Interleukin 4 induces cultured monocytes/macrophages to form giant multinucleated cells.

Giant multinucleated cells (GMCs) are associated with granulomatous lesions that form in response to various infectious and noninfectious agents. The present study shows that mouse IL-4 induces the in vitro formation of GMCs by factor-dependent bone marrow and alveolar monocytes via cell fusion. GMCs appear 2 d after incubation of cell cultures with 20 U/ml or more of IL-4. Anti-IL-4 mAbs block the appearance of GMCs in these cultures, indicating that IL-4 acts directly on monocytes to promote fusion and does not secondarily induce the production of other soluble fusion factors. In soft agar cultures, IL-4 also causes the aggregation of macrophages and diminishes their migration. The role of IL-4 in a granulomatous inflammatory response is discussed.

Role of interleukin 2, interleukin 4, and alpha, beta, and gamma interferon in stimulating macrophage antibody-dependent tumoricidal activity.

Pretreatment of murine peritoneal exudate macrophages with 1-5 U/ml rIFN-gamma or rIL-2, or higher concentrations of IFN-alpha or IFN-beta greatly stimulated ADCC to Rl lymphoma targets. The assay was direct counting of viable target cells after 9 and 24 h using an E/T ratio of 5:1. 2d of pretreatment was optimal for enhancing ADCC. rIL-4 was inactive and IL-4-depleted Con A-induced spleen lymphokine retained its ADCC-stimulating activity. Antibody to IFN-gamma blocked the ADCC-promoting effect of the lymphokine, suggesting a major role for this factor.

Proliferation and differentiation of highly enriched mouse hematopoietic stem cells and progenitor cells in response to defined growth factors.

Three distinct hematopoietic populations derived from normal bone marrow were analyzed for their response to defined growth factors. The Thy-1loT- B- G- M-population, composing 0.2% of bone marrow, is 370-fold enriched for pluripotent hematopoietic stem cells. The two other populations, the Thy-1- T- B- G- M- and the predominantly mature Thy-1+ T+ B+ G+ M+ cells, lack stem cells. Thy-1loT- B- G- M- cells respond with a frequency of one in seven cells to IL-3 in an in vitro CFU-C assay, and give rise to many mixed colonies as expected from an early multipotent or pluripotent progenitor. The Thy-1- T- B- G- M- population also contains progenitor cells which responded to IL-3. However, colonies derived from Thy-1- T- B- G- M- cells are almost exclusively restricted to the macrophage/granulocyte lineages. This indicates that IL-3 can stimulate at least two distinct clonogenic early progenitor cells in normal bone marrow: multipotent Thy-1loT- B- G- M- cells and restricted Thy-1- T- B- G- M- cells. Thy-1loT- B- G- M-cells could not be stimulated by macrophage colony-stimulating factor (M-CSF), granulocyte CSF (G-CSF) or IL-5 (Eosinophil-CSF). The hematopoietic precursors that react to these factors are enriched in the Thy-1- T- G- B- M- population. Thus, multipotent and restricted progenitors can be separated on the basis of the expression of the cell surface antigen Thy-1.

Interleukin-10 is a natural suppressor of cytokine production and inflammation in a murine model of allergic bronchopulmonary aspergillosis.

We have used interleukin-10 (IL-10) gene knockout mice (IL-10-/-) to examine the role of endogenous IL-10 in allergic lung responses to Aspergillus fumigatus Ag. In vitro restimulated lung cells from sensitized IL-10-/- mice produced exaggerated amounts of IL-4, IL-5, and interferon-gamma (IFN-gamma) compared with wild-type (WT) lung cells. In vivo, the significance of IL-10 in regulating responses to repeated A. fumigatus inhalation was strikingly revealed in IL-10-/- outbred mice that had a 50-60% mortality rate, while mortality was rare in similarly treated WT mice. Furthermore, IL-10-/- outbred mice exhibited exaggerated airway inflammation and heightened levels of IL-5 and IFN-gamma in bronchoalveolar lavage (BAL) fluids. In contrast, the magnitude of the allergic lung response was similar in intranasally (i.n.) sensitized IL-10-/- and wild-type mice from a different strain (C57BL/6). Using a different route of priming (intraperitoneal) followed by one i.n. challenge we found that IL-10-/- C57BL/6 mice had heightened eosinophilic airway inflammation, BAL-IL-5 levels, and numbers of alphabetaT cells in the lung tissues compared with WT mice. We conclude that IL-10 can suppress inflammatory Th2-like lung responses as well as Th1-like responses given the constraints of genetic background and route of priming.

