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Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation in vivo by reducing lipid peroxides to the respective alcohols thereby stabilizing oxidation products of unsaturated fatty acids. During reticulocyte maturation, lipid peroxidation mediated by 15-lipoxygenase in humans and rabbits and by 12/15-lipoxygenase (ALOX15) in mice was considered the initiating event for the elimination of mitochondria but is now known to occur through mitophagy. Yet, genetic ablation of the Alox15 gene in mice failed to provide evidence for this hypothesis. We designed a different genetic approach to tackle this open conundrum. Since either other lipoxygenases or non-enzymatic autooxidative mechanisms may compensate for the loss of Alox15, we asked whether ablation of Gpx4 in the hematopoietic system would result in the perturbation of reticulocyte maturation. Quantitative assessment of erythropoiesis indices in the blood, bone marrow (BM) and spleen of chimeric mice with Gpx4 ablated in hematopoietic cells revealed anemia with an increase in the fraction of erythroid precursor cells and reticulocytes. Additional dietary vitamin E depletion strongly aggravated the anemic phenotype. Despite strong extramedullary erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. Gpx4-deficiency in hematopoietic cells led to systemic hepatic iron overload and simultaneous severe iron demand in the erythroid system. Despite extremely high erythropoietin and erythroferrone levels in the plasma, hepcidin expression remained unchanged. Conclusively, perturbed reticulocyte maturation in response to Gpx4 loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation., Haematologica, 105 (4), ISSN:0390-6078, ISSN:1592-8721

Maintaining cellular redox balance is vital for cell survival and tissue homoeostasis because imbalanced production of reactive oxygen species (ROS) may lead to oxidative stress and cell death. The antioxidant enzyme glutathione peroxidase 4 (Gpx4) is a key regulator of oxidative stress-induced cell death. We show that mice with deletion of Gpx4 in hematopoietic cells develop anemia and that Gpx4 is essential for preventing receptor-interacting protein 3 (RIP3)-dependent necroptosis in erythroid precursor cells. Absence of Gpx4 leads to functional inactivation of caspase 8 by glutathionylation, resulting in necroptosis, which occurs independently of tumor necrosis factor α activation. Although genetic ablation of Rip3 normalizes reticulocyte maturation and prevents anemia, ROS accumulation and lipid peroxidation in Gpx4-deficient cells remain high. Our results demonstrate that ROS and lipid hydroperoxides function as not-yet-recognized unconventional upstream signaling activators of RIP3-dependent necroptosis.

Signaling through tumor necrosis factor receptor 1 (TNFR1) controls bacterial infections and the induction of inflammatory Th1 cell–mediated autoimmune diseases. By dissecting Th1 cell–mediated delayed-type hypersensitivity responses (DTHRs) into single steps, we localized a central defect to the missing TNFR1 expression by endothelial cells (ECs). Adoptive transfer and mast cell knockin experiments into KitW/KitW-v, TNF−/−, and TNFR1−/− mice showed that the signaling defect exclusively affects mast cell–EC interactions but not T cells or antigen-presenting cells. As a consequence, TNFR1−/− mice had strongly reduced mRNA and protein expression of P-selectin, E-selectin, ICAM-1, and VCAM-1 during DTHR elicitation. In consequence, intravital fluorescence microscopy revealed up to 80% reduction of leukocyte rolling and firm adhesion in TNFR1−/− mice. As substitution of TNF−/− mice with TNF-producing mast cells fully restored DTHR in these mice, signaling of mast cell-derived TNF through TNFR1-expressing ECs is essential for the recruitment of leukocytes into sites of inflammation.

In this article, we review and analyze different modes of exposure to ultrafine particles in order to assess particle-induced inflammatory responses and the underlying mechanisms in vitro and in vivo. Based on results from monocytic cells cultured under submerged conditions, we discuss (1) the impact of particle properties such as surface area and oxidative potential on lipid metabolism as a highly sensitive regulatory pathway and (2) the interference of diesel exhaust particles with toll-like receptor-mediated inflammatory responses. Furthermore, new developments of air-liquid interface exposure used as an alternative approach to simulate cell particle interactions are presented. In addition to the in vitro approaches, animal exposure studies are described that apply selected mouse models to elucidate potential allergic and inflammatory pulmonary responses and mast-cell-related mechanisms after particle exposure. Long-term inhalation of ultrafine particles might lead to irreversible changes in lung structure and function. Clinical studies addressing the characteristics of inflammatory airway cells are a promising approach to understand underlying pathophysiological mechanisms in chronic obstructive pulmonary disease. Finally, a potential outcome of human particle exposure is chronic cough in children. Here, discrimination between asthmatic and nonasthmatic cough by means of immunological parameters appears to be an important step toward improving diagnosis and therapy.

