Preventive effect of G-CSF on acute lung injury via alveolar macrophage regulation.

BACKGROUND: Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) are devastating clinical syndromes associated with a high mortality rate. We examined the preventive effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in a mouse ALI/ARDS model induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: Mice were injected with LPS (10-20 mg/kg) with or without rhG-CSF pretreatment (250 mg/kg/d). Survival rate, cytokine mRNA expression, and pathologic findings were examined. RESULTS: The 96-h survival rate of the control group was 20%. Survival was significantly increased to 80% in rhG-CSF-treated animals. LPS-induced destruction of the alveolar structure was not observed in the rhG-CSF group. Pretreatment with rhG-CSF led to significantly lower mRNA expression of TNF-α and IL-1ß in the lung 24 h after LPS administration and significantly higher IL-10 expression 96 h after LPS administration. Immunohistochemical analysis revealed that treatment with rhG-CSF also prevented the up-regulation of TNF-α and IL-1ß protein expression in alveolar macrophages. CONCLUSION: Treatment with rhG-CSF prevents the development of ALI/ARDS induced by LPS by affecting the production of pro- and anti-inflammatory cytokines and may be promising in clinical applications.

Relationship between the cAMP levels in leukocytes and the cytokine balance in patients surviving gram negative bacterial pneumonia.

Lipopolysaccharide-stimulated leukocytes secrete proinflammatory cytokines including tumor necrosis factor-α and interleukin-12. Over-activation of host defense systems may result in severe tissue damage and requires regulation. Granulocyte colony-stimulating factor and interleukin-10 are candidate cytokines for inducing tolerance to lipopolysaccharide re-stimulation. We compared cytokines secreted by lipopolysaccharide-stimulated blood cells from patients who had survived gram negative bacterial pneumonia (Pseudomonas aeruginosa, Escherichia coli or Proteus mirabilis, n = 26) and age-matched healthy volunteers (n = 18). Interleukin-12p70 and tumor necrosis factor-α expression was significantly lower in patients (p = 0.0039 and p<0.001) compared to healthy controls, while granulocyte colony-stimulating factor production was markedly higher in patients (p<0.001). Levels of interleukin-10 were comparable. Granulocyte colony-stimulating factor expression was inversely correlated with interleukin-12p70 (R = -0.71, p<0.001) and tumor necrosis factor-α (R = -0.64, p<0.001) expression; interleukin-10 showed no significant correlation. In unstimulated leukocytes from patients, cAMP levels were significantly raised (p = 0.020) and were correlated inversely with interleukin-12p70 levels (R = -0.81, p<0.001) and directly with granulocyte colony-stimulating factor (R = 0.72, p = 0.0020), matrix metalloproteinase-9 (R = 0.67, p = 0.0067) and interleukin-10 (R = 0.54, p = 0.039) levels. Our results demonstrate that granulocyte colony-stimulating factor production by lipopolysaccharide-stimulated leukocytes is a useful indicator of tolerance induction in surviving pneumonia patients and that measuring cAMP in freshly isolated leukocytes may also be clinically significant.

The neutrophil/Th1 lymphocyte balance and the therapeutic effect of granulocyte colony-stimulating factor in TNBS-induced colitis of rat strains.

Intramucosal neutrophil infiltration is related to the activity of ulcerative colitis, and Th1 immunity is responsible for the onset of Crohn's disease. We examined the therapeutic effects of recombinant human granulocyte colony-stimulating factor (rHuG-CSF) in the two types of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis of five rat strains. SD and DA rats showed much lower mRNA expression levels of endogenous G-CSF in lipopolysaccharide (LPS)-stimulated splenocytes than did Lewis, F344, and BN rats. On day 7 after anal instillation of TNBS, SD and DA rats demonstrated massive lymphocyte infiltration with an interferon-gamma (IFN-gamma) mRNA upregulation, whereas Lewis, F344, and BN rats showed an intense submucosal neutrophil accumulation with high tumor necrosis factor-alpha (TNF-alpha) mRNA levels. A 5-day course of rHuG-CSF pretreatment (250 microg/kg/day, s.c.) reduced the elevated levels of both cytokines. The treatment improved the survival rate of DA and reduced the degree of body weight loss of SD, while not significantly influencing the wasting disease of other strains. Interleukin-10 (IL-10) mRNA levels were highly upregulated by rHuG-CSF treatment on day 1 in the neutrophil-dominant lesions of F344 but not in the Th1-type lesions of SD, and IL-12p35 mRNA levels were downregulated in both. A supply of G-CSF prevents the onset of Th1-type TNBS colitis and does not deteriorate neutrophil-dominant chronic colitis in hosts showing higher expression of endogenous G-CSF.

