Cynthia A. Ellison

Effect of recombinant human keratinocyte growth factor (rHuKGF) on the immunopathogenesis of intestinal graft-vs.-host disease induced without a preconditioning regimen.

We studied the effect of rHuKGF on acute, lethal graft- vs.-host disease (GVHD) in the C57BL/6→(C57BL/6 X DBA/2)F1-hybrid model. rHuKGF-treated recipients did not develop intestinal GVHD despite elevated levels of intestinal NO and TNFα, did not develop endotoxemia, and did not die. LPS augmented serum TNFα release and intestinal NO production, but did not induce intestinal epithelial cell apoptosis, a phenomenon associated with acute GVHD. These data suggest that KGF prevents the development of acute lethal GVHD by protecting epithelial cell injury mediated by TNF-α, NO, and other potential cytotoxic factors. We noted a moderate reduction in intestinal KGFR mRNA expression in untreated GVH mice on day 8, when IFN-γ mRNA levels were highest. This reduction in KGFR mRNA levels was not seen in recipients of IFN-γ gene knockout grafts, suggesting that IFN-γ may be involved in reducing KGFR mRNA expression in the intestine. A similar reduction in intestinal KGFR mRNA expression was also seen in rHuKGF-treated recipients, suggesting that rHuKGF does not mediate its protective effect by maintaining KGFR at control levels. KGF-treatment also redirected the cytokine response in acute GVH mice from Th1 to a mixed pattern of both Th1 and Th2 cytokines. This was associated with histopathologic changes resembling chronic GVHD.

The role of interferon‐γ, nitric oxide and lipopolysaccharide in intestinal graft‐versus‐host disease developing in F1‐hybrid mice

(C57BL/6 × DBA/2)F1‐hybrid mice injected with lymphoid cells from wild‐type, C57BL/6 donors develop acute, lethal graft‐versus‐host disease (GVHD) in which the intestine is a major target. In its destructive phase intestinal GVHD is characterized by apoptosis of intestinal crypt epithelial cells and the development of endotoxaemia. Injection of as little as 10 μg endotoxin is lethal in mice with acute GVHD, and associated with the release of large amounts of tumour necrosis factor‐α (TNF‐α) into the serum. To explore the role of interferon‐γ (IFN‐γ) in the pathogenesis of intestinal GVHD we used IFN‐γ gene knockout (gko) mice as donors. Recipients of grafts from these donors did not develop intestinal GVHD and, unlike recipients of wild‐type grafts, did not die when injected with lipopolysaccharide (LPS). We also found that injection 10 μg LPS into recipients of wild‐type grafts induced apoptosis of intestinal epithelial crypt cells and was associated with a burst of nitric oxide production in the intestine. Administration of Nωnitro l‐arginine methyl ester blocked this response. In contrast, LPS did not induce either intestinal epithelial cell apoptosis or increased nitric oxide production in recipients of IFN‐γ gko grafts. These findings indicate that donor‐derived IFN‐γ is instrumental for the development of intestinal GVHD. In a previous study we showed that recipients of IFN‐γ gko grafts develop high levels of LPS‐induced TNF‐α release. When our current data are viewed in the context of this observation, they suggest that intestinal epithelial cell apoptosis in the parent→F1‐hybrid model of acute GVHD is mediated primarily by nitric oxide rather than TNF‐α, and that this depends on donor‐derived IFN‐γ.

Depletion of natural killer cells from the graft reduces interferon-gamma levels and lipopolysaccharide-induced tumor necrosis factor-alpha release in F1 hybrid mice with acute graft-versus-host disease.

BACKGROUND We wished to determine whether removal of NK1.1+ cells from the graft provides protection against acute graft-versus-host disease (GVHD) by obviating the Th1 immune response that underlies the development of this disease. METHODS Graft-versus-host (GVH) reactions were induced in two groups of (C57BL/6 x DBA/2)F1 hybrid mice. The first received grafts harvested from polyinosinic:polycytidylic acid-stimulated, C57BL/6 donors and depleted in vitro of NK1.1+ cells. This treatment provides protection against GVHD-associated mortality and cachexia. The second received unmodified grafts. We compared interferon-gamma and interleukin-10 production as well as the levels of engraftment in these two groups. Lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-alpha) release was also compared since TNF-alpha levels in GVH mice following injection of a sublethal dose of endotoxin provide an index of macrophage priming by Th1 cytokines. RESULTS Interferon-gamma production was absent in recipients of NK1.1-depleted grafts at the time when high levels were seen in recipients of unmodified grafts. Following lipopolysaccharide injection, high levels of TNF-alpha were observed in recipients of unmodified grafts, whereas negligible amounts were present in recipients of NK1.1-depleted grafts. The use of NK1.1-depleted grafts did not result in a reduced level of engraftment of CD4+ or CD8+ cells. CONCLUSIONS These results suggest that NK1.1 depletion of the graft confers protection against mortality by interfering with an immunoregulatory mechanism that results in the development of a Th1 response in GVH mice, and does not result in abortion of the graft. Because macrophage priming is prevented, recipients are also protected from the exaggerated sensitivity to endotoxin seen in mice with acute GVHD.