Objective Induction of arthritis with autoantibodies against glucose-6-phosphate isomerase (GPI) is entirely independent of T cells and B cells but is strictly dependent on the presence of mast cells. Here, we used this disease model to analyze whether exclusive intraarticular mast cell reconstitution is sufficient for disease induction and whether targeted mast cell silencing can prevent neoangiogenesis and joint destruction, 2 hallmarks of rheumatoid arthritis. Methods Ankle swelling and clinical index scores were determined after injection of either K/BxN mouse–derived serum or control serum in wild-type Kit+/Kit+ mice, congenic mast cell–deficient KitW/KitW-v mice, or mast cell–deficient KitW/KitW-v mice reconstituted with mast cells, either by intraperitoneal or selective intraarticular injection. Angiogenesis was quantified in vivo by measuring activated αvβ3 integrin using 18F–galacto-RGD and positron emission tomography. In addition, staining of joint tissue with hematoxylin and eosin, Giemsa, β3, and α-actin was performed. The effect of mast cell stabilization by treatment with cromolyn or salbutamol was investigated in C57BL/6 or BALB/c mice. Results Comparing wild-type mice, mast cell–deficient KitW/KitW-v mice, and mast cell–reconstituted KitW/KitW-v mice, we first showed that intraarticular and intraperitoneal mast cell engraftment fully restores susceptibility to antibody-induced arthritis, angiogenesis, and αvβ3 integrin activation. Importantly, selective mast cell silencing with either salbutamol or cromolyn prevented αvβ3 integrin activation, angiogenesis, and joint destruction. Conclusion Mast cell engraftment fully restores susceptibility to αvβ3 integrin activation, angiogenesis, and joint destruction in GPI antibody–induced arthritis. Importantly, selective mast cell stabilization prevents αvβ3 integrin activation, angiogenesis, and joint destruction.

Over a decade ago endothelin-1 (ET-1) was described as belonging to the assortment of mast cell-derived and mast cell-bound cytokines. Mast cell subtype- and environment-dependent, ET-1 acts as a potent non-immunological mast-cell activator, which leads to degranulation and mediator release. The biological significance of this phenomenon remained obscure; however, a recent report sheds new light on this delicate mast cell-ET-1 connection. The authors of this report use mast cell-deficient mice to provide convincing evidence that mast cells counteract the toxicity induced by high concentrations of ET-1, and thereby promote homeostasis.

Streptolysin O (SLO), a major virulence factor of pyogenic streptococci, binds to cholesterol in the membranes of eukaryotic cells and oligomerizes to form large transmembrane pores. While high toxin doses are rapidly cytocidal, low doses are tolerated because a limited number of lesions can be resealed. Here, we report that at sublethal doses, SLO activates primary murine bone marrow-derived mast cells to degranulate and to rapidly induce or enhance the production of several cytokine mRNAs, including tumor necrosis factor alpha (TNF-α). Mast cell-derived TNF-α plays an important protective role in murine models of acute inflammation, and the production of this cytokine was analyzed in more detail. Release of biologically active TNF-α peaked ∼4 h after stimulation with SLO. Production of TNF-α was blunted upon depletion of protein kinase C by pretreatment of the cells with phorbol-12 myristate-13 acetate. Transient permeabilization of mast cells with SLO also led to the activation of the stress-activated protein kinases p38 mitogen-activated protein (MAP) kinase and c-jun N-terminal kinase (JNK), and inhibition of p38 MAP kinase markedly reduced production of TNF-α. In contrast, secretion of preformed granule constituents triggered by membrane permeabilization was not dependent on p38 MAP kinase or on protein kinase C. Thus, transcriptional activation of mast cells following transient permeabilization might contribute to host defense against infections via the beneficial effects of TNF-α. However, hyperstimulation of mast cells might also lead to overproduction of TNF-α, which would then promote the development of toxic streptococcal syndromes.

Polymorphonuclear leukocytes (PMNs) characterize the pathology of T cell-mediated autoimmune diseases and delayed-type hypersensitivity reactions (DTHRs) in the skin, joints, and gut, but are absent in T cell-mediated autoimmune diseases of the brain or pancreas. All of these reactions are mediated by interferon gamma-producing type 1 T cells and produce a similar pattern of cytokines. Thus, the cells and mediators responsible for the PMN recruitment into skin, joints, or gut during DTHRs remain unknown. Analyzing hapten-induced DTHRs of the skin, we found that mast cells determine the T cell-dependent PMN recruitment through two mediators, tumor necrosis factor (TNF) and the CXC chemokine macrophage inflammatory protein 2 (MIP-2), the functional analogue of human interleukin 8. Extractable MIP-2 protein was abundant during DTHRs in and around mast cells of wild-type (WT) mice but absent in mast cell-deficient WBB6F(1)-Kit(W)/Kit(W-)(v) (Kit(W)/Kit(W)(-v)) mice. T cell-dependent PMN recruitment was reduced >60% by anti-MIP-2 antibodies and >80% in mast cell-deficient Kit(W)/Kit(W)(-v) mice. Mast cells from WT mice efficiently restored DTHRs and MIP-2-dependent PMN recruitment in Kit(W)/Kit(W)-(v) mice, whereas mast cells from TNF(-/)- mice did not. Thus, mast cell-derived TNF and MIP-2 ultimately determine the pattern of infiltrating cells during T cell-mediated DTHRs.

