Expression of interferon-γ receptors on bladder cancer cells: does it correlate with biological response?

Summary Previously we have shown a differential biological response of three human bladder cancer cell lines (RT4, RT112 and MGH-U1) to gamma interferon (IFN-). The present study examines the relationship between the biological response and the expression of the interferon- receptor on the tumour cell surface. Using a competitive radioligand binding assay and Scatchard analysis, we measured the number and affinity of the IFN- receptors on each of the above cell lines. Individual cells from each line expressed large numbers (29,100–41,800) of high-affinity receptors (k _d =2.4–3.9×10¹⁰M). There was no statistically significant difference in either of these parameters between the three lines. We therfore conclude that the biological response of these bladder lines to IFN- does not relate to the number or affinity of its receptor on the plasma membrane of these tumour cells.     

Autologous mesenchymal stem cells prevent transplant arteriosclerosis by enhancing local expression of interleukin-10, interferon-γ, and indoleamine 2,3-dioxygenase

Transplant arteriosclerosis (TA) remains the major limitation of long-term graft survival in heart transplantation despite the advances in immunosuppressants. Mesenchymal stem cells (MSCs) have been demonstrated to suppress allogeneic immune responses by numerous in vitro studies. However, the immunomodulatory effects of MSCs in vivo are controversial and the underlying molecular mechanisms are not conclusive. In this study, we investigated the therapeutic potential of autologous bone marrow-derived MSCs on TA in a porcine model of femoral artery transplantation. MSCs or saline were injected into the soft tissue surrounding the arterial grafts immediately postanastomosis. Four weeks after transplantation, neointimal formation increased significantly in untreated allografts compared with the MSC-treated grafts as assessed by intravascular ultrasound (maximum luminal area stenosis: 40 ± 12% vs. 18 ± 6%, p < 0.001). Grafts harvested at 4 weeks showed dense perivascular lymphocyte infiltration accompanied by significant intimal hyperplasia in the untreated but not in the MSC-treated allografts. Serial angiographic examination showed that all of the untreated allografts became occluded at the 8th week whereas the majority of the MSC-treated grafts remained patent at the 12th week posttransplantation (n = 12 each group, p < 0.001). Quantitative PCR analysis revealed that Foxp3 expression was comparable between the untreated and the MSC-treated groups. However, expression of interleukin-10 (IL-10), interferon-γ (IFN-γ), and indoleamine 2,3-dioxygenase (IDO) was increased significantly in the MSC-treated allografts compared with that in the allograft controls (p = 0.021 for IL-10, p = 0.003 for IFN-γ, and p = 0.008 for IDO). In conclusion, local delivery of autologous MSCs alleviates TA by inducing allograft tolerance via enhanced expression of IL-10, IFN-γ, and IDO but not Foxp3-positive cells in the vessel wall. These results suggest that MSCs induce immune tolerance by activating the type 1 regulatory T-like cells.     

Soluble FGL2 induced by tumor necrosis factor-α and interferon-γ in CD4 T cells through MAPK pathway in human renal allograft acute rejection

Background

Acute rejection (AR), initiated by alloreactive CD4⁺ T cells, hampers allograft survival. Soluble fibrinogen-like protein 2 (sFGL2) is a novel effector of CD4⁺ T cells. We previously found that serum sFGL2 significantly increased in renal allograft recipients with AR. In this study, sFGL2 secretion by CD4⁺ T cells and its mechanism were further explored both in vivo and in vitro.

Materials and methods

Forty cases of living-related renal transplant recipients with biopsy-proven AR or stable renal function were collected and detected serum sFGL2, tumor necrosis factor (TNF)-α and interferon (IFN)-γ, and peripheral CD4⁺ T cells. In vitro, the isolated human CD4⁺ T cells were stimulated by TNF-α or IFN-γ. sFGL2 in the supernatant and mitogen-activated protein kinase (MAPK) proteins in the CD4⁺ T cells were investigated. Approval for this study was obtained from the Ethics Committee of Fudan University.

Results

sFGL2, TNF-α, IFN-γ, and CD4⁺ T cells were significantly increased in the peripheral blood of renal allograft recipients with AR. Stimulation with 1000 U/mL TNF-α or 62.5 U/mL IFN-γ for 48 h provided an optimal condition for CD4⁺ T cells to secrete sFGL2 in vitro. Phosphorylated (p-) c-Jun N-terminal kinase was remarkably upregulated in the activated CD4⁺ T cells, whereas no significant changes were found in p-p38 MAPK or p-ERK1/2 expression. Furthermore, inhibition of c-Jun N-terminal kinase significantly reduced sFGL2 secretion by CD4⁺ T cells.

Conclusions

sFGL2 secretion by CD4⁺ T cells can be induced with TNF-α and IFN-γ stimulation through MAPK signaling in renal allograft AR. Our study suggests that sFGL2 is a potential mediator in the pathogenesis of allograft rejection.     