Facilitation of survival in a rat fulminant hepatic failure model by combination therapy using recombinant G-CSF and tacrolimus.

The mortality rate of fulminant hepatic failure (FHF) is high because of retarded liver regeneration. Recombinant human granulocyte colony-stimulating factor (rHuG-CSF) and tacrolimus are known to be immunosuppressive and supportive to liver regeneration. We investigated the effects of their combination therapy in a rat FHF model with a 68% partial hepatectomy and 24% liver necrosis. All rats without drug pretreatment died within 55 h. The median time was prolonged from 37 to 52 h by rHuG-CSF (250 microg/kg/day s.c. on days -5 to 0) and to 46 h by tacrolimus (0.5 mg/kg/day i.m. on days -2 to 0). Notably, the combination therapy facilitated DNA biosynthesis and survival prolongation, with a median of 77 h. The interferon-gamma (IFN-gamma) protein levels and natural killer cell (NK) activity in the liver were low at 12 h, and no further inhibition was detected by any treatment. Tacrolimus significantly upregulated the mRNA levels of insulin receptors and transforming growth factor-alpha (TGF-alpha), whereas rHuG-CSF did not. Regarding tissue remodeling-related factors, rHuG-CSF upregulated mRNA levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase- 9 (MMP-9), whereas tacrolimus did not. The combination treatment upregulated protein levels of both insulin receptors and VEGF. These results suggest that tacrolimus improves the hepatocyte replication and rHuG-CSF contributes to tissue reconstitution, and this combination therapy directly facilitates liver regeneration in the FHF model.

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.

Inhibition of tumor growth in immunocompromised hosts by restoring type-2 immunity using infusion of G-CSF-treated allogeneic CD8+ leukocytes.

Since recombinant human granulocyte colony-stimulating factor (rhG-CSF)-treated donor leukocyte infusion (G-DLI) has been shown to downregulate type-1 immunity in a heart transplant model, we examined influences of G-DLI on tumor growth in immunosuppressed hosts. F344 rats were treated with tacrolimus (8 mg/kg i.m.) and syngeneic colon adenocarcinoma RCN-9 cells (3 x 10(6)) were inoculated subcutaneously. For G-DLI, allogeneic DA rats were pretreated with rhG-CSF (250 microg/kg, days -5 to 0) and isolated leukocytes or sorted CD8(+) cells were injected intravenously to the hosts on day 0. Tumors in tacrolimus-treated hosts continuously grew over 5 weeks. G-DLI of 100 x 10(6) leukocytes attenuated tumor growth rate while direct host pretreatment with rhG-CSF did not. Notably, G-DLI of 10 x 10(6) CD8(+) cells blocked tumor expansion after day 14. Tacrolimus-induced inhibition of lymphocyte infiltration into tumors was recovered by the G-DLIs. Flow cytometry showed no detectable donor-type T cells in the tumor and circulation. Quantification of intratumor transcription levels using reverse transcription-real-time polymerase chain reaction showed recovery from tacrolimus-induced downregulation of interleukin-4 but not interferon-gamma levels. In vivo rhG-CSF-treated CD8(+) allogeneic cells demonstrate potent anti-tumor effects by restoring type-2 immunity of immunosuppressed hosts.

Studies on post-transplant dyslipidemia in kidney transplant patients.

