Plasma levels of IL-7 and IL-15 after reduced intensity conditioned allo-SCT and relationship to acute GVHD.

To assess the impact of homeostatic expansion on the occurrence of acute GVHD after reduced intensity conditioning (RIC) transplantation, systemic levels of IL-7 and IL-15 and expression of their specific receptor chains were prospectively investigated in 45 fully HLA-matched allograft recipients. IL-7 and IL-15 levels peaked at four- to fivefold over pre-conditioning values. IL-7 levels were inversely correlated to absolute T-cell counts. Peak IL-15 levels positively correlated to concurrent CRP levels, but normalized earlier than IL-7. These results indicate that the kinetic course of IL-7 depends mainly on initiation of T-cell recovery, while IL-15 depends more on peri-transplant inflammation after RIC. Longer duration of the rise in IL-7 levels was associated with preservation of a normal CD4/CD8 ratio. In all, 16 (35%) patients developed grade 2-4 acute GVHD at a median of 42 days post graft, preceded by higher IL-7 levels and more downregulation of IL-7 receptor α chain on CD4(+) T cells than in patients without acute GVHD, suggesting enhanced homeostatic expansion. In multivariate analysis, IL-7 level measured on day +30 was the foremost predictive factor for grade 2-4 acute GVHD (P=0.002). Measurement of IL-7 level after RIC transplantation might help predict risk of subsequent acute GvHD.

Plasma levels of IL-7 and IL-15 in the first month after myeloablative BMT are predictive biomarkers of both acute GVHD and relapse.

T-cell reconstitution after allo-SCT initially depends on homeostatic peripheral expansion of donor T cells, the level of which may promote the differentiation of alloreactive and tumor-reactive effectors. IL-7 and IL-15 exert their effect as key homeostatic cytokines. We prospectively investigated plasma levels of IL-7 and IL-15 in a homogeneous group of 40 patients in CR of their hematologic malignancy undergoing myeloablative, fully (10/10) HLA-matched BMT. IL-7 and IL-15 proceeded along similar kinetic courses, peaking at wide ranges (3.8-30.2 and 14.3-66 pg/ml, respectively) on day +14 when all patients were profoundly lymphopenic. Occurrence and grade of subsequent acute GVHD were significantly associated with heightened day +14 IL-7 and IL-15 levels. Association of peak IL-7 level to grade 2-4 acute GVHD was confirmed by Cox multivariate analysis (hazard ratio (HR)=5.38; P=0.022). Malignancy relapse was significantly associated with reduced day +14 levels of IL-15 (Cox multivariate analysis: HR=0.93; P=0.035). Plasma IL-7 and IL-15 levels in the early post transplantation period are therefore biomarkers that can help predict subsequent development of acute GVHD and malignancy relapse.

Cytokine concentrations in exhaled breath condensates in systemic sclerosis.

BACKGROUND: Pulmonary fibrosis in systemic sclerosis (SSc) involves inflammatory processes in the lower respiratory tract. Analysis of exhaled breath condensate (EBC) is a non-invasive method for studying inflammatory mediators, such as cytokines, which are of interest from both physiological and therapeutic perspectives. The aim of this study was to assess and compare cytokine concentrations in the EBC of SSc patients and controls. MATERIAL AND METHODS: EBC was collected from 19 SSc patients and 19 controls. We used a multiplex assay test kit to assay interleukin (IL)-2, -4, -6, -10, tumour necrosis factor-alpha, and interferon-gamma in samples concentrated by lyophilization. RESULTS: Cytokine concentrations in EBC were higher in SSc patients than in controls. The stepwise analyses showed that IL-4 was the biomarker which contributed most to the discrimination between controls and patients (Wilk's Lambda = 0.55, p < 0.001). We observed significant negative correlations of EBC cytokines with total lung capacity and diffusion capacity of the lung for carbon monoxide. CONCLUSIONS: These findings suggest that EBC sampling permits the non-invasive study of inflammation in SSc patients, and may be correlated with the severity of interstitial lung disease.