The trioma approach is a new immunotherapeutic strategy for treating B-cell lymphomas. It is based on converting the tumour idiotype to a bispecific immunoglobulin that redirects the idiotype to antigen-presenting cells. We show here that even pre-existing tumours can be eradicated by trioma vaccination, that the trioma approach is superior to vaccination with cytokine gene-modified autologous tumour cells and that there is a synergism between trioma immunisation and GM-CSF gene transfer. Furthermore, we show that the immunising potential of GM-CSF gene-modified autologous lymphoma cells is not as dependent on the cytokine expression level as described for other tumour models, such that even minute expression rates are effective. IL-4 gene transfer in the lymphoma model is considerably less efficient or even ineffective when more sensitive systems are used. Remarkably, trioma-mediated effects are extinguished when IL-4 is expressed by the trioma cell.

The monocyte/macrophage (Mphi is central in the regulation of the immune response in states of trauma and sepsis. Because monocyte subsets, characterized by expression of the Fc-receptor (FcR), were shown to play distinct immunologic roles in trauma, it was the objective of this study to assess insights into the functional role of FcR positive (FcR+) and negative (FcR-) subclasses in surgical sepsis. In a prospective study, peripheral blood Mphi from 20 septic patients and 10 healthy volunteers were evaluated on consecutive days after the onset of sepsis. FcR+/- subsets were separated by rosetting with antibody-coated human erythrocytes. Receptor expression and synthesis of proinflammatory cytokines were used to evaluate the functional role of these cells. We demonstrated a significant monocytosis (350%; p

Mast cells are thought to contribute significantly to the pathology and mortality associated with anaphylaxis and other allergic disorders. However, studies using genetically mast cell–deficient WBB6F1-KitW/KitW-v and congenic wild-type (WBB6F1-+/+) mice indicate that mast cells can also promote health, by participating in natural immune responses to bacterial infection. We previously reported that repetitive administration of the c-kit ligand, stem cell factor (SCF), can increase mast cell numbers in normal mice in vivo. In vitro studies have indicated that SCF can also modulate mast cell effector function. We now report that treatment with SCF can significantly improve the survival of normal C57BL/6 mice in a model of acute bacterial peritonitis, cecal ligation and puncture (CLP). Experiments in mast cell–reconstituted WBB6F1-KitW/KitW-v mice indicate that this effect of SCF treatment reflects, at least in part, the actions of SCF on mast cells. Repetitive administration of SCF also can enhance survival in mice that genetically lack tumor necrosis factor (TNF)-α, demonstrating that the ability of SCF treatment to improve survival after CLP does not solely reflect effects of SCF on mast cell– dependent (or –independent) production of TNF-α. These findings identify c-kit and mast cells as potential therapeutic targets for enhancing innate immune responses.

GTP cyclohydrolase I controls the de novo pathway for the synthesis of tetrahydrobiopterin, which is the essential cofactor for tryptophan 5-monooxygenase and thus, for serotonin production. In mouse bone marrow-derived mast cells, the kit ligand selectively up-regulates GTP cyclohydrolase I activity (Ziegler, I., Hultner, L., Egger, D., Kempkes, B., Mailhammer, R., Gillis, S., and Rodl, W. (1993) J. Biol. Chem. 268, 12544–12551). Immunoblot analysis now confirms that this long term enhancement is caused by increased expression of the enzyme. Furthermore we show that GTP cyclohydrolase I is subject to modification at the post-translational level. In vivolabeling with [32P]orthophosphate demonstrates that in primary mast cells and in transfected RBL-2H3 cells overexpressing GTP cyclohydrolase I, the enzyme exists in a phosphorylated form. Antigen binding to the high affinity receptor for IgE triggers an additional and transient phosphorylation of GTP cyclohydrolase I with a concomitant rise in its activity, and in consequence, cellular tetrahydrobiopterin levels increase. These events culminate 8 min after stimulation and can be mimicked by phorbol ester. The hyperphosphorylation is greatly reduced by the protein kinase C inhibitor Ro-31-8220. In vitro phosphorylation studies indicate that GTP cyclohydrolase I is a substrate for both casein kinase II and protein kinase C.