This study was performed to retrospectively compare changes in the levels of total cholesterol, non-HDL cholesterol, triglycerides, and immunosuppressive drugs, cyclosporine A and steroids in patients with living-relation renal transplants with those from non-heart-beating donors. We experienced 11 cases of kidney transplants from non-heart-beating donors during the period from April 1995 to May 2003. We evaluated 13 cases of kidney transplants from living-relation donors during the same period. The immunosuppressants used included mainly cyclosporine A as well as mycophenolate mofetil or azathioprine, steroid and ALG, or basiliximab. Over-night fasting lipids (total cholesterol, triglycerides and HDL cholesterol) were studied before renal transplantation and repeated after renal transplantation at 1, 3, 6 and 12 months. The levels of total cholesterol and triglycerides remained in the normal range before transplantation. However, the levels of total cholesterol increased siginificantly 1 and 3 months after transplantation from non-heart-beating donors and remained at higher levels up to 12 months after transplantation. A similar pattern in the levels of triglycerides was observed. The levels of HDL cholesterol remained unchanged and stayed in the normal range before and 1, 3, 6, and 12 months after transplantation from non-heart-beating donors. On the other hand, significant increases in non-HDL cholesterol were observed 3 and 6 months after transplantation from non-heart-beating donors. After transplantation from living-relation donors, levels of total cholesterol, triglycerides, and non-HDL cholesterol remained unchanged and remained in the normal range up to 12 months after transplantation. Although there were no significant differences in the total dosage of cyclosporine A between the patients with living-relation donors and those with non-heart-beating donors, a significant increase in the total dosage of methylprednisolone was observed in patients with non-heart-beating donors compared with those in the patients with living-relation donors. Renal function recovery in patients with living-relation donors was better than in those with non-heart-beating donors. These results may suggest that significant increases in total cholesterol, especially non-HDL cholesterol and triglycerides, were probably partly due to an increased use of immunosuppressants, steroids. It is necessary to aggressively control post-transplant hyperlipidemia and important to reduce or withdraw steroids in the selected, low-risk recipients as early as possible from the viewpoint of preventing post-transplant hyperlipidemia.

Studies on eleven kidney transplants from non-heart-beating donors.

This study was performed to analyze postoperative courses and complications, retrospectively, following transplants from non-heart-beating donors and to examine the correlation between early graft function and clinical parameters. We experienced 11 cases of kidney transplants from non-heart-beating donors during the period from April 1995 to May 2003. Warm ischemic time was less than 30 min in all cases, and total ischemic time ranged from 8.4 hours to 27.9 hours. Rejection reactions occurred in seven cases, two of which were vascular rejections. Infectious disease complications included CMV in two cases, interstitial pneumonia in one case and fungal infection in one case. One patient died from interstitial pneumonia, and three patients had to be restarted on dialysis due to loss of function of the grafted kidney. The remaining seven patients all made full recoveries. All of the 16 patients who underwent living related kidney transplantations during the same period made full recoveries. Both the donor's gender and the latest creatinine level of the donor influenced the posttransplant dialysis period. The posttransplant dialysis period significantly influenced the creatinine level one month after transplant. These results suggest that patients who undergo kidney transplants from non-heart-beating donors have higher rates of complications than patients who undergo living related kidney transplantation. It is important that, in cases where the donor's creatinine level is high, especially when the donor is male, the kidney is carefully retrieved and transported to the recipent hospital to shorten the ischemic period as much as possible.

Allochronic overlapping malignancies after renal transplantation in a patient with p53 gene mutation: report of a case.

We report a rare case of the development of various tumors over a 16-year period after renal transplantation. A 56-year-old woman underwent renal transplantation using a US kidney. Immunosuppressive treatment consisted of a triple regimen of methylprednisolone, azathioprine, and mizoribine. Left breast cancer was diagnosed 9 years after the renal transplantation, then colon cancers and meningeal epidermal meningioma were diagnosed, 10 years and 12 years post-transplant, respectively. During the investigations for the breast and colon cancers, a p53 gene mutation was detected. A deterioration of renal function was found 16 years after the transplant and graft biopsy confirmed chronic rejection. We suggest that the effects of the immunosuppressive drugs combined with the p53 gene abnormality accelerated tumor development in this patient.