Marked elevation of interferon-γ in acute focal bacterial nephritis

Acute focal bacterial nephritis (AFBN) is a localized, interstitial bacterial infection of the renal parenchyma. In this study, we measured the serum levels of several cytokines in patients with AFBN. A total of 11 children were enrolled in the study and classified into two groups of patients: an AFBN group and a control group. There was no significant difference in the serum levels of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, granulocyte-colony stimulating factor, or tumor necrosis factor-α among the patients in the two groups. However, the serum levels of interferon-γ among the patients in the AFBN group were significantly higher than those among the patients in the control group. The current results suggest that the bacterial kidney infection in the AFBN group is localized and that interferon-γ may be produced locally in response to the infection.     

FTY720 treatment of kidney transplant patients: a differential effect on B cells, naïve T cells, memory T cells and NK cells

FTY720 alters lymphocyte recirculation and homing by interfering with S1P receptors on lymphocytes, possibly in combination with chemokine receptors, and induces a decrease in PBL counts. In fresh, whole blood samples of 14 kidney transplant patients, we analyzed by flow cytometry the effect of FTY on the number of NK cells, monocytes, na?ve (CCR7+) T cells, memory (CCR5+) T cells and B cells. Patients treated with 0.5, 2.5 or 5mg FTY/day showed a strong decrease in T and B cell numbers. NK cells and monocytes were not affected. FTY reduced primarily na?ve T cells. From the memory T cells (CCR5+), predominantly CD8 cells, 40-60% remained in the circulation. The majority of the CCR7+ cells disappeared from the circulation within 3-6h, while a further reduction was achieved later. The more slowly decrease in na?ve CCR7+ T cell numbers was also observed in the group treated with 0.25mg FTY/day. Elispot assays revealed no IL-4 producing cells and a low frequency of IFN-gamma producing cells. We suggest that both CCR7 dependent and independent mechanisms are involved in the depletion of T cells from peripheral blood.     

Impaired interferon-α production in whole-blood cultures from bladder cancer patients

Summary Interferon- (IFN-) production was investigated in whole-blood cultures of 66 bladder cancer patients and 65 control subjects. IFN synthesis was induced with Sendai virus, and IFN activity was assayed in FL cells challenged with vesicular stomatitis virus (VSV). The mean levels of the IFN- produced were 5,724±2,288 IU/ml in the control subjects and 4,800±2,353 IU/ml in the bladder cancer patients. IFN- production was significantly suppressed in the bladder cancer patients compared with that in the control subjects (P (0.05). The impairment in IFN- production correlated with the tumor grade, and it was shown that the tendency toward decreased IFN- production was closely associated with the advancement of the tumor stage. Our results suggested that the decreased IFN- production may contribute to the disordered immunoregulation in bladder cancer patients.     

Distribution of mouse interferon-β in normal and brain tumour-bearing mice

Summary The distribution of¹²⁵I-labelled recombinant mouse interferon- (rMuIFN-) in normal and glioma (203 glioma) bearing mice was studied by radioassay and macro-autoradiography at 15 and 30 min after a single intravenous injection. The level of rMuIFN- in the spleen was about 20-fold higher than in serum. Concentrations higher than the serum level was detected in the lung, liver and kidney. The concentration of rMuIFN- in the brain was 8% of the serum level and the concentration in the glioma 30 min after administration was about 10-fold higher than in normal mouse brain. Macro-autoradiographic study demonstrated a wide distribution range and selective uptake in glioma tissue. Furthermore, we found that mouse gliomas were sensitive to mouse IFN-. Our findings demonstrate that in the mouse glioma model, intravenously administered interferon reaches the tumour.     

Trophic effect of adipose tissue–derived stem cells on porcine islet cells

Background

Adipose tissue–derived stem cells (ADSCs), which are widely known as multipotent progenitor cells, release several cytokines that support cell survival and repair. The aim of this study was to investigate whether ADSC-secreted molecules could induce a trophic effect in pancreatic islet culture conditions in vitro.

Materials and methods

We cocultured porcine islet cells with ADSCs using a transwell system for 48 h and evaluated the viability of islet cells. We also determined the concentration levels of cytokines and insulin in the supernatant of the culture medium. We used anti-vascular endothelial growth factor (VEGF) and anti-interleukin (IL)-6 receptor antibodies to investigate the effect of VEGF and IL-6 on islet cells.

Results

ADSCs improved the viability of islet cells in the absence of cell-cell contact (P < 0.05). VEGF and IL-6 levels in the culture medium increased when islet cells were cocultured with ADSCs (P < 0.05). Furthermore, inhibition of VEGF decreased the viability of islet cells (P < 0.05); however, inhibition of IL-6 did not affect islet cell viability.

Conclusions

These results suggested that trophic factors, particularly VEGF, secreted by human ADSCs enhanced the survival and function of porcine islet cells.     