A high proportion of donor CD4+ T cells expressing the lymph node-homing chemokine receptor CCR7 increases incidence and severity of acute graft-versus-host disease in patients undergoing allogeneic stem cell transplantation for hematological malignancy.

CC-chemokine receptor 7 (CCR7), a chemokine receptor required for transmigration into lymphoid organs, is only expressed by naive and central memory T cells. T cells with a capacity of homing into lymphoid organs can initiate acute graft-versus-host disease (GVHD) in mice and respond vigorously in vitro to alloantigens in humans, but their impact on clinical outcomes is unknown. We evaluated prospectively the distribution of naive, central memory and CCR7neg memory T-cell subsets in 39 bone marrow and 23 granulocyte colony-stimulating factor-mobilized peripheral blood stem cell allografts and investigated their impact on patient outcomes. Ranges of the relative proportions of CCR7+ cells within CD4+ and CD8+ T-cell populations were broad, but did not differ between the two sources of allografts. By multivariate analysis, high percentage of donor-derived CD4+CCR7+ T cells (>73.5%) significantly correlated with incidence, earliness of onset and severity of acute GVHD, conferring the highest adjusted hazard ratio (HR=3.9; 95% confidence interval 1.4-10.8; P=0.008) without interfering in other clinical events, especially chronic GVHD and relapse. Determination of the percentage of CD4+CCR7+ T cells in the graft provides a predictive indicator of acute GVHD. Partial depletion of this subset may reduce the risk of acute GVHD while preserving immunotherapeutic effects.

Low ileal interleukin 10 concentrations are predictive of endoscopic recurrence in patients with Crohn's disease.

BACKGROUND: Endoscopic recurrence after surgery in Crohn's disease is frequent and unpredictable. Abnormal intestinal production of pro- (interleukin (IL)-1 beta, tumour necrosis factor alpha (TNF-alpha)) and anti- (IL-10) inflammatory cytokines has been associated with severe outcome in experimental models of colitis. PATIENTS AND METHODS: We evaluated if ileal TNF-alpha, IL-1 beta, or IL-10 mRNA levels measured at the time of surgery predict endoscopic recurrence, and if ileal IL-10 levels are associated with particular IL-10 promoter alleles. Ileal biopsies were obtained peroperatively from the healthy neoileum of patients undergoing a right ileocolectomy for Crohn's disease. Mucosal TNF-alpha, IL-1 beta, and IL-10 mRNA levels were quantified by competitive polymerase chain reaction. A cut off value was determined using a receiver operating curve. IL-10.G promoter haplotypes were analysed using a polymorphic dinucleotide repeat in the IL-10 promoter region. RESULTS: Three months after surgery, 53% of patients had endoscopic recurrence while 47% remained free of disease. The risk of endoscopic recurrence correlated with ileal IL-10 mRNA concentrations (r(2)=0.81). Endoscopic recurrence occurred more frequently in patients classified as low IL-10 producers than in those that were high producers (80% v 40%) (p=0.02). Patients with at least one of the two alleles G7-8 or G10-13 produced, respectively, higher (p=0.006) and lower (p=0.029) ileal IL-10 mRNA. The distribution of IL-10.G microsatellite genotypes was similar in patients with or without endoscopic recurrence. CONCLUSION: Low ileal IL-10 mRNA concentration is a good marker of endoscopic recurrence in Crohn's disease but the distribution of IL-10.G haplotypes cannot predict the postoperative evolution of the disease.

Human endothelial-cell specific molecule-1 binds directly to the integrin CD11a/CD18 (LFA-1) and blocks binding to intercellular adhesion molecule-1.