Regulation of T helper type-1 immunity in hapten-induced colitis by host pretreatment with granulocyte colony-stimulating factor.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is an immunoregulatory drug whose effects include modulation of antigen-presentation. We investigated the potential ameliorative effect of pretreatment with rhG-CSF in a hapten-induced colitis animal model. Sprague-Dawley rats were given rhG-CSF (125 microg/kg subcutaneously twice a day for 5 days) before a colonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in 50% ethanol. Consequent colonic damage was evaluated pathologically, and cytokine mRNA expression levels in macroscopically inflamed sites were measured by real-time quantitative reverse transcription-polymerase chain reaction. Pretreatment with rhG-CSF remarkably attenuated both the loss of body weight and colonic wall thickening due to progressive transmural inflammation. In the control, treatment with TNBS led to a statistically significant (p < 0.05) upregulation of IFN-gamma mRNA expression in the inflammatory sites measured at post-treatment day 7. In the experimental group, pretreatment with rhG-CSF abrogated transcription of IFN-gamma (p < 0.05), but was not, however, associated with an upregulation of IL-4 or the regulatory cytokines TGF-beta and IL-10. Furthermore, transcription of IL-12p35 (a rate-limiting factor for the production of IL-12) was significantly (p < 0.05) downregulated by rhG-CSF at 24h post-TNBS instillation, whereas IL-12p40 was not affected. Pretreatment with rhG-CSF drastically attenuated the degree of TNBS-induced colitis through selective downregulation of Th1-associated cytokines.

Downregulation of interleukin-12p35 and upregulation of interleukin-12p40 mRNA expression in heart allografts by blood transfusion from granulocyte colony-stimulating factor-treated donors.

Pretransplant treatment of recipients with recombinant human granulocyte colony-stimulating factor (rhG-CSF, 250 microg/kg/day s.c. for 5 days) facilitates heart allograft acceptance in tacrolimus-treated rat recipients. We examined effectiveness of transfusion of in vivo rhG-CSF-treated blood since rhG-CSF induces immunoregulatory cells in human blood. DA heart grafts were transplanted into tacrolimus (2 mg/kg i.m. on day 0)-treated Lewis recipients. Although graft survival prolongation by blood transfusion on day 0 from rhG-CSF-treated syngeneic Lewis was comparable to that in directly rhG-CSF-pretreated recipients (p = 0.22), transfusion of rhG-CSF-treated allogeneic DA blood was much more effective (p = 0.0016). Intragraft cytokine mRNA levels were measured by reverse transcription and real-time polymerase chain reaction at 24 h after transplantation. IL-12p35 expression was downregulated by both treatments. Notably, IL-12p40 was upregulated by rhG-CSF-treated DA blood transfusion but downregulated by transfusion of rhG-CSF-treated isogeneic blood. Differential expression of IL-12 subunits was associated with facilitation of graft acceptance by rhG-CSF-treated donor blood transfusion.

Facilitation of tacrolimus-induced heart-allograft acceptability by pretransplant host treatment with granulocyte colony-stimulating factor: interleukin-12-restricted suppression of intragraft monokine mRNA expression.

BACKGROUND: Because recombinant human granulocyte colony-stimulating factor (rhG-CSF) is known to modulate function of antigen-presenting cells, we examined effects of pretransplant host treatment with rhG-CSF on allograft survival. METHODS: In DA-to-Lewis rat heart transplantation, hosts were given pretransplant injections of rhG-CSF (250 microg/kg/day subcutaneously from day -5-0) and/or posttransplant injections of tacrolimus (2 mg/kg/day intramuscularly from day 0-3). Cytokine mRNA levels in grafts were measured by real-time reverse-transcription polymerase chain reaction. RESULTS: rhG-CSF pretreatment was effective in prolonging allograft survival only in tacrolimus-treated hosts (P <0.001). Intragraft mRNA expression of interleukin (IL)-12 subunits (p35, p40) at 24 hours after transplantation was significantly (P <0.05) down-regulated by the addition of rhG-CSF and was associated with suppression of interferon-gamma levels on day 6, although other proinflammatory cytokines (tumor necrosis factor -alpha, IL-1beta, IL-6, IL-18) and anti-inflammatory cytokines (IL-10, transforming growth factor-beta) were not. CONCLUSIONS: rhG-CSF pretreatment down-regulates intragraft expression of the type-1 T-helper cell (Th1)-driving cytokine IL-12 and facilitates tacrolimus-induced graft acceptance.

