clinical trial registered with www.clinicaltrials.gov (NCT00252915).

The glutathione-S-transferase Mu 1 null genotype modulates ozone-induced airway inflammation in human subjects.

BACKGROUND: The glutathione-S-transferase Mu 1 (GSTM1) null genotype has been reported to be a risk factor for acute respiratory disease associated with increases in ambient air ozone levels. Ozone is known to cause an immediate decrease in lung function and increased airway inflammation. However, it is not known whether GSTM1 modulates these ozone responses in vivo in human subjects. OBJECTIVE: The purpose of this study was to determine whether the GSTM1 null genotype modulates ozone responses in human subjects. METHODS: Thirty-five healthy volunteers were genotyped for the GSTM1 null mutation and underwent a standard ozone exposure protocol to determine whether lung function and inflammatory responses to ozone were different between the 19 GSTM1 wild type and 16 GSTM1 null volunteers. RESULTS: GSTM1 did not modulate lung function responses to acute ozone. Granulocyte influx 4 hours after challenge was similar between GSTM1 normal and null volunteers. However, GSTM1 null volunteers had significantly increased airway neutrophils 24 hours after challenge, as well as increased expression of HLA-DR on airway macrophages and dendritic cells. CONCLUSION: The GSTM1 null genotype is associated with increased airways inflammation 24 hours after ozone exposure, which is consistent with the lag time observed between increased ambient air ozone exposure and exacerbations of lung disease.

Effects of IL-17 on human gingival fibroblasts.

Interleukin (IL)-17 is present in inflammatory periodontal lesions, thus suggesting a role in mediating inflammation. We tested the hypothesis that IL-17, especially when combined with interferon (IFN)-gamma, may modulate the responses of human gingival fibroblasts (HGFs). IL-17 induced IL-8 and minimal intercellular adhesion molecule (ICAM)-1 expression. It had no effect on expression of HLA-DR, CD40, or the immune-suppressive enzyme indoleamine 2,3-dioxygenase (IDO). The effects of IL-17 on HGFs were compared with those of IFN-gamma. Unlike IL-17, IFN-gamma augmented the expression of HLA-DR, ICAM-1, and IDO, but not IL-8. Thus, IL-17 and IFN-gamma induce different HGF responses when administered separately. Interestingly, when IL-17 and IFN-gamma were combined, marked enhancement of ICAM-1, IL-8, and IDO expression by HGFs was observed. These findings suggest that IL-17, especially when combined with IFN-gamma, could play an important role in immune modulation through stimulation of HGFs in periodontal disease.

[Efficacy of local use of recombinant interferon alpha-2 preparations in combined treatment of recurrent respiratory papillomatosis].

Clinical-immunological examination of 41 patients with recurrent respiratory papillomatosis (RRP) included determination of phenotype CD3, CD4, CD8, CD16, CD25, CD56, HLA-DR in peripheral blood by flow cytofluorimetry, the levels of IFN-gamma, TNF-alpha, GM-CSF, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13 in the laryngeal secretion by multiplex immunoassay. Interferon inhalation therapy was conducted to prevent recurrence in 23 patients after surgical treatment and in 18 patients as monotherapy. The efficacy of the monotherapy was 45.5%. Treatment with IFN-alpha raised the levels of cytokines modulating an immune response by Th1-type (IFN-gamma, IL-12, IL-2) and GM-CSF, and reduced the levels of IL-4, IL-10 and IL-13. Local treatment with recombinant IFN-alpha is effective in aggressive RRP. As prognostic markers of the treatment efficacy may serve baseline high levels of TNF-alpha and IL-4/IFN-gamma index in laryngeal secretion. Treatment efficacy can be assessed by raise of IFN-gamma, IL-2 and IL-12 in combination with reduction of IL-4/IFN-gamma index.

Insulin in the critically ill with focus on cytokines, reactive oxygen species, HLA-DR expression.

