Activation of macrophage cytostatic effector mechanisms during acute graft-versus-host disease: release of intracellular iron and nitric oxide-mediated cytostasis.

During acute graft-versus-host disease (GVHD) the activation of macrophages (Mphi) is mediated by 2 signals, interferon (IFN)-gamma and bacteria-derived lipopolysaccharide (LPS). A cascade of inflammatory responses that includes the release of mediators of tissue injury follows Mphi activation. Among the tissues characteristically targeted during acute GVHD are epithelial tissues of the skin and gastrointestinal tract that normally undergo continuous proliferation and are therefore sensitive to cytostatic processes. We have investigated whether Mphi can mediate cytostatic mechanisms capable of interrupting cell proliferation during acute GVHD. GVHD was induced in nonirradiated C57BL/6XAF(1) (B6AF(1)) mice by the injection of 60 x 10(6) (acute GVHD) or 30 x 10(6) (nonlethal GVHD) C57BL/6 (B6) lymphoid cells. Mphi from animals undergoing acute GVHD could be triggered by normally insignificant quantities of LPS to mediate a cytostatic effect on target cells, resulting in the complete shutdown of cellular proliferation. The same amounts of LPS had no effect on Mphi from normal or syngeneically transplanted animals. Mphi mediated the release of significant quantities of intracellular iron from target cells undergoing cytostasis. Reversal of cytostasis occurred following inhibition of nitric oxide (NO) production by N(G)-monomethyl-L-arginine (NMMA). Production of NO by LPS-triggered Mphi reflected the severity of GVHD. NO release increased significantly during acute GVHD but was only transiently increased during nonlethal GVHD. The results provide evidence that, as a result of activation during acute GVHD, Mphi produce NO and induce the release of iron from target cells, resulting in a potent cytostatic effect that inhibits cellular proliferation. (Blood. 2000;96:1836-1843)

Progressive accumulation of bacterial lipopolysaccharide in vivo during murine acute graft-versus-host disease.

In a previous report the authors demonstrated that acute graft-versus-host disease (GVHD) was associated with pathologic amounts of tumour necrosis factor alpha (TNF-alpha) and the appearance of lipopolysaccharide (LPS) in the blood of GVH reactive mice just prior to death. In this study the authors have investigated the kinetics of LPS accumulation in different organs during the course of acute GVHD using a murine model. Unirradiated C57BL/6 x AF1 (B6AF(1)) mice were transplanted with C57BL/6 (B6) lymphoid cells and killed at predetermined times after transplantation for LPS analysis. Control animals were injected with either 60 x 10(6) B6AF1 lymphoid cells (syngeneic) or 60 x 10(6) irradiated (2000 rad) CBA lymphoid cells (allogeneic). Lipopolysaccharide began to appear in the liver and the spleen of GVH reactive mice from day 2 post-transplant and by day 10 all GVH reactive mice tested positive for hepatic and splenic LPS. Low levels of LPS were detected in some control mice from days 2 to 10 post-transplant but LPS was never detected after day 10 in control groups. Total hepatic and splenic LPS in acute GVH reactive mice peaked at a time coincident with the appearance of LPS in the serum and with the onset of mortality. These results demonstrate that tissue levels of LPS increase throughout the course of acute GVHD and are sufficient to trigger the release of pathologic amounts of TNF-alpha from primed macrophages resulting in the cachexia and mortality associated with acute GVHD in this model.

RS cyclophilins: identification of an NK-TR1-related cyclophilin.

We report the isolation of a large cyclophilin protein containing RS (arginine-serine) repeats from a yeast two-hybrid screen using ClK (CDC28/cdc2-like kinase) as a probe. This Clk associating RS-cyclophilin (CARS-Cyp) possesses 39% homology to the NK-TR1 (natural killer tumor recognition protein-1) we have previously characterized (Anderson et al. (1993) Proc. Natl. Acad. Sci. USA 90 (1993) 542-546). CARS-Cyp is expressed in a variety of tissues and cell types, and codes for a protein with a predicted mass of 89 kDa containing a cyclophilin-related domain, two Nopp140 (nucleolar phosphoprotein of 140 kDa)-related domains, and a large RS domain. The RS-cyclophilins, a novel class of proteins, may play an important role in the regulation of pre-mRNA splicing.

IL-12 p40 messenger RNA expression in target organs during acute graft-versus-host disease. Possible involvement of IFN-gamma.