Murine graft-versus-host disease induced using interferon-γ-deficient grafts features antibodies to double-stranded DNA, T helper 2-type cytokines and hypereosinophilia

Acute, lethal graft‐versus‐host disease (GvHD) develops in B6D2F1 hybrid recipients of wild‐type, C57BL/6, parental strain grafts; however, when interferon‐γ (IFN‐γ) gene knockout (gko) donors are used, the disease is prolonged and associated with a higher level of engraftment, particularly of T cells. Lesions containing large, mixed cellular infiltrates develop in the skin, liver, pancreas, salivary gland, lung and kidney. In our current study, we wished to determine whether GvHD features a preponderance of T helper 2 (Th2) cytokines in the absence of donor‐derived IFN‐γ, and whether autoantibody production, commonly associated with chronic GvHD, also occurs. Because mitogen responsiveness is consistently suppressed in mice with acute GvHD, we wished to measure this response in recipients of IFN‐γ gko grafts. Our findings indicate that spleen cells from the latter produce interleukin (IL)‐4, IL‐5 and IL‐13 in culture, but respond poorly to concanavalin A (Con A) and lipopolysaccharide (LPS). Their sera contain anti‐nuclear antibodies (ANA), some of which are specific for double‐stranded (ds)DNA and are predominantly immunoglobulin (Ig)M and IgG1. We also noted the presence of numerous eosinophils in the infiltrates developing within the target organs. In some respects, this syndrome bears resemblance to both systemic lupus erythematosus (SLE) and chronic GvHD. However, histological evidence of glomerulonephritis is lacking and proteinuria fails to develop in recipients of IFN‐γ gko grafts, suggesting that IFN‐γ may be necessary for the development of lupus nephritis. On a broader scope, our findings underscore the importance of IFN‐γ in the pathogenetic mechanism of GvHD, and demonstrate that the absence of this cytokine promotes the development of chronic GvHD and autoimmunity.

Palifermin Mediates Immunoregulatory Effects in Addition to its Cytoprotective Effects in Mice with Acute Graft-Versus-Host Disease

IntroductionTreating recipient mice with palifermin (recombinant human keratinocyte growth factor) prevents the development of acute, lethal, graft-versus-host disease (GVHD). This is due, at least in part, to the ability of palifermin to protect epithelial cells from injury. Using the C57BL/6→(C57BL/6 × DBA/2)F1-hybrid model, we previously showed that the protective effect of palifermin was also associated with redirection of the cytokine profile from Th1 to Th2.DiscussionTo study this immunoregulatory effect more directly, we induced acute GVH reactions in which we treated the donors rather than the recipients with palifermin. The recipient mice were protected from GVHD-associated morbidity, and their cytokine profile was predominantly Th2. The palifermin-treated donor mice alone showed a similar Th2 cytokine profile, and we observed elevated levels of thymic stromal lymphopoietin mRNA in the thymus. We further demonstrated that treating the donor mice with palifermin protects against GVHD-associated morbidity, even if the donors are deficient in Vα14i natural killer T cells. Our findings clearly show that palifermin mediates immunoregulatory effects in addition to its cytoprotective effects and that both are likely to be involved in the mechanism through which palifermin provides protection from acute murine GVHD.

In vivo Direction of CD4 T Cells to TH1 and TH2-Like Patterns of Cytokine Synthesis

Factors that influence the initial development, and continued maintenance, of Th1 or Th2-like responses in vivo play a pivotal role in determining immune effector mechanisms and clinical outcome. Here, we review recent developments in this area with particular emphasis on (i) the ability of chemically modified exogenous antigens to preferentially activate Th1-dominated responses in vivo and (ii) the role played by NK cells in initial commitment of naive exogenous antigen-specific T cells to Th1 or Th2-like cytokine synthesis. We find that NK cell depletion of naive mice prior to immunization with OVA (which induces balanced Th0 like responses), or a high Mr polymer (that preferentially elicits OVA-specific Th1-dominated responses), fails to influence the development of cytokine or specific antibody responses. The results argue that NK cells do not play an essential role in shaping induction of immune responses to exogenous antigens, the most common class of inhalant allergen.

Lack of functional TSLP receptors mitigates Th2 polarization and the establishment and growth of 4T1 primary breast tumours but has different effects on tumour quantities in the lung and brain.

The 4T1 mammary carcinoma cell line produces TSLP. We had hypothesized that TSLP promotes the development of a permissive environment for the growth and metastasis of primary tumour and that this is associated with a Th2‐polarized antitumour immune response. We found that, in Tslpr−/− mice, the mean tumour diameters were smaller from days 27 to 40, and relatively fewer tumour cells were present in the lung, compared with wild‐type mice. Polarization of the Th2 cytokine profile was also diminished in Tslpr−/− mice. These findings confirmed those reported previously by others. Here, we further show that primary tumours are established less often in Tslpr−/− mice and that, unexpectedly, the relative number of tumour cells in the brain is greater in Tslpr−/− mice compared with wild‐type mice. Findings from our cytotoxicity assays show that 4T1‐directed lysis is undetectable in both WT and Tslpr−/− mice, ruling out the possibility that altered cytotoxic responses in Tslpr−/− mice are responsible for the differences we observed. In a human tissue microarray, positive staining for TSLP was seen in tumour cells from breast cancer tissue, but it was also seen in normal glandular epithelial cells from normal breast tissue, which has not been shown before. Thus, our findings provide new insight into the effects of TSLP in metastatic breast cancer.

