Stromal Macrophages Research Articles

Regulation of interleukin-6, and macrophage colony-stimulating factor mRNA levels by histamine in stromal cell line (MC3T3-G2/PA6).

The aim of this study was to examine the role of histamine in regulation of IL-6 and M-CSF gene expression in bone marrow stromal cells. Total cellular synthesis was also estimated for IL-6. The effects of the amine were evaluated using the stromal cell line, MC3T3-G2/PA6 (PA6). Histamine caused a distinct accumulation of IL-6 mRNA in the cells, whereas it decreased M-CSF transcripts. Both pyridylethylamine, a H1 agonist, and dimaprit, a H2 agonist, caused a large increase in the level of IL-6 mRNA in PA6 cells. The histamine-induced expression of IL-6 mRNA was associated with enhanced secretion of IL-6, as determined by ELISA. These results, together with the results of our previous studies, suggest that histamine produced by stromal macrophages differentially regulates the production of IL-6 and M-CSF in other kinds of stromal cells and hence promotes differentiation and/or proliferation of hematopoietic progenitor cells.

Induction of inducible nitric oxide synthase expression in human secretory endometrium.

The endometrial secretory phase is characterized by stromal oedema, a premenstrual increase in stromal macrophages and an increased cytokine production as menstruation approaches. Nitric oxide (NO) is a mediator of vasodilatation and cytotoxicity which is synthesized from L-arginine by NO synthases (NOS). These enzymes are either constitutively expressed or induced by lipopolysaccharides and/or cytokines. The presence and function of the inducible isoform of NOS (iNOS) in normal human endometrium has not been fully elucidated until recently. Frozen tissue sections taken from 22 women who underwent hysterectomy and adnexectomy for benign disease were immunostained with antibodies raised against the different NOS isoforms to investigate the presence of NOS in human endometrium. iNOS stained positive in the glandular epithelial cells of the secretory endometrium. Staining was either weak or absent in the proliferative and inactive endometrium, as well as in the oviduct and the glandular epithelium of the endocervix. The stroma remained uniformly negative. Immunoreactivity for endothelial constitutive NOS (eNOS) was confined exclusively to endothelial cells. Furthermore, epithelial cells from endometrium, oviduct and endocervix and all endothelial cells showed positive staining for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, which is a histochemical marker for NOS activity. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed in order to assess the presence of NOS mRNA. Abundant expression of iNOS mRNA was detected in the secretory phase endometrium only. The strong expression of inducible NO synthase in human secretory phase endometrium suggests that the increased production of NO, probably induced by cytokines, may be relevant to the process of menstruation.

Role of nitric oxide in tumour progression: lessons from human tumours.

Varied cellular expression and localisation of nitric oxide synthase (NOS) isoforms has been shown in human cancers, including tumours of the breast, ovary, stomach, cervix and central nervous system. Mapping of NOS expression within tumour tissue from breast and gastric cancers shows inducible NOS (iNOS) is expressed predominantly in stromal (macrophage and endothelial) cells, although the level of NOS activity is at least 1-2 orders of magnitude lower than the enzyme activity associated with cytotoxicity and apoptosis. There is evidence that the intratumoural environment may provide chemoattractant signals for monocyte-macrophage recruitment and their subsequent activation via expression of interleukin-4, IgE, and CD23. Such signals lead to induction of iNOS in human macrophages in vitro. The correlation between NOS activity and grade for breast cancer suggests that NO may provide a positive growth signal within the tumour microenvironment. In vivo studies showing increased growth rate, vascular density and invasiveness of a human tumour cell line transfected to constitutively express iNOS support this. Furthermore, in vivo administration of a highly selective inhibitor of iNOS limited invasion and growth rate of iNOS transfected tumours and other murine tumours expressing this isoform. Inhibition of NO generation in the intratumoural microenvironment may prove a useful cancer therapy by preventing angiogenesis, invasion and metastasis.

Establishment and characterization of murine bone marrow stromal macrophage line

The authors established a murine bone marrow stromal cell line QXMSC1 by discarding the suspensible hematopoietic cells and passaging the adherent stromal cells many times in long-term bone marrow culture. QXMSC1 cells have been passaged 65 times for 15 months and immortalized. The mean number of QXMSC1 chromosomes is 66 ± 4. The appearance of the cells is elliptical. There are many pseudopods and mononuclei under the optical microscope. Many lisosomes and phagosomes in cytoplasm exist under the transmission electron microscope. There are no desmosome junction between the cells, no lipid drops, no intermediate filament. Vimentin is positive and keratin is negative by stain of immunocytochemistry. Nonspecific lipase staining is positive. These results indicate that QXMSC1 cells are murine bone marrow macrophages.