Upregulation of intragraft interleukin-10 by infusion of granulocyte colony-stimulating factor-mobilized donor leukocytes.

We examined the effects of granulocyte colony-stimulating factor (G-CSF)-mobilized donor leukocyte infusion (G-DLI) on facilitation of allograft survival using heart transplantation from DA to Lewis rats that were transiently treated with tacrolimus (2 mg/kg i.m. on day 0). Other DA rats were given G-CSF (250 microg/kg/day s.c. from days -5 to 0), and isolated leukocytes were infused into Lewis recipients after surgery. Cytokine mRNA levels were quantified by reverse transcription and real-time polymerase chain reaction. After G-CSF treatment, leukocytes in circulation increased by 7.6 times and secreted in-vitro 6.0-times-higher levels of IL-10 after lipopolysaccharide stimulation than did untreated leukocytes. G-DLI facilitated graft survival dose-dependently. Significant IL-10 mRNA upregulation was detected in grafts 24 h after surgery but not in the recipient's heart, spleen, or liver. On day 6, IFN-gamma and IL-2 mRNA levels were approximately half those of the control levels. Allograft-restricted IL-10 upregulation followed by type-1 cytokine downregulation can be achieved by the use of G-DLI.

Augmentation of local antitumor immunity in liver by interleukin-2 gene transfer via portal vein.

Metastasis to the liver remains an important problem in the treatment of patients with gastrointestinal cancer. We examined the mechanism and effect on liver metastasis of in vivo interleukin-2 (IL-2) gene transfer to the liver. RCN-9 cells derived from F344 rat colon adenocarcinoma were injected into syngeneic rats via the ileocecal vein to induce liver tumors. A total of 2.5x10(9) pfu of adenovirus vector harboring the human IL-2 gene (AdCMVhIL-2), or 2.5x10(9) pfu of control vector encoding beta-galactosidase was administered before RCN-9 cell challenge. On day 14, mean tumor weight was 4.0+/-2.4 g in the control group, whereas IL-2-transduced livers had no tumors. Survival of AdCMVhIL-2-treated rats was significantly longer than that of control rats (P<.01). Flow cytometry demonstrated that the proportion of natural killer (NK) cells had increased among sinusoidal cells collected from IL-2-transduced livers. These cells were highly cytotoxic to RCN-9 cells in vitro in the presence of a physiological high concentration of recombinant IL-2. Preventative effects of IL-2 transduction of the liver against liver metastasis were lost after depletion of NK cells by treatment with anti-asialo GM1 antibodies. Our results indicate that IL-2 gene transfer to the liver prevents liver metastasis by continuously providing physiological high concentrations of IL-2 in the liver, thereby activating sinusoidal NK cells.

Downregulation of both interleukin-12 and interleukin-2 in heart allografts by pretransplant host treatment with granulocyte colony-stimulating factor and tacrolimus.

Since recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been reported to induce immune deviation, we examined the effects of pretransplant treatment of recipients with rhG-CSF on heart allograft survival. Before heterotopic heart transplantation from DA to Lewis rats, recipients were given rhG-CSF (125microg/kg s.c. twice a day from day -5 to 0) and/or tacrolimus (2mg/kg i.m. on day 0). Combined treatment with both rhG-CSF and tacrolimus prolonged graft survival significantly (P<0.05), while rhG-CSF or tacrolimus alone did not. At 24h after transplantation, cytokine mRNA levels in the grafts were measured by reverse transcription and real-time polymerase chain reaction. IL-12 p35 expression was highly inhibited by rhG-CSF treatment, but tacrolimus did not change the levels. Conversely, rhG-CSF treatment did not affect IL-2 levels, while tacrolimus completely blocked its expression. Combined pretreatment was effective for suppressing acute rejection reaction by downregulating these two key type-1 cytokines, IL-12 and IL-2, with unchanged levels of IL-10.