Interleukin 10: a novel stimulatory factor for mast cells and their progenitors.

We have characterized the mast cell stimulating activity of murine cytokine synthesis inhibitory factor, referred to as interleukin 10 (IL-10). It was found that IL-10 alone failed to support the growth of mast cell lines and mast cell progenitors. Nevertheless, it dramatically enhanced their growth when combined with IL-3 or IL-4. Moreover, IL-4 plus IL-10 supported the proliferation of mast cells as well as IL-3, suggesting that these two factors may provide a pathway for their development independent of IL-3. However, optimal mast cell growth was stimulated by the combination of IL-10, IL-4, and IL-3. This particular set of cytokines are coordinately produced by activated T cells and may constitute an effective network regulating early and late stages of mast cell development during certain immune responses.

Interleukin 10 but not interleukin 4 is a natural suppressant of cutaneous inflammatory responses.

We have examined the role of endogenously produced interleukin (IL) 4 and IL-10 in the regulation of inflammatory and immune reactions in the skin. In these experiments, irritant and contact hypersensitivity (CH) responses were elicited in mice with targeted disruptions of the IL-4 (IL-4T) or IL-10 (IL-10T) gene. Our study showed that IL-4T and wild-type (wt) mice exhibited equivalent responses to the irritant croton oil. In contrast, the response of IL-10T mice challenged with croton oil was abnormally increased. When IL-10T mice were exposed to a higher dose of irritant, irreversible tissue damage occurred. By comparison, any treatment of wt mice with croton oil resulted in far less tissue damage and resolution of inflammation. Neutralizing antibody studies demonstrated that the necrosis that occurred in IL-10T mice was due to the overproduction of tumor necrosis factor. The anti-tumor necrosis factor antibody treatment of IL-10T mice did not significantly reduce the edema or the influx of inflammatory cells, suggesting that these changes were due to the uncontrolled production of other proinflammatory cytokines. T cell-dependent immune responses were also evaluated using the contact sensitizer oxazolone. The response of IL-4T mice did not differ from wt mice. In contrast, IL-10T mice mounted an exaggerated CH response, increased in both magnitude and duration as compared with wt mice. Based on these studies, we have concluded that IL-10, but not IL-4, is a natural suppressant of irritant responses and of CH, and it limits immunopathologic damage in the skin.

T helper cell 1-type CD4+ T cells, but not B cells, mediate colitis in interleukin 10-deficient mice.

Mice rendered deficient in the production of interleukin 10 (IL-10-/-) develop a chronic inflammatory bowel disease (IBD) that predominates in the colon and shares histopathological features with human IBD. Our aim was to identify which cell type(s) can mediate colitis in IL-10-/- mice. We detected an influx of immunoglobulin-positive cells into the colon and the presence of colon-reactive antibodies in the serum of IL-10-/- mice. To assess a pathogenic role for B cells, we generated a B cell-deficient (B-/-) strain of IL-10-/- mice. B-/-IL-10-/- mice acquired a severe colitis analogous to that IL-10-/- mice, implying that B cells were not the primary mediator of IBD in this model. A series of cell transfer experiments was performed to assess a pathogenic role for T cells. When IL-10-/- T cell-enriched lamina propria lymphocytes (LPL) or intraepithelial lymphocytes (IEL) were transferred into immunodeficient recombinase-activating gene (RAG)-2-/- recipients, a mild to severe colitis developed, depending on the cell number transferred. Lymphocytes recovered from the colon of transplanted RAG-2-/- mice with colitis were predominantly alpha beta TCR+CD4+, including a large proportion of CD4+CD8 alpha + cells. These cells were also CD45RB-/low and CD44+, indicative of an activated/memory population. Individual populations of CD4+CD8 alpha-, CD4+CD8 alpha + and CD4-CD8 alpha + T cells were then isolated from the lamina propria compartment of IL-10-/- mice and transferred into RAG-2-/- recipients. Only IL-10-/- CD4-expressing LPL, including both the CD4+CD8 alpha- and CD4+CD8 alpha + populations, induced colitis in recipient mice. Interferon-gamma, but little to no IL-4, was produced by CD4+CD8 alpha- and CD4+CD8 alpha + LPL recovered from the inflamed colons of RAG-2-/- recipients implicating alpha T helper cell 1 (TH1)-mediated response. We thus conclude that colitis in IL-10-/- mice is predominantly mediated by TH1-type alpha beta TCR+ T cells expressing CD4 alone, or in combination with the CD8 alpha molecule.