Insulin is essential for glucose homeostasis. Insulin/glucose-insulin-potassium (GIK) regimen suppresses the production of tumor necrosis factor-alpha (TNF-alpha), interleukins-6 (IL-6), macrophage migration inhibitory factor (MIF) and other pro-inflammatory cytokines and reactive oxygen species (ROS), enhances the synthesis of endothelial nitric oxide (eNO), and anti-inflammatory cytokines interleukins-4 (IL-4) and interleukin-10 (IL-10). In subjects who are critically ill, monocyte HLA-DR expression was significantly decreased with a concomitant increase in plasma IL-10 and IL-4 concentrations. Large increases in the plasma concentrations of TNF-alpha, IL-6, sustained increase in the expression of leukocyte CD11b/CD18, and ROS generation following surgery and infections were found to be associated with increased mortality. By virtue of its actions on pro- and anti-inflammatory cytokines and ROS, insulin may have the ability to alter HLA-DR expression in the critically ill and thus bring about its beneficial actions in sepsis/septic shock, myocardial recovery following acute myocardial infarction, improve prognosis of those who are critically ill, and suppress inflammation.

Effects of dexmedetomidine on CD42a+/CD14+, HLADR+/CD14+ and inflammatory cytokine levels in patients undergoing multilevel spinal fusion.

OBJECTIVE: To assess the effects of dexmedetomidine (Dex) on CD42a+/CD14+，HLADR+/CD14+ and inflammatory cytokine levels in patients undergoing multilevel spinal fusion. Patients and methods Forty ASA I-II patients undergoing multilevel spinal fusion were randomly divided into Dex and control groups (n=20). A continuous intravenous infusion of Dex (0.5µg/kg/h) or normal saline was started 10min prior to induction and was stopped 15min before operation completion. Serum levels of CD42a+/CD14+, HLADR+/CD14+, WBC, PLT, CRP, IL-6, IL-10, and TNF-α were measured before induction (T1), 30min (T2) after operation initiation, and 60min (T3), 1d (T4), 3d (T5), and 5d (T6) post-operation. VAS values were obtained at T3, T4, T5 and T6, as well as hospital days. RESULTS: Treatment with Dex significantly decreased CD42a+/CD14+ at T2, T3, and T4, and markedly increased HLADR+/CD14+ at T4 and T5 when compared with controls. CRP and WBC were markedly decreased at T2, T3, T4 and T5 (P<0.01 or P<0.05). Serum IL-6 and TNF-α level in Dex group was significantly increased at T3 and T4 (P<0.05), and IL-6 and TNF-α level in control group was significantly increased at T2, T3, T4 and T5 (P<0.05) when compared with their respective preoperative levels (T1). IL-6 and TNF-α levels at T2, T3, T4 and T5 in Dex group were significantly lower than those in control group (P<0.05). There were no significant differences in operation time, hospital days or VAS values between the two groups (P>0.05). CONCLUSION: Dex can inhibit the inflammatory response and reduce immunosuppression in patients undergoing multilevel spinal fusion.

Second-generation peptidomimetic inhibitors of antigen presentation effectively treat autoimmune diseases in HLA-DR-transgenic mouse models.

Peptidomimetic compounds that bind to major histocompatibility complex class II molecules and are resistant to cathepsins can competitively inhibit the presentation of processed protein antigens. Therefore, compounds that bind to autoimmune disease-associated class II molecules are expected to compete with autoantigens for presentation and thereby interrupt the disease process. The first generation of such competitors developed for rheumatoid arthritis-associated HLA-DR molecules, although resistant to cathepsins, has remained sensitive to plasma proteases, and was thus unlikely to be effective in vivo. We have therefore produced a second generation of compounds that are resistant to cathepsins and stable in plasma while maintaining binding affinity for HLA-DR molecules associated with rheumatoid arthritis and multiple sclerosis. Selected compounds of this series are shown to inhibit antigen presentation in vivo, as well as effectively treat collagen induced arthritis and experimental autoimmune encephalomyelitis in HLA-DR transgenic mouse models.

Phase I trial of interferon gamma to potentiate cyclosporine-induced graft-versus-host disease in women undergoing autologous bone marrow transplantation for breast cancer.

