Insights from knock-out models concerning postischemic release of TNFalpha from isolated mouse hearts.

The inflammatory cytokine tumor necrosis factor alpha (TNFalpha) is controversially discussed in ischemia/reperfusion damage of the heart. Purpose of this study was to elucidate cellular sources of TNFalpha and parameters which possibly influence its release in the heart following ischemia. Isolated hearts of mice were subjected to 15 min of global ischemia and 90 min of reperfusion. We employed hearts of various mice knock-out strains (interleukin-6(-/-), matrix metalloprotease-7(-/-), mast-cell deficient WBB6F1-Kit(W)/Kit(W-v), TNF-R1(-/-)) and wildtype mice, the latter perfused without and with infusion of cycloheximide or TNFalpha-cleaving-enzyme inhibitor (TAPI-2). Normoxic control hearts showed basal release of TNFalpha during the whole experiment. Immunohistology identified cardiac mast cells, macrophages and endothelial cells as main sources. TNFalpha release was stimulated during postischemic reperfusion, occurring in a two-peak pattern: directly after ischemia (0-10 min) and again after 60-90 min. The first peak mainly reflects tissue washout of TNFalpha accumulated during ischemia. The second, protracted peak arose continuously from the basal level and was abolished by protein synthesis inhibitor cycloheximide. Both properties are characteristic for de novo synthesis of TNFalpha, e.g., in cardiac muscle cells. However, immunohistological staining for TNFalpha failed in cardiomyocytes after 90 min of reperfusion. In contrast to hearts of TNF-R1(-/-) and Kit(W/W-v)-mice, those of IL-6(-/-) and MMP-7(-/-) mice lacked the late TNFalpha peak. TAPI did not suppress release of TNFalpha. While autostimulation via TNF-R1 also does not seem obligatory and mast cell can be ignored as source of the second peak, IL-6 may support de novo synthesis of TNFalpha. Additionally, TNFalpha release may essentially involve cleavage of membrane bound TNFalpha by MMP-7.

IL-9 and IL-13 production by activated mast cells is strongly enhanced in the presence of lipopolysaccharide: NF-kappa B is decisively involved in the expression of IL-9.

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-LPS(d), 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-kappa B, which is shown by an NF-kappa B-driven reporter gene construct. In the presence of an inhibitor of NF-kappa B 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-kappa B drives the expression of IL-9 via three NF-kappa B 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 control neutrophil recruitment during T cell-mediated delayed-type hypersensitivity reactions through tumor necrosis factor and macrophage inflammatory protein 2.

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.

Murine bone marrow-derived mast cells as potent producers of IL-9: costimulatory function of IL-10 and kit ligand in the presence of IL-1.

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.

In activated mast cells, IL-1 up-regulates the production of several Th2-related cytokines including IL-9.

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 (alpha or beta) 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.

Protection from septic shock by neutralization of macrophage migration inhibitory factor.

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.

Guanosine-rich oligodeoxynucleotides induce proliferation of macrophage progenitors in cultures of murine bone marrow cells.

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.

Gene therapy of B-cell lymphoma with cytokine gene-modified trioma cells.

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.

Evaluation of Fc-receptor positive (FcR+) and negative (FcR-) monocyte subsets in sepsis.

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<.01) and suppression of human lymphocyte antigen (HLA-DR) expression (35%; p<.05). Synthesis of Interleukin-1beta (IL-1beta; e.g., Day 1: 230+/-30 pg/mL) and Interleukin-6 (IL-6; e.g., Day 1: 1920+/-350 U/mL) were significantly higher (p<.05) in FcR+ subsets than in controls (IL-1beta: 100+/-5 pg/mL; IL-6: 353+/-75 U/mL). Tumor necrosis factor-alpha (TNF-alpha) was elevated in FcR+ monocytes but did not reach a significant value. Interleukin-8 (IL-8) synthesis showed only on Day 1 and in controls significant differences in FcR+ and FcR- cells (Day1: FcR-: 19.6+/-4.1 nM; FcR+: 9+/-4.3 nM). Sepsis results in a significant shift toward FcR+ monocytes. This cell population is characterized by high proinflammatory cytokine synthesis. The extent of this shift seems to identify a group of high risk septic patients that might benefit from immunomodulatory therapy.