Prostaglandin (PG) D2, PGJ2and Δ12-PGJ2are antiproliferative eicosanoids. We investigated the production of PGD2by murine bone marrow-derived mast cells (BMMC) taking into consideration metabolism of PGD2to PGD2and Δ12-PGJ2. PG-metabolites were quantified by high performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA). Stimulated with calcium ionophore A23187 BMMC released eight-fold more PGJ2and Δ12-PGJ2than PGD2. Conversion of endogenously produced PGD2to PGJ2and Δ12-PGJ2proceeded rapidly in contrast to metabolism of exogenously added PGD2. The antiproliferative potency of these prostaglandins is demonstratedin vitro. We conclude that determination of PGD2production by mast cells must take into consideration rapid conversion to active derivatives, which may play a significant role in growth regulation.

Activation of naive dense CD4+ T cells by plate-bound anti-CD3 antibodies favors the development of Th1 cells which, upon re-stimulation, produce significant amounts of IFN-gamma but no IL-4. However, co-activation of such naive T cells in the presence of IgE [anti-dinitrophenyl (DNP)]-loaded bone marrow-derived mast cells (BMMC) on plates coated with anti-CD3 antibodies and DNP-BSA led to the development of IL-4-producing Th2 cells. The same result could be observed if irradiated (800 rad) BMMC were applied as co-stimulators. Moreover, BMMC could be replaced by the supernatant of IgE-activated BMMC suggesting that a soluble mediator, presumably IL-4, was responsible for this effect. This assumption was substantiated using neutralizing anti-IL-4 antibodies which abolished the BMMC-mediated Th2 development in all cases. Addition of IL-12, a cytokine that was shown to antagonize the Th2-promoting effect of IL-4 in vivo, could not inhibit the development of IL-4-producing T cells, but gave rise to a T cell population which produced relatively high amounts of IL-4 and IFN-gamma. Since BMMC represent the in vitro equivalent of mucosal mast cells these data suggest that IgE-activated mucosal mast cells can bias an emerging T cell dependent immune response towards a Th2 dominated reaction by the initial production of IL-4.

Immunologists are just beginning to understand the early signals required to recruit memory T cells to sites of antigen-specific inflammation. Now it seems that activation of κ light chain–sensitized mast cells by antigen-specific recognition initiates local immune responses (pages 694–701).

The object of this prospective, randomized trial was to study the dysregulation effects of cardiopulmonary bypass on the synthesis pattern of interleukin-1, tumor necrosis factor, and interleukin-6, which have been identified as the key mediators of acute phase response. In addition, the counterregulation achieved by administration of indomethacin, which blocks the downregulating mediator prostaglandin E 2 , or indomethacin combined with thymopentin, which enhances T-lymphocytic reactivity, was investigated. Sixty patients who had undergone open heart operations were included in the study. These patients were divided into three groups: group A (n = 20) received intravenous indomethacin alone, group B (n = 20) received both indomethacin and thymopentin, and group C (n = 20) served as control. In control patients interleukin-1 and tumor necrosis factor synthesis were suppressed postoperatively. This effect was significantly counteracted by indomethacin with no further improvement by adding thymopentin. Interleukin-6 synthesis increased in all groups. Although indomethacin treatment alone had little effect on this phenomenon, additional administration of thymopentin significantly reduced elevated interleukin-6 synthesis. Corresponding differences in clinical outcome could not be detected due to small patient numbers. This study was, however, able to demonstrate that an immunomodulatory therapy can influence alterations in immune mechanisms after cardiopulmonary bypass.

It has long since been recognized that mast cells are critical effectors of anaphylactic reactions, and the existence of these potentially hazardous cells has solely been justified due to their beneficial role in some infections with extracellular parasites. A novel understanding of mast cells as sentinels of the immune system has been made possible by taking advantage of mast cell-deficient mice in order to study the roles of mast cells in vivo and by detailed analyses of mast cell activation in vitro. Collectively, these experiments have revealed a variety of IgE-independent stimuli, which lead to the activation of mast cells as crucial initiators of an inflammatory response. Besides their effector function, a variety of studies reviewed herein point towards important regulatory roles for mast cells, especially regarding their interaction with other cell types.

Mast cells, due to their ability to produce a large panel of mediators and cytokines, participate in a variety of processes in adaptive and innate immunity. Herein we report that in primary murine bone marrow-derived mast cells activated with ionomycin or IgE-Ag the bacterial endotoxin LPS strongly enhances the expression of IL-9 and IL-13, but not IL-4. This costimulatory effect of LPS is absent in activated mast cells derived from the LPS-hyporesponsive mouse strain BALB/c-LPSd, although in these cells the proinflammatory cytokine IL-1 can still substitute for LPS. The enhanced production of mast cell-derived IL-13 in the presence of IL-1 is a novel observation. Coactivation of mast cells with LPS leads to a synergistic activation of NF-κB, which is shown by an NF-κB-driven reporter gene construct. In the presence of an inhibitor of NF-κB activation, the production of IL-9 is strongly decreased, whereas the expression of IL-13 is hardly reduced, and that of IL-4 is not affected at all. NF-κB drives the expression of IL-9 via three NF-κB binding sites within the IL-9 promoter, which we characterize using gel shift analyses and reporter gene assays. In the light of recent reports that strongly support critical roles for IL-9 and IL-13 in allergic lung inflammation, our results emphasize the potential clinical importance of LPS as an enhancer of mast cell-derived IL-9 and IL-13 production in the course of inflammatory reactions and allergic diseases.