PURPOSE: We investigated if interferon gamma (IFN-gamma) could augment cyclosporine (CSA)-induced graft-versus-host disease (GVHD) following autologous bone marrow transplant in women with metastatic breast cancer and defined the toxicities of this therapy. PATIENTS AND METHODS: Thirty-six women with advanced breast cancer were treated with CSA 2.5 mg/kg daily for 28 days and IFN-gamma 0.025 mg/m2 subcutaneously (SC) every other day, days 7 to 28 following autologous bone marrow transplantation and monitored for induction and severity of GVHD and toxicity of therapy. RESULTS: GVHD was induced in 56% of patients. The severity of GVHD was greater than in a historic control population treated with CSA alone. Stage III rash was seen in 36% of patients, compared with 3% in the historic control population. Fourteen of 36 patients required therapy with topical corticosteroids and two of 36 required systemic treatment. Only three of 31 historic controls needed topical corticosteroids and no patient was treated systemically. There was no severe visceral GVHD. Hematopoietic recovery was not delayed. There were three toxic deaths. CONCLUSION: CSA-induced GVHD can be safely augmented by IFN-gamma in women treated with high-dose alkylating agents and autologous bone marrow transplantation. There is little evidence of increased toxicity. Evidence of antitumor efficacy awaits further investigation.

Association between human leukocyte antigen (HLA) and interferon- induced thyroid diseases in four patients with HCV-related chronic hepatitis.

OBJECTIVES: The interferon-alpha (IFN-alpha) therapy for HCV hepatitis may exacerbate or induce underlying thyroid disorders. Besides viral factors, the human leukocyte antigen (HLA) may be an independent risk factor. METHODS: We evaluated fifteen patients with HCV chronic hepatitis during a period of 40 months. At the enrollment, all the patients were negative for thyroid disorders, excluding one patient with subclinical hypothyroidism. Eleven patients received IFN-alpha therapy. The HLA system was examined in every patient, evaluating antigens (n=40) of locus A, B and Cw and alleles (n=19) of locus DRB1* and DQB1*. The HLA system was also examined in healthy subjects (n=107) as a control group. RESULTS: The HCV genotype distribution in patients was: 1b=20%, 2a=60%, 3a=20%. Four IFN-treated patients presented clinical thyroid disorders, including autoimmune hypothyroidism (n=2), transient thyrotoxicosis (n=1) and subacute thyroiditis (n=1). The HLA susceptibility to thyroid disorders (antigen/allele frequency) in the whole group of patients was not different in respect to controls and normal Italian population. The patients with HCV chronic hepatitis that developed thyroid diseases after IFN- treatment had a double and specific association with the HLA system (Mantel-Haenszel X(c)(2)=4.706, p<0.05). CONCLUSIONS: This case report suggests that HLA system examination is an important and promising diagnostic aspect that may be considered in order to evaluate the appearance of thyroid disorders during the IFN-alpha treatment for HCV-related chronic hepatitis.

Hepatitis C virus genotypes, HLA-DRB alleles and their response to interferon-alpha and ribavirin in patients with chronic hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) is a worldwide common disease. Some predictive factors influencing the response to interferon alpha (IFN-alpha) therapy have been identified, but the conclusions differ in various counties and areas. The aim of this study was to study the associations between HCV genotypes, HLA-DRB alleles and their response to IFN-alpha and ribavirin in Chinese patients with chronic hepatitis C in Northeast China. METHODS: HCV genotypes of 113 patients with HCV were investigated. Gene chips were used to analyze the frequency of HLA-DRB in 25 of these patients and their response to IFN-alpha and ribavirin. The associations of HCV genotypes, HLA-DRB alleles and their response to IFN-alpha and ribavirin were also studied. RESULTS: The response rates differed in several types of HCV, with HCV 2b being the highest (57.78%), HCV 1a and 2a lower (46.15% and 47.62%) and HCV 1b the lowest (11.76%). The response rates to IFN-alpha and ribavirin in patients with DRB1*07 were higher than those with DRB1*04. Sex, HCV type and HLA-DRB were all related to the response. Most female patients with HCV 2b and HLA-DRB1*07 presented complete response, whereas male patients with HCV 1b and HLA-DRB1*04 usually demonstrated no response. DRB1*07 allele and HCV 2b were the factors closely related to the response. CONCLUSIONS: The response rate of HCV 1b may be the lowest even IFN-alpha and ribavirin are combined in treatment. Not only virus but also the host plays an important role in anti-virus therapy. Thus, it is necessary to adjust the host's immune status to accelerate the clearance of HCV.