ICAMs are ligands for LFA-1, a major integrin of mononuclear cells involved in the immune and inflammatory processes. We previously showed that endothelial cell specific molecule-1 (ESM-1) is a proteoglycan secreted by endothelial cells under the control of inflammatory cytokines. Here, we demonstrate that ESM-1 binds directly to LFA-1 onto the cell surface of human blood lymphocytes, monocytes, and Jurkat cells. The binding of ESM-1 was equally dependent on Ca(2+), Mg(2+), or Mn(2+) divalent ions, which are specific, saturable, and sensitive to temperature. An anti-CD11a mAb or PMA induced a transient increase in binding, peaking 5 min after activation. Direct binding of ESM-1 to LFA-1 integrin was demonstrated by specific coimmunoprecipitation by CD11a and CD18 mAbs. A cell-free system using a Biacore biosensor confirmed that ESM-1 and LFA-1 dynamically interacted in real time with high affinity (K(d) = 18.7 nM). ESM-1 consistently inhibited the specific binding of soluble ICAM-1 to Jurkat cells in a dose-dependent manner. These results suggest that ESM-1 and ICAM-1 interact with LFA-1 on binding sites very close to but distinct from the I domain of CD11a. Through this mechanism, ESM-1 could be implicated in the regulation of the LFA-1/ICAM-1 pathway and may therefore influence both the recruitment of circulating lymphocytes to inflammatory sites and LFA-1-dependent leukocyte adhesion and activation.

CD28+ intraepithelial lymphocytes with long telomeres are recruited within the inflamed ileal mucosa in Crohn disease.

Crohn disease is a chronic inflammatory bowel disease that involves all the intestine but predominantly alters the ileum. The disease largely depends on T cells, but the biologic role of intestinal intraepithelial lymphocytes (IEL) in transmural inflammation remains poorly characterized. To address this issue, a comparison of IEL and lamina propria lymphocytes (LPL) isolated from the uninvolved and the inflamed ileal mucosa of Crohn disease patients was performed. More CD8+ IEL (26% versus 8%) from the inflamed ileal mucosa expressed the CD28 receptor and the CD11a integrin than IEL from the uninvolved ileal mucosa, which were mostly CD28-. IEL had longer telomeres in the inflamed than in the uninvolved areas and a TCR Vbeta repertoire more similar to circulating T cells, suggesting that the increased proportion of CD28+ TCRalphabeta+ IEL within the inflamed mucosa is more likely due to recruited lymphocytes from the periphery that populate the epithelial layer than to the acquisition of the CD28 molecule by activated resident lymphocytes. In the uninvolved ileal mucosa, IEL from Crohn disease patients had shorter telomeric lengths than IEL from control patients, suggesting that they have been chronically stimulated. Such perturbation of the IEL population within the ileal mucosa could contribute to the inflammation in Crohn disease.

Pentoxifylline prevents upregulation of monocyte tissue factor in renal transplant recipients undergoing post-graft complications.

Pentoxifylline (PTX) has been demonstrated to improve graft survival in renal transplant recipients undergoing post graft complications. As activated monocytes are possible initiators of vascular damage through tissue factor (TF) expression, we evaluated the monocyte TF expression and endothelium activation markers in 140 consecutive patients receiving cadaveric kidney grafts, randomized in a double-blind study comparing PTX versus placebo. Monocyte TF expression and plasma von Willebrand factor, tissue plasminogen activator, thrombomodulin and tumor necrosis factor-alpha (TNF-alpha) levels were determined before transplantation and each month after. Additional samplings were realized in case of acute rejection. TF and TNF-alpha expression were significantly modified after graft. In patients with complications, PTX prevented the increase of TF expression at month one, and after rejection episodes. Endothelium activation markers were significantly modified after graft and in patients with complications but PTX had no significant effect on their plasma levels. These results suggest that the protective effect of PTX on graft survival could be related to the prevention of monocyte TF upregulation associated with complications.

Prolongation of cardiac allograft survival by selective injection of donor liver leukocytes in non-immunosuppressed rats.