Antibody to interleukin-5 inhibits helminth-induced eosinophilia in mice.

When rodents are infected with the nematode Nippostrongylus brasiliensis, large numbers of eosinophils appear in their blood and lungs and their serum immunoglobulin E (IgE) is increased. Injection of a monoclonal antibody to interleukin-5 completely suppressed the blood eosinophilia and the infiltration of eosinophils in the lungs of parasitized mice but had no effect on serum IgE. In contrast, an antibody to interleukin-4 inhibited parasite-induced IgE but not the eosinophilia. These results show that interleukin-5 is important in eosinophil production in vivo and that IgE and eosinophil production are regulated by different cytokines produced by the TH2 subset of CD4-expressing T cells.

Regulation of expression of the interleukin-2 receptor on hematopoietic cells by interleukin-3.

Remarkable similarities in the intracellular and genetic events occur when lymphoid and hematopoietic cells are exposed to their specific growth factors. The interleukin-2 (IL-2) receptor, whose cell-surface expression is an absolute requirement for the growth and differentiation of lymphoid cells, was detected on various nonlymphoid hematopoietic cell types in this study. Cell lines consisting either of granulocyte-macrophage precursors or mast cells, which are dependent on interleukin-3 (IL-3) for their growth, expressed high levels of the IL-2 receptor on their surface. Analysis of the binding characteristics of these receptors with 125I-labeled recombinant IL-2 revealed that only receptors with low affinity for IL-2 were present on these cells. Addition of purified recombinant IL-3 to these cell lines led to an increase in IL-2 receptor gene expression within 1 hour in isolated nuclei. This IL-3--induced increase in the number of IL-2 receptors on the cell surface is maximal within 24 hours. Addition of 10,000 units of IL-2 to these cells had no apparent effect on their growth or differentiation. The presence of the receptor with only low affinity for IL-2 on hematopoietic cells and the regulation by IL-3 suggest that this receptor is involved in some important metabolic event in hematopoiesis.

Requirement for IL-13 independently of IL-4 in experimental asthma.

The pathogenesis of asthma reflects, in part, the activity of T cell cytokines. Murine models support participation of interleukin-4 (IL-4) and the IL-4 receptor in asthma. Selective neutralization of IL-13, a cytokine related to IL-4 that also binds to the alpha chain of the IL-4 receptor, ameliorated the asthma phenotype, including airway hyperresponsiveness, eosinophil recruitment, and mucus overproduction. Administration of either IL-13 or IL-4 conferred an asthma-like phenotype to nonimmunized T cell-deficient mice by an IL-4 receptor alpha chain-dependent pathway. This pathway may underlie the genetic associations of asthma with both the human 5q31 locus and the IL-4 receptor.

Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance.

Previous studies in vivo have shown that IL-10 infusion can prevent lethal endotoxic shock. Mice deficient in the production of IL-10 (IL10T) were used to investigate the regulatory role of IL-10 in the responses to LPS in three experimental systems. In a model of acute endotoxic shock, it was found that the lethal dose of LPS for IL10T mice was 20-fold lower than that for wild type (wt) mice suggesting that endogenous IL-10 determines the amount of LPS which can be tolerated without death. The high mortality rate of IL10T mice challenged with modest doses of LPS was correlated to the uncontrolled production of TNF as treatment with anti-TNF antibody (Ab) resulted in 70% survival. Additional studies suggested that IL-10 mediates protection by controlling the early effectors of endotoxic shock (e.g., TNF alpha) and that it is incapable of directly antagonizing the production and/or actions of late appearing effector molecules (e.g., nitric oxide). We also found that IL10T mice were extremely vulnerable to a generalized Shwartzman reaction where prior exposure to a small amount of LPS primes the host for a lethal response to a subsequent sublethal dose. The priming LPS dose for IL10T mice was 100-fold lower than that required to prime wt mice implying that IL-10 is important for suppressing sensitization. In agreement with this assumption, IL-10 infusion was found to block the sensitization step. Interestingly, IL-10 was not the main effector of endotoxin tolerance as IL10T mice could be tolerized to LPS. Furthermore, IL-10 infusion could not substitute for the desensitizing dose of LPS. These results show that IL-10 is a critical component of the host's natural defense against the development of pathologic responses to LPS although it is not responsible for LPS-induced tolerance.

Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4(+) TH1-like responses.

We have characterized the progressive stages of chronic intestinal inflammation that develops spontaneously in specific pathogen-free (SPF) mice with a targeted disruption in the IL-10 gene (IL-10-/-). Our longitudinal studies showed that inflammatory changes first appear in the cecum, ascending and transverse colon of 3-wk-old mutants. As the disease progressed, lesions appeared in the remainder of the colon and in the rectum. Some aged IL-10-/- mice also developed inflammation in the small intestine. Prolonged disease with transmural lesions and a high incidence of colorectal adenocarcinomas (60%) was observed in 6-mo-old mutants. Mechanistic studies have associated uncontrolled cytokine production by activated macrophages and CD4+ Th1-like T cells with the enterocolitis exhibited by IL-10-/- mice. A major role for a pathogenic Th1 response was further suggested by showing that anti-IFNgamma antibody (Ab) treatment significantly attenuated intestinal inflammation in young IL-10-/- mice. When weanlings were treated with IL-10, they failed to develop any signs of intestinal inflammation. Interestingly, IL-10 treatment of adults was not curative but did ameliorate disease progression. Our studies have also shown that inheritable factors strongly influence the disease susceptibility of IL-10-/- mice. In 3-mo-old mutants, intestinal lesions were most severe in IL-10-/- 129/SvEv and IL-10-/- BALB/c strains, of intermediate severity in the IL-10-/- 129 x C57BL/6J outbreds, and least severe in the IL-10-/- C57BL/6J strain.

Studies with IL-10-/- mice: an overview.

Our studies have elucidated, in part, the mechanism whereby persistent stimulation by normal enteric antigens leads to the development of chronic enterocolitis in interleukin 10-deficient (IL-10-/-) mice. This disease is mediated by IL-10-/- CD4+ T cells as evidenced by their ability to transfer colitis to immunodeficient RAG-2-/- mice. Furthermore, the CD4+ T cells recovered from the affected colons of IL-10-/- mice consisted of a highly polarized Th1-like population because they produced interferon-gamma (IFN-gamma) but not IL-4. We found that enterocolitis could be prevented if 3-week-old mutants were treated for 6-8 weeks with either anti-IL-12 or anti-IFN-gamma monoclonal antibodies (mAb). These results were consistent with the findings of in vitro studies suggesting that IFN-gamma and, in particular, IL-12 direct the differentiation of naive T cells toward a Th1 phenotype. Apparently, the uncontrolled production of IL-12 and IFN-gamma by accessory cells and T cells, respectively, in IL-10-/- mice ultimately resulted in the excessive generation and activation of Th1 cells, hence, immunopathology. IL-10-/- mice have also been used to evaluate the importance of IL-10 in regulating immune responses outside of the gastrointestinal (GI) tract. In these studies, IL-10-/- mice were challenged with a variety of foreign antigens using different routes of administration. In general, the results repeatedly demonstrated that a major function of IL-10 is to protect the host from the harmful side effects of an overly zealous immune-inflammatory response. However, other studies have confirmed speculations that the potent immunosuppressive activities of IL-10 may, under certain circumstances, increase the host's susceptibility to infection with certain types of pathogenic organisms.

The antibody response to a single antigenic determinant of the tobacco mosaic virus protein: analysis using monoclonal antibodies, mutant proteins and synthetic peptides.