Differentiation of adult human bone marrow mesenchymal stem cells into Schwann-like cells in vitro

Bonemarrowmesenchymalstemcells, alsocalledbonemarrowstromalcells (BMSCs), areisolatedfrombonemarrow, andtheycanmultiplyinvitroanddifferentiateintoosteogeniccells,chondrocytes, adipocytes, musclecellsandneuralcells.1 5 RecentstudieshavedemonstratedthatBMSCsfromadultratscoulddifferentiateintoSchwann likecellsinspecificconditions.6, 7 BMSCsmayhavepotentialapplicationforautologousneuraltransplantation. Inthisstudy, wetrytoinvestigatethedifferentiativecapabilityofadulthumanBMSCsintoSchwann l…     

Activation of human dendritic cells by porcine aortic endothelial cells: transactivation of na?ve T cells through costimulation and cytokine generation.

BACKGROUND: Dendritic cells (DC) are the most potent antigen-presenting cells in the immune system. To define the role of human DC in human anti-porcine immune responses, we defined the interaction of human DC with porcine aortic endothelial cells (PAEC). METHODS: To determine the immune responses, both monocyte-derived and peripheral blood DC were cultured with porcine and human endothelial cells. We analyzed the role of CD11a, CD11b, and CD54 in a cell-to-cell adhesion assay using antibodies against these molecules. The expression pattern of costimulatory molecules (CD40, CD80, CD86), adhesion molecules (CD54), and intracellular cytokines (interleukin-12p70 and tumor necrosis factor [TNF]-alpha) in DC after interaction with endothelial cells was determined by immunofluorescence. RESULTS: Human DC significantly adhered to PAEC (38-40%), and this adhesion was augmented (>50%) upon treatment with either recombinant swine interferon-gamma or recombinant human TNF-alpha. Addition of human DC to PAEC was blocked by pretreatment of DC with antibodies specific to human leukocyte function-associated antigen-1 or CD54. Adhesion of DC to PAEC also resulted in the activation of DC, which was manifested by up-regulation of costimulatory molecules (CD40, CD80, CD86), adhesion molecules (CD54), and HLA-DR. PAEC-activated human DC provided proliferative signals to the na?ve autologous CD4+ T cells and synthesized interleukin-12p70 and TNF-alpha. However, activated DCs failed to lyse PAEC in such interaction. CONCLUSION: Human DC effectively adhered to PAEC and were activated by xenoantigen, resulting in highly efficient antigen presentation and proliferation of CD4+ T cells. Further, this interaction of human DC to PAEC is regulated by the participation of costimulatory and adherence molecules and cytokines.     

Intracellular interferon-γ staining analysis of donor-specific T-cell responses in liver transplant recipients

Objective

Biomarkers that accurately reflect, detect, and/or predict detrimental immune responses to grafts are important in organ transplantation. We established a new detection method for alloreactive T cells on the basis of intracellular staining for interferon (IFN)-γ, using CD40-activated B cells as stimulators, and assessed temporal changes in alloreactive T-cell frequencies in patients who received liver transplantation.

Methods

Peripheral blood mononuclear cells and CD40-activated B cells were used as responder and stimulator cells, respectively. The responder cells were cultured with the stimulator cells for 7 days, restimulated for 5 hours, and flow cytometrically tested by intracellular staining for IFN-γ.

Results

The relative postoperative-preoperative ratio of donor-specific CD8⁺ T cells in the nonrejection group was significantly lower than that in the rejection group and found to be <1 in most individuals of the group throughout the postoperative periods, indicating an induction of donor-specific suppression of the CD8⁺ T-cell responses. In contrast, such differences were not found in the donor-specific CD4⁺ T cells. These results suggest that the relative postoperative-preoperative ratio of the donor-specific CD8⁺ T cells is a good indicator of graft rejection.

Conclusion

We established a new flow cytometric method for the detection of alloreactive T cells by intracellular staining for IFN-γ, using CD40-activated B cells as stimulator cells. Using this system, we found that the relative postoperative-preoperative ratio of the donor-specific CD8⁺ T cells is a possible evaluative indicator of the risk for graft rejection.     

Nephrotic syndrome associated with interferon-β-1b therapy for multiple sclerosis

A 43-year-old woman with multiple sclerosis (MS) had nephrotic syndrome 21 months after starting treatment with interferon (IFN)-β-1b (subcutaneous administration). She had taken no drug except for the IFN-β-1b. Because nephrotic syndrome may be induced by IFN therapy, the IFN was stopped. Percutaneous renal biopsy revealed that she had minimal change nephrotic syndrome. As nephrotic-range proteinuria, hypoalbuminemia, and general edema were worsening even 2 weeks after cessation of the drug, oral corticosteroid therapy (prednisolone 40 mg/day) was started. The nephrotic syndrome was treated successfully with prednisolone. The dosage of prednisolone was tapered, without a relapse, and then the corticosteroid therapy was stopped. IFN-β-1b therapy was then resumed, and the patient is in remission for both nephrotic syndrome and MS. Though proteinuria and nephrotic syndrome is a rare adverse effect of IFN-β-1b therapy, physicians treating MS patients with this agent should pay careful attention to new clinical symptoms and laboratory findings.