Mast cells can play detrimental roles in the pathophysiology and mortality observed in anaphylaxis and other Th2-dominated allergic diseases. In contrast, these cells contribute to protective host defense mechanisms against parasitic worm infections. After IgE/Ag activation, mast cells can produce multiple cytokines that may enhance allergic inflammations, while a similar panel of Th2-related cytokines may support immunological strategies against parasites. Here we report that in primary mouse bone marrow-derived mast cells activated by ionomycin or IgE/Ag, the proinflammatory mediator IL-1 (α or β) up-regulated production of IL-3, IL-5, IL-6, and IL-9 as well as TNF, i.e., cytokines implicated in many inflammatory processes including those associated with allergies and helminthic infections. IL-1 did not induce significant cytokine release in the absence of ionomycin or IgE/Ag, suggesting that Ca-dependent signaling was required. IL-1-mediated enhancement of cytokine expression was confirmed at the mRNA level by Northern blot and/or RT-PCR analysis. Our study reveals a role for IL-1 in the up-regulation of multiple mast cell-derived cytokines. Moreover, we identify mast cells as a novel source of IL-9. These results are of particular importance in the light of recent reports that strongly support a central role of IL-9 in allergic lung inflammation and in host defense against worm infections.

Recently, the Th2-type cytokine IL-9 was identified by genetic mapping analyses as a key mediator that determines the susceptibility to asthma. This has been further supported by data from IL-9-transgenic mice in which the overexpression of IL-9 in the lung causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. In an accompanying paper, we demonstrate that murine bone marrow-derived mast cells (BMMC) after stimulation with either ionomycin, a combination of ionomycin and IL-1, or via IgE-Ag complexes and IL-1 are very potent producers of IL-9. Herein we show that a dramatic increase of IL-9 production is observed when BMMC activated with ionomycin/IL-1 or with IgE-Ag complexes/IL-1 are treated with either additional kit ligand (KL) or IL-10. Both KL and IL-10 considerably enhance the production of IL-9 mRNA and protein. We were also able to demonstrate that the production of endogenous IL-10 by activated mast cells acts on the production of IL-9. Half-life measurements of IL-9 mRNA revealed no significant effect by KL, but a 2-fold increase of mRNA stability under the influence of IL-10. Reporter gene assays of transfected BMMC showed an enhanced transcriptional activity of the IL-9 promoter in the presence of either IL-10 or KL compared with cells stimulated only with a combination of IL-1 and ionomycin. The influence of KL and IL-10 might be of physiological importance, because it is known that both cytokines are produced by bronchial epithelial cells.

Identification of new therapeutic targets for the management of septic shock remains imperative as all investigational therapies, including anti-tumor necrosis factor (TNF) and anti-interleukin (IL)-1 agents, have uniformly failed to lower the mortality of critically ill patients with severe sepsis. We report here that macrophage migration inhibitory factor (MIF) is a critical mediator of septic shock. High concentrations of MIF were detected in the peritoneal exudate fluid and in the systemic circulation of mice with bacterial peritonitis. Experiments performed in TNFalpha knockout mice allowed a direct evaluation of the part played by MIF in sepsis in the absence of this pivotal cytokine of inflammation. Anti-MIF antibody protected TNFalpha knockout from lethal peritonitis induced by cecal ligation and puncture (CLP), providing evidence of an intrinsic contribution of MIF to the pathogenesis of sepsis. Anti-MIF antibody also protected normal mice from lethal peritonitis induced by both CLP and Escherichia coli, even when treatment was started up to 8 hours after CLP. Conversely, co-injection of recombinant MIF and E. coli markedly increased the lethality of peritonitis. Finally, high concentrations of MIF were detected in the plasma of patients with severe sepsis or septic shock. These studies define a critical part for MIF in the pathogenesis of septic shock and identify a new target for therapeutic intervention.

