Microchimerism, donor dendritic cells, and alloimmune reactivity in recipients of Flt3 ligand-mobilized hemopoietic cells: modulation by tacrolimus

Flt3 ligand (FL) is a potent hemopoietic growth factor that strikingly enhances stem cells and dendritic cells (DC) in vivo. We examined the impact of infusing FL-mobilized bone marrow (BM) cells on microchimerism and anti-donor reactivity in normal and tacrolimus-immunosuppressed, noncytoablated allogeneic recipients. BM from B10 (H2b) mice given FL (10 microg/day; days 0-8; FL-BM) contained a 7-fold higher incidence of potentially tolerogenic immature CD11c+ DC (CD40low, CD80low, CD86low, MHC IIlow) that induced alloantigen-specific T cell hyporesponsiveness in vitro. C3H (H2k) mice received 50 x 106 normal or FL-BM cells (day 0) and tacrolimus (2 mg/kg/day; days 0-12). On day 15, enhanced numbers of donor (IAb+) cells were detected in the thymi and spleens of FL-BM recipients. Tacrolimus markedly enhanced microchimerism, which declined as a function of time. Ex vivo splenocyte proliferative and CTL responses and Th1 cytokine (IFN-gamma) production in response to donor alloantigens were augmented by FL-BM infusion, but reduced by tacrolimus. Systemic infusion of purified FL-BM immature DC, equivalent in number to that in corresponding whole BM, confirmed their capacity to sensitize, rather than tolerize, recipient T cells in vivo. In vitro, tacrolimus suppressed GM-CSF-stimulated growth of myeloid DC from normal BM much more effectively than from FL-BM without affecting MHC class II or costimulatory molecule expression. Infusion of normal B10 BM cells at the time of transplant prolonged C3H heart allograft survival, whereas FL-BM cells did not. A therapeutic effect of tacrolimus on graft survival was observed in combination with normal, but not FL-BM cells. These findings suggest the need for alternative immunosuppressive strategies to calcineurin inhibition to enable the engraftment, survival, and immunomodulatory function of FL-enhanced, immature donor DC.     

G-CSF-induced evacuation of sinusoidal NK cells and the facilitation of liver regeneration in a partial hepatectomy

Since liver regeneration after partial hepatectomy (PHx) is known to improve by pretreatment with recombinant human G-CSF (rhG-CSF), we investigated the mechanism by evaluating the distribution and activity of sinusoidal NK cells. F344 rats were treated with rhG-CSF (250 microg/kg/day) for 5 days before PHx. Pretreatment with rhG-CSF improved the serum ALT levels and DNA biosynthesis of the remnant liver tissues at 20 h after PHx. Notably, the rhG-CSF pretreatment decreased the number of NK cells in the liver determined by immunohistochemistry using anti-NKR-P1A mAb before and at 20 h after PHx with no significant change in the NK activity per cell base, while also increasing the number of NK cells in the peripheral blood detected by flow cytometry. The rhG-CSF induced a pre-PHx downregulation of the IL-12p70 protein levels, while also promoting the post-PHx reduction of the protein levels of IL-12p70 and IFN-gamma. Conversely, rhG-CSF had no effect on the pre-PHx mRNA levels or the PHx-induced upregulation of mRNA levels of TNF-alpha, IL-1beta, IL-6, TGF-beta, IL-10, HGF, and c-Met determined by real-time RT-PCR. These results strongly suggest that rhG-CSF-induced facilitation of liver regeneration is achieved by immunoregulation through the intrahepatic IL-12 downregulation and evacuation of sinusoidal NK cells.     

The associations of IL-18 serum levels and promoter polymorphism with tacrolimus pharmacokinetics and hepatic allograft dysfunction in Chinese liver transplantation recipients

Interleukin 18 (IL-18) is a potent proinflammatory cytokine, which promotes the secretions of TNF-α, IL-1β, IL-2 and IFN-γ. All those inflammatory cytokines can influence the CYP450 and MDR dependent drug disposition. On the other side, those cytokines can induce hepatic allograft dysfunction. We investigated the effects of serum IL-18 and IL-18 gene promoter polymorphisms on tacrolimus pharmacokinetics and hepatic allograft dysfunction in liver transplant recipients. A total of 155 liver transplant recipients were enrolled into this study (34 females and 121 males). The mean follow-up was 52 months (range 16-96 months).The total liver transplant recipients were divided into hepatic allograft dysfunction (N = 14) and no hepatic allograft dysfunction (N = 141). We studied two single-nucleotide polymorphisms in the promoter region of IL-18 gene at the position G-137C (rs187238) and A-607C (rs1946518) by HRM analysis (high-resolution melting curve analysis). Tacrolimus dosage, tacrolimus blood concentration, serum levels of IL-18 and IFN-γ were also investigated. We found the recipients with higher IL-18 and IFN-γ serum levels had lower tacrolimus concentration/dose (C/D) ratios (P < 0.05). In the mean time, after transplantation hepatic allograft dysfunction was more likely to happen to those recipients. However, there was no significant difference in the frequencies of A-607C and G-137C allelic distribution in recipients' tacrolimus concentration/dose (C/D) ratios. This study identifies IL-18 reduced tacrolimus concentration/dose (C/D) ratio through up regulation of P-glycoprotein (P-gp).     