Three hybridomas were selected which secreted monoclonal antibodies specific to a decapeptide determinant representing residues 103-112 of the tobacco mosaic virus protein ( TMVP ). A series of proteins from several strains of TMV which differ in the amino acid sequence in this region of the protein were used as probes for specificity analysis. The fine-specificity analysis was extended by assessing the binding of the antibodies with a panel of synthetic peptide analogues of the native decapeptide with amino acid substitutions at different locations. The binding of each synthetic peptide with each of the monoclonal antibodies was determined by the ability of the radiolabeled peptide to bind with the antibody. The binding of the decapeptide with antibodies was determined by equilibrium dialysis; the relative binding affinity of each peptide of the panel was determined by the capacity of the peptide to inhibit the binding between the antibody and the radiolabeled native decapeptide. The results demonstrated that a panel of synthetic peptide analogues constitutes a powerful tool for discerning the fine specificity of antibodies directed to a given determinant of a protein antigen. The data indicated that, although all of the antibodies recognized the same nominal decapeptide determinant, their binding with the different mutant proteins or with the synthetic peptides of the panel differed greatly, indicating dramatic differences in their fine specificity. The existence of such differences should be taken into consideration when assessing residues of a protein antigen that are involved in antibody binding. The differences which were found in monoclonal antibodies produced following immunization with the whole TMVP reflect differences which occur in heterogeneous serum antibody populations and point out the complexity of antigenic recognition even of as small an epitope as a decapeptide.

Radioprotective effects of flk2/flt3 ligand.

The radioprotective properties of flk2/flt3 ligand (FL) were evaluated in lethally irradiated mice. Optimum survival rates (70-80%) were observed when 5 to 20 microg of FL was administered at both 20 and 2 hours before LD100/30 radiation. Administration of FL well in advance of irradiation was essential for conferring most of the radioprotection, since a single dose given at -20 hours still resulted in a significant survival rate (65%), whereas a single dose given at -2 hours was relatively nonprotective. Histopathologic examination at 7 and 9 days postirradiation revealed significant myelopoietic activity in the bone marrow (BM) of FL-treated mice, suggesting that their survival might be due to sparing of radiosensitive hematopoietic cells. By comparison, the BM of mice treated with phosphate-buffered saline was extremely hypocellular and remained that way until they died of bacterial infection. Hematopoietic assays confirmed a marked stimulation of early white blood cell (WBC) recovery in the BM and blood of FL-protected mice relative to PBS-treated controls. By day 21, FL-protected mice showed circulating WBC numbers that were higher than preirradiation values; however, their BM colony-forming units in culture were still depressed. Moreover, these mice experienced a prolonged anemia and thrombocytopenia. These findings are discussed in light of the restricted subset of hematopoietic progenitors shown to be responsive to FL in vitro.

Interleukin-10 promotes the growth of megakaryocyte, mast cell, and multilineage colonies: analysis with committed progenitors and Thy1loSca1+ stem cells.

The growth-promoting activities of interleukin-10 (IL-10) were assessed in hematopoietic colony-forming assays. We found that IL-10 failed to support the clonal growth of normal and lineage-depleted (Lin-) bone marrow (BM) cells. Furthermore, IL-10 neither enhanced nor suppressed colony formation by eosinophil, neutrophil, or macrophage progenitors when combined with a variety of factors. IL-10 stimulated a modest increase in erythropoietin (Epo)-dependent erythroid colonies but had no effect on the burst-promoting activities of IL-3. However, the combination of IL-10 plus IL-3 resulted in the enhanced growth of mast cell progenitors. In addition to its mast cell stimulating activity, IL-10 promoted the growth of megakaryocyte (Mk) and Mk-mixed colonies when combined with Epo or with Epo plus IL-3, IL-6, or IL-11. Comparative studies showed that the megakaryocyte potentiating activity of IL-10 is roughly equivalent to that of IL-6 and IL-11. In experiments using Thy1loSca1+ stem cells, IL-10 was shown to enhance the number of cells initiating IL-3-dependent colony formation. IL-10 also costimulated increased colony formation when used with IL-3 and another factor such as IL-1, IL-6, and granulocyte colony-stimulating factor (G-CSF). Cellular analysis of the resulting colonies indicated that IL-10 increases the formation of multilineage colonies containing erythrocytes, megakaryocytes, and/or mast cells. The ability of IL-10 to cooperatively regulate various stages of hematopoietic development is discussed.