Immunostimulatory CpG-oligodeoxynucleotides cause extramedullary murine hemopoiesis.

Bacterial DNA and the synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted attention because they activate cells of the adaptive immune system (lymphocytes) and the innate immune system (APCs) in a sequence-dependent manner. Here, we addressed whether CpG-ODNs affect hemopoiesis. Challenging mice with immunostimulatory CpG-ODN sequences led to transient splenomegaly, with a maximum increase of spleen weight at day 6. The induction of splenomegaly by CpG-ODNs was sequence-specific, dose-dependent, and associated with an increase in splenic cell count, in numbers of granulocyte-macrophage CFUs (GM-CFUs), and early erythroid progenitors (burst-forming units-erythroid). The transfer of spleen cells from CpG-ODN-pretreated animals into lethally irradiated syngeneic mice yielded an increase of spleen CFUs. Furthermore, the challenge of sublethally irradiated mice with CpG-ODNs caused radioprotective effects, in that recovery of GM-CFUs and cytotoxic T cell function was enhanced. The increase in GM-CFU and CTL function correlated with an enhanced resistance to Listeria infection in irradiated mice. We conclude from these data that CpG-ODNs trigger extramedullary hemopoiesis, and that this finding could be of therapeutic relevance in myelosuppression.

Release of prostaglandin D2 by murine mast cells: importance of metabolite formation for antiproliferative activity.

Prostaglandin (PG) D2, PGJ2 and delta12-PGJ2 are antiproliferative eicosanoids. We investigated the production of PGD2 by murine bone marrow-derived mast cells (BMMC) taking into consideration metabolism of PGD2 to PGD2 and delta12-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 PGJ2 and delta12-PGJ2 than PGD2. Conversion of endogenously produced PGD2 to PGJ2 and delta12-PGJ2 proceeded rapidly in contrast to metabolism of exogenously added PGD2. The antiproliferative potency of these prostaglandins is demonstrated in vitro. We conclude that determination of PGD2 production by mast cells must take into consideration rapid conversion to active derivatives, which may play a significant role in growth regulation.

The c-kit ligand, stem cell factor, can enhance innate immunity through effects on mast cells.

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)-alpha, 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-alpha. These findings identify c-kit and mast cells as potential therapeutic targets for enhancing innate immune responses.

Phosphorylation of GTP cyclohydrolase I and modulation of its activity in rodent mast cells. GTP cyclohydrolase I hyperphosphorylation is coupled to high affinity IgE receptor signaling and involves protein kinase C.

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., Hültner, L. , Egger, D., Kempkes, B., Mailhammer, R., Gillis, S., and Rödl, 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 vivo labeling 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.

Induction of leukotriene production by bleomycin and asparaginase in mast cells in vitro and in patients in vivo.

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 C4 (LTC4) production by murine bone marrow-derived mast cells (BMMC) was determined by radioimmunoassay (RIA). Leukotriene production in patients was assessed by determining leukotriene E4 and N-acetyl-leukotriene E4 in urine by means of combined HPLC and RIA. Bleomycin induced an up to 2.1-fold increase in LTC4 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 bleomycin or 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.

Bispecific antibodies target operationally tumor-specific antigens in two leukemia relapse models.

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.

Schistosoma mansoni infection in mice augments the capacity for interleukin 3 (IL-3) and IL-9 production and concurrently enlarges progenitor pools for mast cells and granulocytes-macrophages.