Recombinant human granulocyte colony-stimulating factor improves the compromised state of recipient mice without affecting the induction of specific tolerance in the cyclophosphamide-induced tolerance system.

The effects of recombinant human granulocyte CSF (rhG-CSF) on cyclophosphamide (CP)-induced tolerance was studied. In the recipient C57BL/10 Sn Slc (B10) mice given 1 x 10(8) B10.BR Sg Sn Slc (B10.BR) spleen cells (SC) on day -2 followed by 200 mg/kg CP on day 0, the number of leukocytes and neutrophils in the periphery declined to their minimum levels on day 4. When rhG-CSF in a dose of 200 micrograms/kg was given daily for 5 days to the B10 mice, which had been treated with B10.BR SC and CP, starting one day after the administration of CP, the leukocyte and neutrophil counts declined to the same levels as those in the B10 mice treated with B10.BR SC and CP alone on day 2. On day 4, however, the counts recovered to their normal levels. The nucleated cell count of the spleen in the B10 mice given B10.BR SC and CP followed by rhG-CSF decreased less and recovered faster than that in the B10 mice given B10.BR SC and CP. The case was found to be the same in bone marrow, and the difference did not reach statistical significance. When the recipient mice were inoculated i.p. with 4 x 10(4) Pseudomonas aeruginosa (GNB-139) on day 4, the survival of the B10 mice treated with B10.BR SC and CP was markedly improved by rhG-CSF administration. The administration of rhG-CSF did not affect either the prolongation or the specificity of skin allograft survival, as shown in an H-2 mis-matched combination of B10.BR----B10 and in an H-2 identical combination of AKR/J Sea(AKR)----C3H/HeN Crj (C3H). The tolerant state, which was demonstrated by various immune responses, such as CTL, delayed footpad reaction, and antibody, was also not affected by rhG-CSF. Furthermore, the basic mechanisms for inducing a long-lasting skin allograft tolerance in this system--namely, the specific destruction of Ag-stimulated and then proliferating mature T cells in the periphery, the establishment of mixed chimerism, and the intrathymic clonal deletion of immature T cells--were preserved even when rhG-CSF was given to C3H mice previously made tolerant of AKR.     

Intracerebroventricular interleukin-6 treatment decreases body fat in rats

Recently we found that interleukin-6 (IL-6) knockout mice develop mature-onset obesity and that a single intracerebroventricular (ICV) injection of IL-6 increases energy expenditure. In the present study we investigated if chronic ICV treatment with IL-6 can suppress body fat mass. IL-6 was injected ICV daily for two weeks to rats fed a high-fat diet. IL-6 treatment but not saline treatment decreased body weight by 8.4% and decreased the relative weights of mesenteric and retroperitoneal fat pads. Consistent with this, circulating leptin levels were decreased by 40% after IL-6 treatment but not after saline treatment. Average food intake per day was decreased in the IL-6 treated group compared to the saline treated rats. IL-6 treatment did not change hepatic expression of the acute-phase protein haptoglobin, serum levels of insulin or insulin-like growth factor-I, or the weights of the heart, liver, kidneys, adrenals, and spleen. We conclude that centrally administered IL-6 can decrease body fat in rats without causing acute-phase reaction.     