Anti-IL-6 antibodies suppress myeloid cell production and the generation of CFU-c in long-term bone marrow cultures.

Interleukin 6 (IL-6) is one of several hemopoietic growth factors produced by stromal cell lines derived from the adherent layer of long-term bone marrow cultures (LTBMCs). To evaluate the potential role of IL-6 in stromal cell-dependent myelopoiesis, we established LTBMCs and verified that IL-6 mRNA is transcribed by heterogeneous adherent cell layers and that IL-6 protein is present in culture supernatants. Established LTBMCs were then depleted of IL-6 by using a specific neutralizing monoclonal antibody (mAb). Cultures treated for 2-3 weeks with anti-IL-6 mAb showed decreased production of maturing myeloid cells and colony-forming progenitor cells (colony-forming units in culture, CFU-c) but not stem cells (spleen colony-forming units, CFU-s). In parallel experiments, it was also found that the addition of IL-6 to LTBMCs stimulated a marked increase in total cell production, CFU-c, and day-8 CFU-s. In sum, it appears that endogenous production of IL-6, although limiting, is essential for the normal level of myelopoiesis associated with stromal cell function in LTBMCs.

Chronic colitis in IL-10-/- mice: insufficient counter regulation of a Th1 response.

IL-10-deficient (IL-10-/-) mice, generated by a gene-targeted mutation, develop abnormal immune responses as a result of uncontrolled interactions between antigen presenting cells and lymphocytes. The studies reviewed herein have focused on the enterocolitis that spontaneously develops in IL-10-/- mice. Not unexpectedly, heightened production of proinflammatory mediators accompanied pathologic changes in the gastrointestinal tract of young mutants. In a series of studies, the proinflammatory mediators responsible for initiating the pathogenic response were distinguished from those that were elicited as a consequence of persistent inflammation. We have also investigated the possibility that different mediators are involved in the inductive versus the maintenance phase of disease. The findings of these mechanistic studies as they relate to our understanding of progressive inflammatory disease and the role of IL-10 in controlling the acute and chronic stages are discussed.

Accelerated alveolar bone loss in mice lacking interleukin-10.

Interleukin-10 regulates pro-inflammatory cytokines, including those implicated in alveolar bone resorption. We hypothesized that lack of interleukin-10 leads to increased alveolar bone resorption. Male interleukin-10(-/-) mice, on 129/SvEv and C57BL/6J background, were compared with age-, sex-, and strain-matched interleukin-10(+/+) controls for alveolar bone loss. Immunoblotting was used for analysis of serum reactivity against bacteria associated with colitis and periodontitis. Interleukin-10(-/-) mice had significantly greater alveolar bone loss than interleukin-10(+/+) mice (p = 0.006). The 30-40% greater alveolar bone loss in interleukin-10(-/-) mice was evident in both strains, with C57BL/6J interleukin-10(-/-) mice exhibiting the most bone loss. Immunoblotting revealed distinct interleukin-10(-/-) serum reactivity against Bacteroides vulgatus, B. fragilis, Prevotella intermedia, and, to a lesser extent, against B. forsythus. The results of the present study suggest that lack of interleukin-10 leads to accelerated alveolar bone loss.

Immunochemical studies on the tobacco mosaic virus protein.

The decapeptide having the amino acid sequence Thr-Thr-Ala-Glu-Thr-Leu-Asp-Ala-Thr-Arg has been shown to be a major antigenic determinant of the tobacco mosaic virus protein in rabbits, mice and guinea pigs. The antigenic specificity of the decapeptide is attributed to its C-terminal tripeptide Ala-Thr-Arg. Although this tripeptide has no demonstrable binding with antibodies to the protein, its N-octanoylated derivative exhibits specific binding with antibodies as well as the capacity to elicit delayed skin reactions in guinea pigs immunized with the protein. The latter results suggest that both B cells and T cells have antigen receptors of identical specificities. Although all mouse strains tested responded equally to TMVP, with the production of anti-protein, the response to the decapeptide was shown to be correlated (albeit not absolutely) with Ig allotype Iga exhibiting generally high responsiveness while Igb exhibiting generally low responsiveness. The low responsiveness could not be attributed to suppression of the secondary immune response.