Interferon-gamma-stimulated human keratinocytes express the genes necessary for the production of peptide-loaded MHC class II molecules.

Keratinocytes exposed to interferon (IFN)-gamma synthesize major histocompatibility complex class II antigens both in vivo and in vitro; however, the expression of class II accessory genes has not yet been investigated. In this study, we examined the capacity of normal human keratinocytes activated with IFN-gamma to express HLA-DR, HLA-DM, and invariant chain genes as well as two major transcription regulatory genes, class II transactivator and RFX5. Cultured keratinocytes were shown to synthesize low levels of DM alpha, invariant chain p33, and RFX5 transcription factor. Upon treatment with IFN-gamma, expression of RFX5, DM alpha, and invariant chain p33 mRNA increased, whereas class II transactivator mRNA appeared de novo, followed by the expression of DR alpha, DMbeta, and invariant chain p41 genes. Western blot analysis showed that both p33 and p41 invariant chain forms and DM became detectable in keratinocytes after stimulation with IFN-gamma, with a higher p41/p33 ratio compared with Raji B cells. Finally, HLA-DR molecules present on IFN-alpha-treated keratinocytes were shown to be remarkably resistant to sodium dodecyl sulfate denaturation at room temperature, a feature that class II molecules acquire when their groove is properly loaded with peptide. These results suggest that human keratinocytes activated with IFN-gamma possess the biochemical requirements for the generation of functional class II peptide complexes.

B7 expression and antigen presentation by human brain endothelial cells: requirement for proinflammatory cytokines.

Interaction between systemic immune cells with cells of the blood-brain barrier is a central step in development of CNS-directed immune responses. Endothelial cells are the first cells of the blood-brain barrier encountered by migrating lymphocytes. To investigate the antigen-presenting capacity of human adult brain endothelial cells (HBECs), we used HBECs derived from surgically resected temporal lobe tissue, cocultured with allogeneic peripheral blood derived CD4+ T lymphocytes. HBECs in response to IFN-gamma, but not under basal culture conditions, expressed HLA-DR, B7.1 and B7.2 antigens. Despite such up-regulation, these IFN-gamma-treated HBECs, in contrast to human microglia and PB monocytes, did not sustain allogeneic CD4+ cell proliferation, supported only low levels of IL-2 and IFN-gamma production, and did not stimulate IL-2 receptor expression. CD4+ T cell proliferation and increased IL-2 receptor expression could be obtained by addition of IL-2. Our data suggests that, although HBECs cannot alone support T cell proliferation and cytokine production, HBECs acting in concert with cytokines derived from a proinflammatory environment could support such a response.

Induction of RANTES, HLA-DR, and intercellular adhesion molecule-1 on highly purified distal tubular cells from human kidney.

BACKGROUND: Expression of proinflammatory molecules by tubular epithelial cells plays an important role in renal allograft rejection and inflammatory kidney diseases. Different studies from patients with acute rejection point to the involvement of distal tubular segments. At present no in vitro system for the human distal tubule is established. METHODS: Human distal tubular cells were isolated immunomagnetically. Cultured cells were stimulated with cytokines (interferon-gamma, tumor necrosis factor-alpha, interleukin-1beta, or a cytokine mix). Secretion of RANTES (regulated upon activation, normal T-cell expressed and secreted) was evaluated with an enzyme-linked immunoassay. Expression of HLA-DR and intercellular adhesion molecule (ICAM)-1 was assessed by flow cytometric analysis and immunofluorescence studies. RESULTS: Our data clearly indicate that distal tubular cells express RANTES, HLA-DR, and ICAM-1 in response to a mixture of specific cytokines. Dexamethasone inhibited the induced expression of RANTES and HLA-DR significantly, but not that of ICAM-1. CONCLUSIONS: We demonstrate an appropriate in vitro system for the human distal tubule. The present study proves the involvement of the distal tubular segment during inflammatory kidney diseases.

Immunoregulatory effects of interferon-beta and interacting cytokines on human vascular endothelial cells. Implications for multiple sclerosis autoimmune diseases.