Widely used to specifically inhibit gene expression, synthetic oligodeoxynucleotides (ODN) can exert a plethora of non-antisense effects. Immunostimulation by CpG-ODN has attracted particular attention. ODN rich in the nucleotide guanosine (G-rich ODN) constitute another type of sequences displaying non-antisense-mediated effects. We have examined the effects of CpG- and G-rich ODN on primary mouse bone marrow cells (BMC) in vitro. CpG-ODN induced rapid proliferation of B cells and production of IL-6 and IL-12p40. However, when tested in agar colony assays, CpG-ODN failed to promote the formation of colonies. In marked contrast, G-rich non-CpG-ODN led to sustained proliferation of macrophage-like cells without inducing cytokines or hemopoietic growth factors. Unlike CpG-ODN, G-rich ODN effectively induced the formation of macrophage colonies in agar assays, indicating a direct action on progenitor cells. Electrophoretic mobility shift assays revealed specific binding of G-rich ODN to a non-nuclear protein. The ability of a panel of ODN to compete for binding correlated with their potential to induce proliferation of macrophage-like cells from primary mouse BMC. As such, these data reveal a so far unrecognized potential of G-rich ODN to signal directly outgrowth of macrophage progenitors from BMC.

Bleomycin and asparaginase are widely used antineoplastic agents which may induce allergic or inflammatory side-effects. Mast cells are implicated as effector cells in allergic and inflammatory responses. The aim of this study was to establish whether bleomycin or asparaginase modulate leukotriene production in vitro and in vivo . Leukotriene C 4 (LTC 4 ) production by murine bone marrow-derived mast cells (BMMC) was determined by radioimmunoassay (RIA). Leukotriene production in patients was assessed by determining leukotriene E 4 and N -acetyl-leukotriene E 4 in urine by means of combined HPLC and RIA. Bleomycine induced an up to 2.1-fold increase in LTC 4 production both in unstimulated and in calcium ionophore-stimulated mast cells. In 3 of 7 patients treated with bleomycin, a greater than 2-fold increase in endogenous leukotriene production was observed. This effect was associated with febrile responses and was most pronounced in a patient who developed an Adult Respiratory Distress Syndrome (ARDS). Asparaginase increased leukotriene production up to 10-fold in stimulated but not in unstimulated BMMC. In a patient who developed an anaphylactic reaction after treatment with asparaginase, a pronounced increase in urinary leukotriene concentration was observed. In contrast to bleomycinor asparaginase, a number of other cytostatic agents did not significantly change leukotriene production by BMMC. Our data indicate that some of the inflammatory and allergic side-effects of bleomycin and asparaginase could be mediated by leukotrienes, a possible source of which may be mast cells.

Despite improved procedures in chemotherapy and bone marrow transplantation (BMT), post-BMT leukemia relapse rates have remained rather constant in the last decade. Immunotherapy with monoclonal or bispecific antibodies (bsAb) is a promising approach to improve this situation, but is hampered by the absence of tumor-specific antigens on the majority of tumors. To evade this problem, we developed a new tumor-specific approach in which bispecific antibodies exploit chimerism after allogeneic BMT by redirecting donor T cells against recipient-specific antigens on tumor cells. Two different leukemia relapse models were established using a T-cell lymphoma (ST-1) and a B-cell lymphoma (BCL1) to evaluate the efficiency of such a therapy. In these experiments, irradiated BALB/c (Thy-1.2+, I-Ad) mice were transplanted with C57BL/6 Thy-1.1 (I-Ab) BM cells under the protection of graft- versus-host disease-preventing monoclonal antibodies. Forty-five days after BMT, the chimeric mice were injected with either 2 x 10(4) recipient-type, Thy-1.2+, CD3- ST-1 cells or major histocompatability complex (MHC) class II+ (I-Ad)-BCL1 cells. Four days later, the mice were treated with 8 microg bsAb G2 (anti-CD3 x anti-Thy-1.2) or 10 microg (+10 microg, day 6) bsAb BiC (anti-CD3 x anti-I-Ad), respectively. These combinations guaranteed exclusive binding of the bsAbs target arms to tumor cells, leaving the surrounding, donor-type hematopoietic cells unbound. Compared with the parental antibodies, the bsAbs markedly reduced tumor mortality. Between 34% and 83% of mice survived in the bsAb groups compared with 0% of the control groups treated with parental antibodies, clearly documenting the benefit of the redirection principle. Furthermore, cytokine release (interleukin- 6) after anti-CD3 antibody or bsAb treatment was decreased by administering a low-dose antibody preinjection. We have shown (1) that 6 weeks after BMT, when donor T-cell reconstitution is still in progress, T-cell-redirecting bsAb are clearly superior to parental antibodies in terms of tumor cell elimination; and (2) that the polymorphism of a common antigen such as Thy-1 or a clinically more relevant target antigen such as MHC class II can be used as an operational tumor-specific antigen after allogeneic BMT.