Host CD40 ligand deficiency induces long-term allograft survival and donor-specific tolerance in mouse cardiac transplantation but does not prevent graft arteriosclerosis

Although interruption of CD40-CD40L interactions via their respective mAbs yields prolonged allograft survival, the relative importance of CD40 or CD40L on donor or host cells remains unknown. Moreover, it is uncertain whether any allospecific tolerance occurring with CD40-CD40L blockade will also prevent allograft arteriopathy, the major long-term limitation to transplantation. Therefore, we performed cardiac transplantations using CD40L-deficient (CD40L-/-) mice to investigate the mechanisms underlying prolonged allograft survival. Without immunosuppression, wild-type (WT) hosts rejected allo-mismatched WT or CD40L-/- heart allografts within 2 wk. Conversely, allografts in CD40L-/- hosts beat vigorously for 12 wk. Anti-CD40 treatment did not induce graft failure in CD40L-/- recipients. Although graft-infiltrating cells were reduced approximately 50% in CD40L-/- hosts, the relative percentages of macrophages and T cell subsets were comparable to WT. IFN-gamma, TNF-alpha, and IL-10 were diminished commensurate with the reduced cellular infiltrate; IL-4 was not detected. CD40L-/- recipients did not develop IgG alloantibodies and showed diminished B7 and CD28 expression on subsets of graft-infiltrating cells. CD40L-/- transplant recipients developed allospecific tolerance to the donor haplotype; second set donor skin grafts engrafted well, whereas third-party skin grafts were vigorously rejected. By MLR, splenocytes from CD40L-/- allograft recipients also demonstrated allo-specific hyporesponsiveness. Nevertheless, allografts in CD40L-/- hosts developed significant graft arteriosclerosis by 8-12 wk posttransplant. Therefore, we propose that early alloresponses, without CD40-CD40L costimulation, induce allospecific tolerance but may trigger allo-independent mechanisms that ultimately result in graft vasculopathy.     

Treatment with interleukin-11 affects plasma leptin levels in inflamed and non-inflamed rabbits

Treatment with the anti-inflammatory cytokine, interleukin-11 (IL-11), in rabbits with TNBS-colitis reduces tissue damage but does not normalize body weight loss despite an increase in plasma levels of motilin, known to stimulate food intake. We investigated whether IL-11 could increase plasma levels of the anorectic peptide, leptin in non-inflamed and inflamed rabbits. In addition, the effect of IL-11 and leptin on motilin mRNA expression in the T84 cell line was tested. Five days post-inflammation, weight loss amounted 10.7+/-1.2%, but plasma leptin and motilin levels were unaffected. During IL-11 treatment, weight loss remained and plasma leptin levels dose-dependently increased with 27+/-5% (4 microg/kg day) and 108+/-7% (720 microg/kg day). Motilin levels increased in parallel with 23+/-12% or 256+/-97%. In non-inflamed animals, a prompt decrease in weight (-11.9+/-1%) was observed after treatment with the highest dose of IL-11 and this was associated with an increase in plasma leptin (70+/-18%) and motilin levels (113+/-7%). Both IL-11 and leptin stimulated motilin mRNA expression in T84 cells with a different time profile. In conclusion, the increase in plasma leptin levels during IL-11 treatment induces wasting in normal rabbits and may be one of the major factors involved in the maintenance of body weight loss in rabbits with colitis. Increase of motilin expression by leptin may be part of a feedback mechanism.     

Introduction of the interleukin-10 gene into mice inhibited bleomycin-induced lung injury in vivo

Interleukin (IL)-10 has been shown to reduce many inflammatory reactions. We investigated the in vivo effects of IL-10 on a bleomycin-induced lung injury model. Hemagglutinating virus of Japan (HVJ)-liposomes containing a human IL-10 expression vector (hIL10-HVJ) or a balanced salt solution as a control (Cont-HVJ) was intraperitoneally injected into mice on day -3. This was followed by intratracheal instillation of bleomycin (0.8 mg/kg) on day 0. Myeloperoxidase activity of bronchoalveolar lavage fluid and tumor necrosis factor-alpha mRNA expression in bronchoalveolar lavage fluid cells on day 7 and hydroxyproline content of the whole lung on day 21 were inhibited significantly by hIL10-HVJ treatment. However, Cont-HVJ treatment could not suppress any of these parameters. We also examined the in vitro effects of IL-10 on the human lung fibroblast cell line WI-38. IL-10 significantly reduced constitutive and transforming growth factor-beta-stimulated type I collagen mRNA expression. However, IL-10 did not affect the proliferation of WI-38 cells induced by platelet-derived growth factor. These data suggested that exogenous IL-10 may be useful in the treatment of pulmonary fibrosis.     

IL-33 expands suppressive CD11b+ Gr-1(int) and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival

IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4(+) Foxp3(+) regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8(+) IFN-γ(+) cells. Also, despite reducing overall CD3(+) cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3(+) cells. Whereas control graft recipients displayed increases in systemic CD11b(+) Gr-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4(+) Foxp3(+) Tregs that underlie IL-33-mediated cardiac allograft survival.     