Liver grafts are spontaneously accepted in several animal combinations and are able to induce acceptance of another organ originating from the same donor, which would be rejected when transplanted alone. However, the exact mechanism of this unique tolerance induction capability remains unclear. The aim of our study was to investigate the ability of nonparenchymal liver cells to induce tolerance when they were separated from their parenchymal environment. In the murine combination we used (BN --> LEW), heart transplants were constantly tolerated after combined liver plus heart grafting, but rejected when transplanted alone. Nonparenchymal liver cells were isolated from BN rat livers by enzymatic digestion and injected, at different times, to LEW rats, which were recipients of BN heart transplants. The average number of mononuclear cells obtained after isolation was 20 x 10(6)/5 g of rat liver. Immediate trypan-blue exclusion test showed more than 95% of viable cells. Phenotypic studies showed a predominant (47%) lymphocyte population, 7% were monocytes and 46% were cellular debris. Among the lymphocyte population, the majority of cells were bearing the NKR-P1 receptor and about 30% CD3 receptors. Inoculation of nonparenchymal liver cells 7 and 30 days prior to heart transplantation significantly prolonged graft survival compared to controls (14.6 and 12.7 vs. 8.1 days; p = 0.0008 and 0.0059, respectively), whereas simultaneous injection (day 0) had no effect. Injection of donor splenocytes or nonparenchymal liver cells from a third party, at any time, had no effect on rejection. These results provide some more evidence about the specific role of liver lymphocytes in allogenic unresponsiveness. They also suggest that the hepatic parenchymal environment is necessary for the optimal development of this phenomenon.

Negative immunohistochemical detection of CD103 (alphaEbeta7 integrin) in the infiltrates of acute rejection in liver and kidney transplantation.

BACKGROUND: The infiltration of epithelium by CD8+ T lymphocytes in human renal or liver allografts is a critical feature of acute rejection. CD103 expression can be acquired in vitro by CD8+ cytotoxic T lymphocytes in response to allogeneic renal epithelial cells and promotes their adhesion to epithelium and subsequent lysis of epithelial cells. We investigated the expression of CD103 in T-cell infiltrates during acute renal or liver rejection (grade < III). METHODS: Immunohistochemical detection of CD103 in 11 liver and 10 kidney transplant biopsies with histopathological diagnosis of acute rejection. RESULTS: None of the infiltrates expressed detectable CD103, although positive controls were stained under our conditions. CONCLUSIONS: Failure to detect CD103 in renal biopsies can be related to the early posttransplantation interval (<6 months) corresponding to a first rejection episode. In our hands, immunohistological detection of CD103 was not possible in the infiltrates of acute rejection in liver or kidney transplantation.

Estradiol-stimulated nitric oxide release in human granulocytes is dependent on intracellular calcium transients: evidence of a cell surface estrogen receptor.

We tested the hypothesis that estrogen acutely stimulates constitutive nitric oxide synthase activity in human granulocytes by acting on a cell surface estrogen receptor (ER). The release of nitric oxide was measured in real time with an amperometric probe. Exposure of granulocytes to 17beta-estradiol stimulated NO release within seconds in a concentration-dependent manner. The NO release was also stimulated by 17beta-estradiol conjugated to bovine serum albumin (E(2)-BSA), which suggests mediation by a cell surface receptor. Tamoxifen, an ER inhibitor, antagonized the action of both 17beta-estradiol and E(2)-BSA, whereas ICI 182,780, an inhibitor of the nuclear ER, had no effect. Using dual emission microfluorometry in a calcium-free medium, the 17beta-estradiol-stimulated release of NO from granulocytes was shown to be dependent on intracellular calcium ([Ca(2+)]i) transients in a tamoxifen-sensitive process. Exposure to BAPTA-AM (1,2bis-(-aminophenoxy)ethans-N,N,N', N'-tetraacetic acid tetra(acetoxyymethyl) ester), a [Ca(2+)]i chelator, reduced [Ca(2+)]i in response to E(2)-BSA, and depleting [Ca(2+)]i stores abolished the effect of 17beta-estradiol on NO release. Confocal photomicrographs using E(2)-BSA-FITC (fluorescein isothiocyanate) revealed cell membrane reactivity. Estrogen-stimulated NO release had an immunosuppressive effect, and it initiated granulocyte rounding and loss of adherence in a tamoxifen-sensitive manner. Finally, using reverse transcriptase-polymerase chain reaction, human neutrophil granulocytes expressed ERalpha but not ERbeta, suggesting that ERalpha may be the membrane receptor for 17beta-estradiol. The study demonstrated that a physiological dose of estrogen down-regulates granulocyte activity by acutely stimulating NO release via the activation of a cell surface ER which is coupled to increases in [Ca(2+)]i. (Blood. 2000;95:3951-3958)