The mechanism(s) of action responsible for the anti-inflammatory effects mediated by interferon (IFN)-beta are still elusive although suggestions include anti-viral effects, the enhancement of natural killer (NK) or suppressor T cell activity and opposition to the effects of inflammatory cytokines. As vascular endothelial cells are active participants in inflammatory and demyelinating processes, we decided to examine the effects of IFN-beta on the expression of major histocompatibility complex (MHC) gene products and intercellular adhesion molecule (ICAM)-1 on human vascular endothelial cells (ECs). Human umbilical ECs demonstrated constitutive expression of ICAM-1 and MHC class I molecules but did not express MHC class II molecules. Basal expression of ICAM-1 molecules was enhanced by TNF alpha and to a lesser extent by IFN-beta, but was not affected by IFN-gamma. MHC class I expression on ECs was enhanced by IFN-beta, IFN-gamma, and tumor necrosis factor (TNF)-alpha. Furthermore, a synergistic effect was observed to combinations of these interacting cytokines. Incubation of ECs with IFN-gamma, but not IFN-beta, induced class II expression in a dose dependent manner. Moreover, co-incubation of ECs with IFN-beta and IFN-gamma resulted in significant down-regulation of class II molecules expression which was directly dependent on IFN-beta concentration. Northern blot analysis of DR alpha and Beta 2-microglobulin mRNA expression suggested that cytokine-mediated regulation of MHC molecules is at the transcriptional level, while modulation of ICAM-1 expression appears to be at the transcriptional as well as post-transcriptional level. Thus, our study demonstrated that IFN-beta and interacting cytokines exert complex immunoregulatory effects on endothelial cells with differential modulatory effects on various cell surface markers. Understanding the biological significance of these immunomodulatory effects mediated by IFN-beta may have important implications for cytokine-based strategies in the treatment of inflammatory and autoimmune diseases.

Inhibitory effects of nicotinamide on recombinant human interferon-gamma-induced intercellular adhesion molecule-1 (ICAM-1) and HLA-DR antigen expression on cultured human endothelial cells.

Intercellular adhesion molecule-1 (ICAM-1), HLA-A, B, C and HLA-DR antigen on endothelial cells (EC) play important roles in the development of inflammatory processes in autoimmune disorders. In the present study, we investigated the effect of nicotinamide, an inhibitor of poly(ADP ribose) synthetase, on interferon-gamma (IFN gamma)-induced ICAM-1 and HLA-DR antigen expression on the surface of cultured human umbilical vein endothelial cells, assessed by flow cytometry, and EC proliferation by counting cell numbers and [3H]thymidine incorporation assays. Nicotinamide dose-dependently inhibited the IFN-gamma-induced ICAM-1 and HLA-DR antigen expression, but not HLA-A, B, C antigen expression on cultured EC. Furthermore, nicotinamide significantly inhibited endothelial cell proliferation, as assessed by [3H]thymidine incorporation assay. Our findings suggest that nicotinamide may suppress mononuclear cell infiltration, antigen presentation and angiogenesis in the lesions of autoimmune disorders by reducing both IFN gamma-induced ICAM-1 and HLA-DR antigen expression on EC, and EC proliferation. Therefore, nicotinamide can be used for the treatment and prevention of the development of autoimmune disorders.

Expression of HLA-DR in granulocytes of polytraumatized patients treated with recombinant human granulocyte macrophage-colony-stimulating factor.

MHC class II determinants are the restriction elements involved in antigen-specific activation of helper T lymphocytes and interaction with CD4 molecules. They are typically expressed on a limited number of cell types, mostly endowed with antigen-presenting capacity. Recently, expression of HLA-DR has been detected on granulocytes stimulated "in vitro" with GM-CSF. However, no evidence of "in vivo" expression in humans has been presented so far. We report here that class II determinant expression is detectable in vivo on peripheral blood granulocytes of polytraumatized patients upon intravenous administration of rhGM-CSF. Expression of these molecules appears to be an early effect of rhGM-CSF treatment, independent from endotoxemia or endogenous production of IL-6 or TNF-alpha, and rapidly declining upon discontinuation of therapy. Thus, this treatment might increase the number of cells potentially capable of presenting class-II-restricted antigens in these patients.