Linked unresponsiveness: early cytokine gene expression profiles in cardiac allografts following pretreatment of recipients with bone marrow cells expressing donor MHC alloantigen

Linked unresponsiveness operates to induce specific unresponsiveness to fully mismatched vascularized allografts in recipients pretreated with anti-CD4 antibody and syngeneic bone marrow cells expressing a single donor MHC class I alloantigen. The aim of the study was to evaluate early post transplant cytokine expression in allografts where linked unresponsiveness was required for long term graft survival. CBA (H2(k)) mice were pretreated with CBK (H2(k)+K(b)) bone marrow cells under the cover of anti-CD4 antibody 28 days before transplantation of a CBK or a C57BL/10 (H2(b)) cardiac allograft. In both cases graft survival was prolonged (MST=100 days). Intragraft expression for interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-10, IL-12(p40), IL-18, iNOS, transforming growth factor (TGF)-beta(1) and C-beta was determined on day 1.5, 3, 7 and 14 after transplantation. Whereas rejecting allografts displayed a sharp peak in IFN-gamma, IL-2, IL-4 and IL-10 expression, non-rejecting allografts were characterized by an initial TGF-beta(1) and IFN-gamma production. An increasing IL-4 expression towards day 14 was a unique feature of linked unresponsiveness. All non-rejecting allografts were characterized by an increasing IL-12(p40) production towards day 14. In summary, the early cytokine expression pattern in allografts after bone marrow induced operational tolerance is influenced by the quantity of donor alloantigens expressed on the graft as well as on the bone marrow inoculum.     

Gut Microbiota and Tacrolimus Dosing in Kidney Transplantation

Tacrolimus dosing to establish therapeutic levels in recipients of organ transplants is a challenging task because of much interpatient and intrapatient variability in drug absorption, metabolism, and disposition. In view of the reported impact of gut microbial species on drug metabolism, we investigated the relationship between the gut microbiota and tacrolimus dosing requirements in this pilot study of adult kidney transplant recipients. Serial fecal specimens were collected during the first month of transplantation from 19 kidney transplant recipients who either required a 50% increase from initial tacrolimus dosing during the first month of transplantation (Dose Escalation Group, n=5) or did not require such an increase (Dose Stable Group, n=14). We characterized bacterial composition in the fecal specimens by deep sequencing of the PCR amplified 16S rRNA V4-V5 region and we investigated the hypothesis that gut microbial composition is associated with tacrolimus dosing requirements. Initial tacrolimus dosing was similar in the Dose Escalation Group and in the Stable Group (4.2±1.1 mg/day vs. 3.8±0.8 mg/day, respectively, P=0.61, two-way between-group ANOVA using contrasts) but became higher in the Dose Escalation Group than in the Dose Stable Group by the end of the first transplantation month (9.6±2.4 mg/day vs. 3.3±1.5 mg/day, respectively, P<0.001). Our systematic characterization of the gut microbial composition identified that fecal Faecalibacterium prausnitzii abundance in the first week of transplantation was 11.8% in the Dose Escalation Group and 0.8% in the Dose Stable Group (P=0.002, Wilcoxon Rank Sum test, P<0.05 after Benjamini-Hochberg correction for multiple hypotheses). Fecal Faecalibacterium prausnitzii abundance in the first week of transplantation was positively correlated with future tacrolimus dosing at 1 month (R=0.57, P=0.01) and had a coefficient±standard error of 1.0±0.6 (P=0.08) after multivariable linear regression. Our novel observations may help further explain inter-individual differences in tacrolimus dosing to achieve therapeutic levels.     

Cytokines regulate the pattern of rejection and susceptibility to cyclosporine therapy in different mouse recipient strains after cardiac allografting

We determined the role of cytokines in regulating the pattern of rejection and recipient susceptibility to cyclosporine (CsA) in a mouse cardiac allograft model. Hearts from C3H mice transplanted into untreated BALB/c (Th2-dominant) and C57BL/6 (Th1-dominant) mice showed different patterns of rejection. C3H allografts in BALB/c mice showed typical acute vascular rejection (AVR) with strong intragraft deposition and high serum levels of anti-donor IgG with predominant IgG1, while C3H allografts in C57BL/6 mice showed typical acute cellular rejection (ACR) with massive intragraft infiltration of CD4(+) and CD8(+) lymphocytes and low serum levels of anti-donor IgG with predominant IgG2a. Elevated intragraft mRNA expression of IL-2, IFN-gamma, and IL-12 mRNA was present in C57BL/6 recipients, whereas allografts in BALB/c mice displayed increased IL-4 and IL-10 mRNA levels. CsA therapy completely inhibited ACR and induced indefinite allograft survival in C57BL/6 recipients, while the same therapy failed to prevent AVR, and only marginally prolonged graft survival in BALB/c recipients. In contrast, rapamycin blocked AVR, achieving indefinite survival in BALB/c recipients, but was less effective at preventing ACR in C57BL/6 recipients. The disruption of the IL-12 or IFN-gamma genes in C57BL/6 mice shifted ACR to AVR, and resulted in concomitant recipient resistance to CsA therapy. Conversely, disruption of IL-4 gene in BALB/c mice markedly attenuated AVR and significantly prolonged allograft survival. These data suggest that the distinct cytokine profiles expressed by different mouse strains play an essential role in regulating the pattern of rejection and outcome of CsA/rapamycin therapy.     