Mast cells play a detrimental role in IgE-dependent allergic reactions. In contrast, a protective function for mast cells has been proposed on the basis of some worm infection models. No reports exist on the in vivo significance of these cells in bacterial infections. Here we use congenitally mast-cell-deficient W/Wv mice and normal +/+ littermates to analyse the role of mast cells in a model of acute septic peritonitis (caecum ligation and puncture (CLP)). Following CLP, W/Wv mice showed a significantly increased mortality compared to +/+ mice. The selective reconstitution of W/Wv mice with cultured +/+ mast cells substantially protected them from the lethal effects of CLP, whereas an anti-tumor-necrosis-factor (TNF) antibody injected immediately after CLP completely suppressed this protection. Our results reveal a previously unrecognized protective role of mast cells and mast-cell-derived TNF in acute bacterial peritonitis.

GTP cyclohydrolase I catalyzes the initial and rate limiting step of the biosynthesis of tetrahydrobiopterin, the cofactor for aromatic amino acid hydroxylation. The mouse mutant HPH-1, previously generated by chemical mutagenesis, shows a phenylketonuria due to decreased hepatic GTP cyclohydrolase I activity. We show that both parameters GTP cyclohydrolase I activity and tetrahydrobiopterin synthesis significantly increase after weaning, but remain reduced during the lifetime. In the wild type mouse (C57BL/6), interferon-γ and kit ligand induce GTP cyclohydrolase I activity in primed T-cells and in bone marrow-derived mast cells, respectively. The same is true for the HPH-1 mutant, but the absolute values remain lower throughout. The open reading frame of GTP cyclohydrolase I is not affected by the hph-1 mutation as shown by sequencing. Northern blot analysis demonstrates a marked decrease in the steady state mRNA level specific for GTP cyclohydrolase I.

(6R)-H4biopterin (BH4) is synthesized from GTP in different types of tissues and in cells of different lineages. It is the natural and immediate electron donor for aromatic amino acid monooxygenases. Thus, it serves as a cofactor in tissues committed to degradation of phenylalanine and the synthesis of the neurotransmitters dopamine, epinephrine, and serotonin (for review, see ref.1). The activity of GTP cyclohydrolase I (GTP-CH), the first and rate limiting enzyme of BH4 synthesis, appears to be constitutively expressed in all competent tissues such as liver, adrenal medulla, and brain2. Changes in its activity may occur in response to physiological conditions and pharmacological treatment3. No specific regulatory factors have been identified yet.

We have previously shown that certain bone marrow-derived mast cell (BMMC) lines proliferate in response to a mast cell growth-enhancing activity (MEA) that is distinct from interleukin (IL) 3 and IL 4. Here we provide evidence that MEA is identical with the recently cloned mouse T cell growth factor P40. The evidence is as follows: (a) recombinant P40 displayed all the biological activities ascribed to MEA: it supported the growth of MEA-sensitive BMMC lines, it induced IL 6 secretion by these cells, and it enhanced survival of primary mast cell cultures; (b) highly purified MEA stimulated the growth of P40-dependent cell lines; (c) a rabbit monospecific antiserum directed against P40 specifically inhibited the action of MEA on BMMC; (d) specific binding sites for P40 were detected on BMMC and (e) MEA competed with P40 for binding to P40-dependent T cells, indicating that the two molecules interact with the same receptor. These observations further extend the range of biological activities ascribed to P40 and warrant its proposed designation as IL9.20

Mast cells are pivotal effector cells in IgE-mediated allergic inflammatory diseases. Central for mast cell activation are signals from the IgE receptor FcεRI, which induce cell degranulation with the release of preformed mediators and de novo synthesis of proinflammatory leukotrienes and cytokines. How these individual mast cell responses are differentially controlled is still unresolved. We identify B cell lymphoma 10 (Bcl10) and mucosa-associated lymphoid tissue 1 (Malt1) as novel key regulators of mast cell signaling. Mice deficient for either protein display severely impaired IgE-dependent late phase anaphylactic reactions. Mast cells from these animals neither activate nuclear factor κB (NF-κB) nor produce tumor necrosis factor α or interleukin 6 upon FcεRI ligation even though proximal signaling, degranulation, and leukotriene secretion are normal. Thus, Bcl10 and Malt1 are essential positive mediators of FcεRI-dependent mast cell activation that selectively uncouple NF-κB-induced proinflammatory cytokine production from degranulation and leukotriene synthesis. JEM © The Rockefeller University Press.

Colony-stimulating factors (CSF) are glycoproteins essential for the formation of colonies of mature haematopoietic cells by single immature progenitor cells (colony-forming cells (CFC)). The number of colonies in semi-solid agar cultures can be used to determine the CSF concentration in biological fluids1. Various murine cells2–6 are able to produce CSF in vitro. Supernatants of spleen cell cultures consisting of a mixture of T cells, B cells and macrophages are found to contain neutrophil-granulocyte–macrophage CSF (GM-CSF), CSF for megakaryocytes (Meg-CSF), eosinophilic granulocytes (Eo-CSF) and erythroid cells (E-CSF)5. The producer cells of these different CSF types, however, have not been defined7. We therefore determined here whether normal T cells produce CSF in vitro and whether GM-CSF, Meg-CSF, Eo-CSF and E-CSF are produced by different T-cell subsets or the progeny of single T-cell clones. Our results indicate that T cells at a high frequency (one out of three) produce CSF at variable amounts even in the absence of accessory cells following mitogen stimulation.