Mast cells and granulocytes-macrophages (GM) are components of the host defense system against worm infections, including schistosomiasis. Here we report the kinetics of changes in the number of colony-forming cells (CFC) for mast cells and GM during the course of a primary experimental infection of mice with Schistosoma mansoni cercariae over a period of 24 weeks postinfection (p.i.). Concurrently, we measured known myelopoietic and/or mast cell-stimulating cytokines (i.e., interleukin 3 [IL-3] and IL-9) in pokeweed mitogen-activated spleen cell-conditioned medium. Our results show that during the acute phase of the hepatic granulomatous reaction, the numbers of both mast-CFC and GM-CFC were significantly elevated in bone marrow. However, while femoral GM-CFC numbers had returned to normal control values at week 16 p.i., femoral and splenic mast-CFC numbers remained significantly elevated until week 20 p.i., which corresponds to the chronic fibrotic phase of hepatic granulomatous inflammation. Increased GM-CFC numbers correlated with elevated IL-3 levels, while increased mast-CFC numbers paralleled the increased IL-9 concentrations in spleen cell-conditioned medium. By the reverse transcription-PCR method, enhanced expression of IL-3 and IL-9 transcripts was found in RNA samples obtained from livers and spleens of infected mice. Our data demonstrate that during the course of infection of mice with S. mansoni, the coordinate need for mast cells and GM is at least partly regulated at the stage of progenitor cell commitment in the bone marrow and spleen. It appears that IL-3 and IL-9 help to promote at this stage the ultimate generation of mature effector cells.

Kinetics of interleukin-6 production after experimental infection of mice with Schistosoma mansoni.

It has been reported that interleukin-6 (IL-6) is expressed in cells of acute inflammatory granulomas experimentally induced in mice by eggs of Schistosoma mansoni. Moreover, in vitro IL-6 was shown to enhance the cytotoxic activity of human platelets against larvae of S. mansoni. To elucidate further a proposed biological significance of this cytokine during the course of schistosomiasis, we studied the kinetics of IL-6 production and concomitantly performed a histopathological analysis of the livers in BALB/c mice subcutaneously infected with S. mansoni cercariae. Over a period of 24 weeks postinfection (p.i.) we monitored serum IL-6 levels, IL-6 production in vitro by pokeweed mitogen (PWM)-stimulated spleen cells as well as IL-6 mRNA expression in livers, spleens and kidneys. We found significantly elevated IL-6 levels in PWM-stimulated spleen cell-conditioned media (SCM) at weeks 6 to 20 p.i., peaking at week 10 p.i. In contrast, serum IL-6 concentrations started to rise not before week 8 but remained significantly elevated above normal control values until week 24 p.i. The time pattern of enhanced IL-6 mRNA expression detected in spleens and livers, but not in kidneys, as well as the rises of IL-6 in SCM and with a delay of 2 weeks in serum samples correlated with the onset of the egg-induced inflammatory reactions as well as the incidence and the number of the granulomas observed histopathologically in the livers of infected mice. Our data emphasize both a local and a systemic role of IL-6 in the host immune response following infection of mice with S. mansoni.

Critical protective role of mast cells in a model of acute septic peritonitis.

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.

Constitutive and modulated cytokine expression in two permanent human bone marrow stromal cell lines.

We present a detailed analysis of cytokine expression patterns of the two permanent human bone marrow stromal cell lines, L87/4 and L88/5. These cell lines, previously established in our laboratory, are highly radiotolerant without cell detachment and support long-term cultures of CD(34+)-enriched human cord blood cells. RT-PCR analysis of 22 different cytokines or cytokine receptor mRNAs showed an almost identical expression pattern in the two stromal cell lines compared to primary human Dexter-type stroma. Since stromal feeder lines employed in long-term cultures usually are irradiated and grown in media containing corticosteroids, we analyzed the impact of irradiation and dexamethasone on cytokine production in the two cell lines by RT-PCR, Northern blot analysis, bioassays, and RIAs. By RT-PCR analysis, constitutive mRNA expression of c-kit, G-CSF, GM-CSF, IL-1 beta, IL-6, IL-7, IL-8, IL-11, Kit ligand (KL), LIF, M-CSF, MIP-1 alpha, TGF-beta, and TNF-alpha was demonstrated in both cell lines, with L87/4 a more potent cytokine producer than L88/5. Northern blot data showed an increase in mRNA levels for GM-CSF, IL-1 beta, and LIF by irradiation and IL-1 alpha treatment in both cell lines. IL-1 alpha-induced GM-CSF, IL-1 beta, IL-6, IL-11, and LIF mRNA levels were reduced by the addition of dexamethasone, whereas dexamethasone had no influence on the amounts of IL-1 alpha-induced G-CSF mRNA. L87/4 and, to a lower extent, L88/5 cells showed dexamethasone-dependent increases in KL mRNA, while KL mRNA levels were not stimulated by IL-1 alpha.