Immunomodulatory effect of pentoxifylline during human allograft rejection: involvement of tumor necrosis factor-alpha and adhesion molecules.

BACKGROUND: Pentoxifylline (PTX), a methylxanthine phosphodiesterase inhibitor, is poorly active as an immunosuppressant but prevents the synthesis of proinflammatory cytokines. In a randomized double-blind study comparing PTX versus placebo in 140 patients receiving cadaveric kidney grafts under cyclosporine and prednisone, we have shown that PTX weakened the consequences of rejection on graft survival. To assess the mechanism underlying the beneficial effect recorded during this trial, we analyzed the impact of PTX on tumor necrosis factor (TNF-alpha) production and expression of cell adhesion molecules. METHODS: Plasma levels of TNF-alpha and its soluble receptors (sTNF-RI, sTNF-RII) and of soluble vascular cell adhesion molecule 1 (sVCAM-1) were monitored over the 6 months postgraft period when PTX or placebo were administered. Expression of VCAM-1 and intercellular cell adhesion molecule 1 was scored by immunohistochemical staining of biopsy specimens from patients who underwent rejection crisis. Lymphocyte subset composition was analyzed longitudinally during cytomegalovirus (CMV) infections. RESULTS: Plasma TNF-alpha levels were significantly reduced in the PTX-treated group over the 6 months of administration, and specifically during isolated rejection episodes and during CMV infections. Plasma levels of sTNFR-I, sTNFR-II, and sVCAM-1 did not differ between the two groups of patients, but a decrease in renal tubular VCAM-1 expression was observed in the PTX group. During CMV infections, CD8 lymphocytosis and expansion of CD57+ (CD28-) CD8+ T cells were similar in the two groups. CONCLUSION: The data collected during this double-blind study point to an immunomodulatory role of PTX, the beneficial effect on graft survival resulting from a restraining effect of the drug on the inflammatory conditions involved in acute graft rejection.

Peripheral human CD8(+)CD28(+)T lymphocytes give rise to CD28(-)progeny, but IL-4 prevents loss of CD28 expression.

At birth, virtually all peripheral CD8(+) T cells express the CD28 co-stimulatory molecule, but healthy human adults accumulate CD28(-)CD8(+) T cells that often express the CD57 marker. While these CD28(-) subpopulations are known to exert effector-type functions, the generation, maintenance and regulation of CD28(-) (CD57(+) or CD57(-)) subpopulations remain unresolved. Here, we compared the differentiation of CD8(+)CD28(bright)CD57(-) T cells purified from healthy adults or neonates and propagated in IL-2, alone or with IL-4. With IL-2 alone, CD8(+)CD28(bright)CD57(-) T cell cultures yielded a prevailing CD28(-) subpopulation. The few persisting CD28(dim) and the major CD28(-) cells were characterized by similar telomere shortening at the plateau phase of cell growth. Cultures from adults donors generated four final CD8(+) phenotypes: a major CD28(-)CD57(+), and three minor CD28(-)CD57(-), CD28(dim)CD57(-) and CD28(dim)CD57(dim). These four end-stage CD8(+) subpopulations displayed a fairly similar representation of TCR V(beta) genes. In cultures initiated with umbilical cord blood, virtually all the original CD8(+)CD28(bright) T cells lost expression of CD28, but none acquired CD57 with IL-2 alone. IL-4 impacted on the differentiation pathways of the CD8(+)CD28(bright)CD57(-) T cells: the addition of IL-4 led both the neonatal and the adult lymphocytes to keep their expression of CD28. Thus, CD8(+)CD28(bright)CD57(-) T cells can give rise to four end-stage subpopulations, the balance of which is controlled by both the cytokine environment, IL-4 in particular, and the proportions of naive and memory CD8(+)CD28(+) T cells.