This report describes the first frequency estimate of immune interferon (IFN-gamma)- and colony-stimulating factor (CSF)-producing human peripheral blood T lymphocytes. Human peripheral blood lymphocytes (HPBL) were activated with T cell mitogens [concanavalin A (Con A) or phytohemagglutinin (PHA)] in bulk culture and subsequently plated in limiting dilution (LD) microcultures in the presence of irradiated allogeneic HPBL filler cells and culture medium supplemented with human interleukin 2 and T cell mitogen (Con A or PHA). HPBL growing in these cultures were T cells as determined by E-rosetting (greater than 85%) and were positive for the OKT8 marker. The growth frequency (f) of HPBL in LD was f = 1/20-1/100. Such cells were induced with T cell mitogens (Con A or PHA) to release lymphokines into the supernatant. Statistical analysis of the data from the LD experiments showed that (a) the precursor cell frequency for IFN-gamma- and CSF-producing T cells was f = 1/63-1/151 and 1/89-1/217, respectively, and (b) most T cell clones released IFN-gamma and CSF simultaneously. In addition, individual T cell clones were shown to be capable of releasing different CSF types. In principle, this experimental system can be used to evaluate the precursor frequency, relationship and activity of normal or pathological human T cells performing any in vitro T cell function.

Murine bone marrow-derived mast cells proliferate in response to interleukin 3. In addition to 6-biopterin, 7-biopterin was identified in these cells by HPLC analysis of iodine oxidized extracts and by alkaline permanganate oxidation to the 6- and 7-carboxylic acids. 7-Biopterin comprised 31.9 (± 7.7)% of the total biopterin. It was absent in cells which were grown with of l-p-chlorophenylalanine, an inhibitor of tryptophan 5-monooxygenase. Both 6- and 7-biopterin were present in the cell as their tetrahydro forms. From these data we conclude that 7-biopterin, in contrast to e.g. brain tissue, regularly occurs as a normal metabolite in primary mast cells and that it is generated during hydroxylation of tryptophan.

1 Institut ftir Immunologie und Genetik, Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany 2 Abteilung far Experimentelle Hgmatologie, der Gesellschaft fiir Strahlenund Umweltforschung, Mtinchen, Federal Republic of Germany 3 Institut ftir Immunotogie und Serologie, Universitiit Heidelberg, Heidelberg, Federal Republic of Germany 4 Medizinische Hochschule, Fachbereich ftir Pharmakologie, Abt. Molekularpharmakologie, Hannover, Federal Republic of Germany

We have analyzed lymphokine release from T cell clones in limiting dilution microcultures or from alloreactive T cell clones in long-term cultures. Our data show that 1) T lyrrphocytes are the producer cells, 2) lymphokines can be released from T cells following mitogenic or antigenic stimulation, 3) the progeny of a particular T cell are capable of releasing several lymphokines simultaneously, and 4) different lynphokines produced by the progeny of particular T cells are not identical. Those data are briefly discussed with respect to release of soluble factors during the course of alloreactivity.

A series of murine interleukin 3 (IL-3)-dependent hemopoietic cell lines was studied for the capacity to produce interleukin 6 (IL-6) in vitro. These included a bone marrow-derived mast cell line (L138.8A) and several early myeloid cell lines described in the literature (DA-1, DA-3, NFS-60, NFS-78, FDC-P1, FDC-P2, FDC-PmixA4, and 32Dcl.23). All of these cell lines produced growth factor activity for IL-6-dependent hybridoma cells (7TD1), which was completely neutralized by the monoclonal anti-IL-6-antibody 6B4. IL-6 expression was also evident at the mRNA level using a murine IL-6-specific cDNA probe. In 32Dcl.23 cells (2 x 10(5)/ml) stimulated for 24 hr with serial dilutions of purified murine IL-3, a positive correlation was found between the IL-3 dose and the amount of IL-6 measured in the conditioned media. At 24 hr this correlation was not evident at the mRNA level. However, prolonged exposure of 32Dcl.23 cells (up to 72 hr) to either a high (60 U/ml) or a low IL-3 concentration (1 U/ml) revealed a time-dependent increase and decrease, respectively, of IL-6 mRNA levels. At both IL-3 concentrations 32Dcl.23 cells remained in a fully viable and proliferative state. The influence of IL-3 on IL-6 release could be specifically counteracted by anti-IL-3-antiserum. IL-6 added alone or in concert with IL-3 did not stimulate 32Dcl.23 proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)