The onset of acute graft-vs-host disease (aGVHD) is accompanied by macrophage (M phi) priming and the presence of bacteria-derived LPS in the sera of transplanted animals. Priming of M phi occurs during aGVHD despite the suppression of T cell function. We have investigated whether IL-12 mediates the continued production of IFN-gamma during the state of T cell immunosuppression that accompanies aGVHD. Acute GVHD was induced in nonirradiated AxC57BL/6F1 mice by the injection of C57BL/6 lymphoid cells. Despite T cell immunosuppression, M phi became primed, as shown by their expression of inducible nitric oxide synthase mRNA and their production of nitric oxide in response to LPS. Continual exposure to IFN-gamma was required to maintain a primed state in M phi during aGVHD. IL-12 p40 peptide mRNA was increased in M phi purified from animals undergoing aGVHD 14 days after transplantation. Target organs of aGVHD, including thymus, salivary gland, and lung, showed increased IFN-gamma mRNA between days 7 and 14 after transplantation. The increase was accompanied by an induction of mRNA for the p40 peptide of IL-12 and inducible nitric oxide synthase within the target organs. These results provide evidence for localized production of IFN-gamma within aGVHD target organs and suggest that it is mediated by LPS-induced production of IL-12 by M phi. Our data elucidate the mechanism of activation of M phi during aGVHD that results in TNF-alpha and nitric oxide production and delineates the effector role of M phi in the pathology of aGVHD.

Macrophage priming and lipopolysaccharide-triggered release of tumor necrosis factor alpha during graft-versus-host disease.

In this report we have investigated macrophage (M phi) activity and tumor necrosis factor alpha (TNF-alpha) production during graft-vs.-host disease (GVHD). TNF-alpha production by M phi requires two signals: priming of M phi by interferon followed by triggering of TNF-alpha production and release by lipopolysaccharide (LPS). The state of M phi activation was examined in nonirradiated B6AF1 recipient mice injected with either 60 x 10(6) (acute GVHD) or 30 x 10(6) (nonlethal GVHD) parental B6 lymphoid cells. During the early phase of acute GVHD, administration of normally sublethal amounts of LPS-triggered release of significant amounts of TNF-alpha into the serum resulting in death of the animals within 36 h. Normal animals treated with the same dose of LPS neither died nor produced detectable amounts of serum TNF-alpha. In vitro studies demonstrated that M phi were primed during GVHD. The level of M phi priming was greater during acute GVHD than nonlethal GVHD since 100-fold less LPS was required to trigger killing of a TNF-alpha-sensitive cell line by M phi from acute GVHD animals. The amount of TNF-alpha released into the serum after LPS injection increased during the course of the GVHD and was significantly greater in acute GVH-reactive mice. Endogenous LPS was detected in the serum of acute GVH-reactive animals coincident with the onset of mortality. The data provide evidence that during GVHD M phi are primed as a result of the allogeneic reaction and that endogenous LPS therefore triggers M phi production of TNF-alpha resulting in the symptoms characteristic of acute GVHD.

Expression of tumor necrosis factor alpha in the developing nervous system.

We present evidence that tumor necrosis factor alpha (TNF-alpha) is transiently expressed at specific times during embryogenesis in precisely defined areas of the nervous system in two different classes of vertebrates. In murine embryos, TNF-alpha was detected in the brain, neural tube and peripheral mixed spinal nerves. In the chick embryo, TNF-alpha was observed in the brain neuroepithelium and in the developing Purkinje neurons of the cerebellum. Western immunoblot analysis revealed that brain tissue from both mouse and chick embryos contained a 50 kDa protein showing immunoreactivity with anti-TNF-alpha antibody. These results suggest that TNF-alpha participates in the normal development of the vertebrate brain and spinal cord.

Lipopolysaccharide-induced fetal resorption in mice is associated with the intrauterine production of tumour necrosis factor-alpha.

Certain strains of mice display an increased frequency of fetal resorption, but little is known about the effector mechanisms involved. We have examined the events associated with lipopolysaccharide (LPS)-induced fetal resorption in mice. Administration of 25 micrograms LPS on Day 12 of gestation resulted in the appearance of tumour necrosis factor-alpha (TNF-alpha) in the amniotic fluid and fetal resorption. Levels of LPS-induced TNF-alpha were reduced by 90% after pretreatment with the TNF-alpha-suppressing drug pentoxifylline (PXF). Treatment of pregnant mice during early gestation with 0.1 micrograms LPS resulted in fetoplacental resorption which was maximal when the LPS was given on Day 8. Resorption induced by 0.1 micrograms LPS on Day 8 of gestation was significantly reduced by pretreatment with PXF. Infiltration of asialo-GM1-positive cells was observed in the decidual-ectoplacental cone area of embryonic units from LPS-treated mice. In addition, treatment with anti-AGM1 antiserum prevented the LPS-induced resorption. Our results suggest that TNF-alpha and asialo-GM1-positive cells are involved in LPS-induced fetal resorption.

The requirement for gamma interferon in resistance of mice to experimental tularemia.

The role of gamma interferon (IFN-gamma) in the host response to experimental tularemia was evaluated in a murine model. C57BL/6 strain mice were given a series of daily intravenous injections of 10(6) units (U) recombinant murine IFN-gamma prior to infection with Francisella tularensis LVS. Three days later, the number of bacteria in the tissues of IFN-gamma-treated mice was found to be less than that in control mice by a factor of 10-20. The effect of IFN-gamma on anti-tularemic resistance was dependent upon the administered dose, with as little as 10(4) U/mouse/day inducing a significant level of enhanced resistance. IFN-gamma was also effective in enhancing resistance to tularemia in the A/J mouse strain which, in comparison with the C57BL/6 strain, is more susceptible to infection. When C57BL/6 mice were treated with a monoclonal antibody directed against murine IFN-gamma, the number of Francisella recovered from their tissues 6 days following infection was increased by as much as 15 times, in comparison with control mice. The results of these experiments clearly indicate that the resolution of experimental murine tularemia is dependent, at least in part, on the participation of IFN-gamma.