Capsicum ethanol extracts and capsaicin enhance interleukin-2 and interferon-gamma production in cultured murine Peyer's patch cells ex vivo

We investigated the effects of red pepper (Capsicum annuum Lin.) extracts (capsicum extract) and its main pungent capsaicin on T helper 1 (Th1) and 2 (Th2) cytokine production in cultured murine Peyer's patch (PP) cells in vitro and ex vivo. Direct administration of capsicum extract (1 and 10 mug/ml) and capsaicin (3 and 30 muM) resulted in suppression of interleukin (IL)-2, interferon (IFN)-gamma, IL-4 and IL-5 production. In an ex vivo experiment using PP cells removed from the mice after oral administration of capsicum extract (10 mg/kg/day for 4 consecutive days), IL-2, IFN-gamma and IL-5 increased in response to concanavalin A (Con A). Oral administration of 3 mg/kg/day capsaicin, one active constituent of the extract, also enhanced IL-2, INF-gamma and IL-4 production in response to Con A stimulation but did not influence the production of IL-5. Orally administered capsazepine (3 mg/kg/day), a selective transient receptor potential vanilloid 1 (TRPV1) antagonist, slightly enhanced IL-2 production also irrespective of Con A stimulation. The capsaicin-induced enhancement of both IL-2 and IFN-gamma production was not reduced by oral administration of capsazepine (3 mg/kg/day), suggesting a TRPV1 receptor-independent mechanism. Flow cytometric analysis revealed that the population of CD3(+) cells in the PP cells was significantly reduced while CD19(+) cells increased after oral administration of capsicum extract (1 and 10 mg/kg/day) and capsaicin (0.3 and 3 mg/kg/day). Capsazepine (3 mg/kg/day) weakly but significantly reversed these effects. Orally administered capsicum extract and capsaicin did not change the T cell subset (CD4(+) and CD8(+)), Th1 (IFN-gamma(+)) and T2 (IL-4(+)) ratio. These findings indicate that capsicum extract and capsaicin modulate T cell-immune responses, and their immunomodulatory effects on murine PP cells are partly due to both TRPV1-dependent and -independent pathway.     

Pharmacodynamics of T-cell function for monitoring immunosuppression

OBJECTIVES: Recent studies show that measuring pharmacodynamic (PD) effects offers a unique possibility to predict immunosuppression. Thus, in this study we have monitored the PD properties of immunosuppressants on diverse T-cell functions in heart transplant (HTx) recipients. MATERIALS: PDs and blood concentrations (PK) of three different basis-immunosuppressive drugs were studied: cyclosporin A (CsA); tacrolimus (TRL) and sirolimus (SRL). T-cell function was analysed by expression of proliferating cell nuclear antigen (PCNA) labelling, expression of cytokines (IL-2, IFN-gamma) and surface antigen (for example, CD25) by FACS analysis. RESULTS: In group I, at time points C0 and C2, increased CsA-PK significantly inhibited expression of IL-2, IFN-gamma, PCNA and CD25 (P < 0.05). Correlations (r(2)) at C2 between inhibition of T-cell functions (PD) with PK and with drug doses were: CsA-PK: 0.71-0.91 and CsA-dose: 0.73-0.87. In group II, increased TRL-PK over time did not further inhibit expression of CD25, but inhibited PCNA expression more on day 3, and IL-2 and IFN-gamma expression was significantly higher on days 2 and 3 compared to PD effects of CsA (P < 0.05). Blood SRL concentrations in C0 group III, increased on day 1 and remained stable at days 3 and 4. Expression of PCNA was not altered in the SRL-PK category, whereas expression of CD25 was higher and expression of cytokines was lower than PD effects of CsA. CONCLUSIONS: Our results show that PD effects on T-cell function can be used to monitor immunosuppression bringing potential to increase the efficacy and safety of immunosuppressive therapy after HTx.