Enhancing the effect of secreted cyclophilin B on immunosuppressive activity of cyclosporine.

BACKGROUND: Cyclophilin B (CyPB) is a cyclosporine (CsA)-binding protein, located within intracellular vesicles and secreted in biological fluids. In previous works, we reported that CyPB specifically interacts with the T-cell membrane and potentiates the ability of CsA to inhibit CD3-induced proliferation of T lymphocytes. METHODS: CyPB levels were measured in plasma from healthy donors and transplant patients. The role of extracellular CyPB on the distribution and activity of CsA was investigated first by studies on the uptake of free and CyPB-complexed drug by blood cells, and second by studies on the inhibitory effects of these two compounds on the CD3-induced proliferation of peripheral blood mononuclear cells. RESULTS: A significant increase in plasma CyPB level was observed for CsA-treated patients (13+/-6.4 nM, n=42) in comparison with untreated donors (4.3+/-2.1 nM, n=34). In vitro, extracellular CyPB dose dependently modified CsA distribution between plasma, erythrocyte, and lymphocyte contents, by both retaining the complexed drug extracellularly and promoting its specific accumulation within peripheral blood mononuclear cells. Moreover, the enhanced ability of CyPB-complexed CsA to suppress CD3-induced T-cell proliferation was preserved in the presence of other blood cells, implying specific targeting of the drug to sensitive cells. Furthermore, although a large interindividual variability of sensitivity to the drug was confirmed for 18 individuals, we found that CyPB potentiated the activity of CsA in restoring a high sensitivity to the immunosuppressant. CONCLUSION: These results suggest that plasma CyPB may contribute to the acceptance and the good maintenance of organ transplantation by enhancing the immunosuppressive activity of CsA through a receptor-mediated incorporation of CyPB-complexed CsA within peripheral blood lymphocytes.

Improvement in the outcome of rejection with pentoxifylline in renal transplantation: a randomized controlled trial.

BACKGROUND: Pentoxifylline (PTX), a methylxantine phosphodiesterase inhibitor commonly used to treat peripheral vascular disease, has been shown to decrease the production of proinflammatory cytokines and reactive oxygen species and to reduce the toxic effects of cyclosporine. Thus, administration of PTX to transplant patients, as an adjunct to immunosuppressive therapy, could prevent numerous posttransplantation complications. METHODS: One hundred forty consecutive patients receiving cadaveric kidney grafts were registered in a randomized double-blind study comparing PTX at a dose of 800 mg/day, then 1200 mg/day, versus placebo during the first 6 months after transplantation. All patients were followed up for 1 year. RESULTS: Rejection episodes were validated as the only independent risk factor for graft loss in this study. We compared graft survival rates in each group according to the presence or absence of acute rejection. Acute rejection reduced graft survival in the control group (graft survival rate at 1 year, 59% vs. 97%, P < 0.001), but this adverse effect was blunted in the PTX group (72% vs. 89%, NS). This improvement was confirmed by multivariate analysis for risk factors, with graft survival rates being described at best as the interaction between rejection and treatment (PTX vs. placebo, P = 0.045). CONCLUSION: Although PTX does not modify the incidence of any posttransplant complications, it weakens the consequences of rejection on graft survival.