Positive selection of NK1.1+ cells on a magnetic cell separator (MACS)

Natural killer (NK) cells are involved not only in resistance to tumors and infection, but also in some transplantation reactions. A specific, inexpensive method for purifying large numbers of NK cells is often required. All NK cells in H-2b mice express the surface marker NK1.1. We report a method for positively selecting NK1.1+ spleen cells from normal and Poly I:C-stimulated C56Bl/6 mice using a magnetic cell separation technique known as MACS. Our results show that cytotoxic activity directed at YAC-1 target cells by normal and Poly I:C-stimulated spleen cells could be increased five-fold using this method. We also found that spleen cells from mice given Poly I:C could lyse NK-resistant, BW1100 target cells, and that this activity could be increased two-fold. Flow cytometry analysis of Poly I:C-stimulated, MACS enriched, NK1.1+ spleen cells revealed the presence of two subpopulations: one consisting of LGL and the other consisting of smaller, agranular lymphocytes (SAL). After enrichment, the percentage of NK1.1+ spleen cells increased from 69% to 91% in the LGL subpopulation and from 33% to 73% in the SAL subpopulation. These results clearly demonstrate the effectiveness of the MACS technique for purifying large numbers of NK1.1+ cells for both flow cytometric and functional analyses.

Graft-versus-host disease in recipients of grafts from natural killer T cell-deficient (Jα281–/–) donors

Vα14i natural killer T cells (NKT cells) are a CD1‐restricted subset of NKT cells that express an invariable Vα14+ Jα281+αβ T‐cell receptor. To determine whether the absence of Vα14i NKT cells from the graft affects the development of acute GVHD, we induced GVH reactions using Jα281–/– mice as donors in the C57BL/6→(C57BL/6 × DBA/2)F1‐hybrid strain combination. Recipients of grafts from Jα281–/– donors were not protected from either the morbidity or the severe wasting syndrome associated with the development of acute GVHD, but the concentrations of some T helper 1 (Th1) and Th2 cytokines were different from those seen in recipients of grafts from wild‐type donors. Interferon‐γ was seen earlier (day 4) in recipients of grafts from Jα281–/– donors but did not reach the concentrations seen in recipients of grafts from wild‐type donors on day 8 (P < 0·02). On day 8, the amount of tumour necrosis factor‐α released into the serum following the injection of a small amount of lipopolysaccharide was lower in recipients of grafts from Jα281–/– donors (P < 0·02). The amount of interleukin (IL)‐5 was also lower in recipients of grafts from Jα281–/– donors, when compared to the concentration seen in recipients of grafts from wild‐type donors (P < 0·002). IL‐13 was seen in recipients of grafts from Jα281–/– donors but not in recipients of grafts from wild‐type donors. Our findings show that the absence of donor Vα14i NKT cells is associated with lower concentrations of some Th1 cytokines. We also observed higher IL‐13 concentrations and lower IL‐5 concentrations in recipients of grafts from Jα281–/– donors indicating a variable effect on Th2 cytokine production.

Immunomodulatory Effects of Palifermin (Recombinant Human Keratinocyte Growth Factor) in an SLE-Like Model of Chronic Graft-Versus-Host Disease

Keratinocyte growth factor (KGF) promotes epithelial cell proliferation and survival. Recombinant human KGF, also known as palifermin, protects epithelial cells from injury induced by chemicals, irradiation and acute murine graft‐versus‐host disease (GVHD). Findings from our studies and others have shown that palifermin also has immunomodulatory properties. In a model of acute GVHD, we showed that it shifts the immune response from one in which Th1 cytokines dominate to mixed Th1 and Th2 cytokine profile. Using the DBA/2→(C57BL/6 × DBA/2)F1‐hybrid model of chronic, systemic lupus erythematosus‐like GVHD, we showed that palifermin treatment is associated with higher levels of Th2 cytokines, the production of anti‐nuclear antibodies, cryoglobulinemia and the development of more severe pathological changes in the kidney. The aim of our current study was to gain a better understanding of the immunobiology of KGF by further characterizing the palifermin‐mediated effects in this model of chronic GVHD. Because the pathological changes we observed resemble those seen in thymic stromal lymphopoietin (TSLP) transgenic mice, we had originally hypothesized that palifermin might augment TSLP levels. Surprisingly, we did not observe an increase in thymic TSLP mRNA expression in palifermin‐treated recipients. We did, however, observe some differences in the percentages of CD4+CD25+Foxp3+ regulatory T cells in the spleen at some time points in palifermin‐treated recipients. Most importantly, we found that TGFβ levels were higher in palifermin‐treated recipients early in the GVH reaction, raising the possibility that KGF might indirectly induce the development of fibrosis and glomerulonephritis through a pathway involving TGFβ.