The effects of polyinosinic:polycytidylic acid (pI:C) on the graft-vs-host (GVH) reaction. II. Increased NK-mediated rejection on C57BL/6 lymphocytes by (C57BL/6 X A)F1 mice.

We previously demonstrated that treatment of (C57BL/6 X A)F1 (F1) recipient mice with polyinosinic:polycytidylic acid (pI:C) before injection with 30 X 10(6) C57BL/6 (B6) lymphocytes prevents both the immunosuppression and pathologic lesions typical of graft-vs-host (GVH) reactions. We now report the further characterization of this phenomenon. Donor spleen and lymph node cells were labeled with fluorescein in vitro and injected into pI:C-treated or untreated mice. Two days later, recipient splenocytes were analyzed for the presence of fluorescein-labeled donor cells by flow microfluorometry. Treatment of F1 mice with pI:C resulted in a sharp reduction in the recovery of labeled B6 but not A strain parental cells. Treatment with pI:C had no effect when syngeneic recipients were used, or when F1 cells were injected into A, B6, or F1 recipients. These results suggest that pI:C treatment induces rejection of B6 but not A or F1 lymphocytes by F1 hybrid mice at least as early as 2 days after donor cell transfer. As F1 cells are not rejected by either parent, rejection does not seem to be directed against classical alloantigens. These observations are compatible with the previously described model of hybrid resistance (HR) against bone marrow grafts. The rapidity of rejection strongly suggested that natural cytotoxic mechanisms were involved, thus, natural killer (NK) cell and macrophage (M phi) cytotoxic activities were tested throughout the time when the parental cell graft was being rejected. Over this period, pI:C treatment increased cytotoxic activity against the NK-sensitive target cell line YAC-1 but had no effect on spontaneous M phi tumoricidal activity against the L5178Y and MDAY-D2 cell lines. The results suggest that NK cells, but not M phi, may be involved in the elimination of B6 parental cells by the pI:C-treated F1 mice. NK cells have been demonstrated to be radioresistant; thus, as a test of our hypothesis, we examined the effects of irradiation on the capacity of pI:C treated F1 mice to reject B6 lymphocytes. The results show that this capacity was not blocked by 750 cGy, a dose of radiation that abrogates most T and B cell functions. Furthermore, rejection of parental cells could be prevented by treatment of recipient F1 mice with antibodies to asialo GM1, a treatment that suppresses NK activity. These data demonstrate that pI:C-mediated protection from GVH-induced changes is due to increased rejection of grafted B6 parental cells by F1 NK cells, a phenomenon very similar, if not identical, to HR to bone marrow grafts.

Alterations in sensitivity to nonspecific cell-mediated lysis associated with tumor progression: characterization of activated macrophage- and natural killer cell-resistant tumor variants.

Certain "membrane-mutant," lectin-resistant (Lecr) variants derived from the highly metastatic and poorly immunogenic DBA/2 mouse tumor MDAY-D2 previously were found to differ substantially in their ability to grow and to metastasize. In the present study, the parental MDAY-D2 tumor and several wheat germ agglutinin-resistant (WGAr) variants were examined for alterations in sensitivity to activated macrophage (M phi)- and natural killer cell (NK)-mediated lysis. The results indicated that selection in WGA after mutagenic treatment of a metastatic parental tumor cell line (MDAY-D2), which was M phi-sensitive (M phi S) and NK-resistant (NKR), can result in the isolation of a significantly M phi-resistant (M phi R) and NK-sensitive (NKS) tumor variant, MDW4. The in vivo hybridization of the M phi R, NKS, Lecr MDW4 variant with a normal host-derived cell within a primary subcutaneous tumor, previously demonstrated to result in the progressive and selective outgrowth and metastasis of hybrid products, was found to be associated directly with reversion to the M phi S, NKR phenotype of the metastatic parental MDAY-D2 cell line. DMA/2 mice given iv injections of 10(5) M phi R, NKS cells (MDW4 or MDW4-110c1, a cloned line isolated from a subcutaneous primary tumor of an MDW4-injected animal) survived for a significantly prolonged period as compared to animals given injections of either the parental tumor or M phi S, NKR hybrid products isolated from a MDW4 subcutaneous primary tumor (MDW4-110c2) or visceral metastases (MDW4-24a, MDW4-24b, and MDW4-24c). The results clearly indicate an inverse relationship among the tumor variants in their ability to be lysed by either M phi or NK and suggest a central role for NK rather than M phi surveillance in this tumor system.