Conjunctival Fibrosis in Ocular Cicatricial Pemphigoid—the Role of Cytokines

Ocular cicatricial pemphigoid (OCP) is a systemic, autoimmune disease characterised by conjunctival scarring that is often progressive. The pathophysiology of the fibrosis is unknown. This study aimed to determine which fibrogenic cytokines are present in the conjunctiva in patients with acute and chronic OCP as a first stage in determining the mechanisms of fibrosis.Conjunctival biopsies from patients with acute, subacute and chronic OCP (n=13) were compared to normal conjunctiva (n=10). Production of mRNA for, and expression of, transforming growth-β1, 2 and 3 (TGF-β), TGF-β receptor, platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) were assessed using in situ hybridisation and immunohistochemistry.Acute disease showed increased levels of mRNA for TGF-β1 and 3, mainly in stromal fibroblasts and macrophages. In the stroma, there were concordant increases in latent and activated TGF-β1 and 3 and TGF-β receptor expression by fibroblasts. There were no significant increases in the expression of TGF-β2, PDGF or FGF in acute disease. No cytokines or receptors were significantly increased in chronic disease.Acutely inflamed conjunctiva in OCP is associated with significant stromal levels of TGF-β1 and 3 but not PDGF or FGF and none were increased in chronic disease. This suggests that TGF-β may have a key role in the pathogenesis of the fibrosis. The absence of fibrogenic cytokines in chronic progressive OCP provides support for the proposal that fibroblasts in OCP conjunctiva may remain functionally and morphologically abnormal after the withdrawal of cytokine influences.

Early complete hydatidiform moles contain inner cell mass derivatives

In four cases of early complete hydatidiform moles, confirmed to be androgenetic in origin by DNA studies, we have identified nonchorionic inner cell mass derived structures which are not commonly observed in specimens of later gestational age. These structures include nucleated red blood cells, endothelial cells, stromal macrophages, amnion and yolk sac. The latter four structures were confirmed by specific immunocytochemical stains. Recognition that such structures can accompany complete hydatidiform moles has both theoretical and practical significance. From a theoretical perspective, it demonstrates that the maternal genome is not required for the initiation of amniogenesis, development of the yolk sac, vasculogenesis, or hematopoiesis. From a practical perspective it emphasizes that complete hydatidiform moles, with their markedly increased risk of subsequent choriocarcinoma, cannot be excluded based on the finding of "fetal structures."

Electron microscopic study of erythroblastic islands obtained by 'tissue-stamp culture' method.

A new 'tissue-stamp culture' method was developed for stamping proliferating erythroblasts of mouse spleens on collagen-coated coverslips after inducing haemolytic anaemia by administration of 1-acetyl-2-phenylhydrazine, and then adherent splenic cells were cultured for a few days. We could obtain many erythroblastic islands, where cultured erythroblasts were located over macrophages and were proliferated synchronously for 10-30 h, and then the erythroblasts were differentiated and enucleated after 30-50 h in the presence of erythropoietin. To observe three-dimensional structures of the erythroblastic islands, a scanning electron microscope was used for the cultured cells treated with critical point-drying method. Immature wrinkled erythroblasts with many micropinocytic pits were attached to the central area of the flattened macrophages with many cytoplasmic projections, though matured erythroblasts were localized on their peripheral areas. Moreover, cytoplasmic projections of underlying macrophages, which were attached to the matured erythroblasts, were decreased in number. At a late stage, deep cytoplasmic invaginations of erythroblasts observed at a middle stage became shallow after their enucleation and flattened to form their concave shapes. This 'tissue-stamp culture' system would be useful for studying specific interaction between stromal macrophages and haematopoietic cells.

Phyllodes tumor. Fine needle aspiration cytology of eight cases.

To elucidate the fine needle aspiration cytology (FNAC) features of phyllodes tumor (PT). Eight FNAC cases of PT (five benign and three malignant) were reviewed. Features examined were cellularity, epithelial/stromal ratio, phyllodes fragments (PFs), blood vessels crossing PFs, bipolar naked nuclei, stromal pleomorphism, stromal cell size, mitosis, macrophages, sarcomatous component and atypical epithelial component. Benign PTs were characterized by cellular smears with both stromal and epithelial elements. All cases had many highly cellular stromal fragment PFs. Stromal cells were twice the size of a lymphocyte and lacked pleomorphism. Bipolar naked nuclei were a constant feature. The epithelial component predominated, demonstrating benign ductal groups and cellular areas with discohesion superficially mimicking adenocarcinoma in three cases. FNAC of primary malignant PT (one case) had two patterns, one mimicking benign PT and the other demonstrating a pure sarcomatous component. FNAC of metastatic PTs (two cases) showed pure sarcomatous elements. The diagnosis of PT is favored over fibroadenoma when many PFs are present. The size and shape of the stromal cell nuclei were not helpful differentiating features in this study. The diagnosis of malignant PT was based on the purely sarcomatous component.

P-Benzoquinone, a Reactive Metabolite of Benzene, Prevents the Processing of Pre-Interleukins-1a and -1b to Active Cytokines by Inhibition of the Processing Enzymes, Calpain, and Interleukin-1b Converting Enzyme

Chronic exposure of humans of benzene affects hematopoietic stem and progenitor cells and leads to aplastic anemia. The stromal macrophage, a target of benzene toxicity, secretes interleukin-1 (IL-1), which induces the stromal fibroblast to synthesize hematopoietic colony-stimulating factors. In a mouse model, benzene causes an acute marrow hypocellularity that can be prevented by the concomitant administration of IL-1 alpha. The ability of benzene to interfere with the production and secretion of IL-1 alpha was tested. Stromal macrophages from benzene-treated mice were capable of the transcription to the IL-1 alpha gene and the translation of the message but showed an inability to process the 34-kDa pre-IL-1 alpha precursor to the 17-kDa biologically active cytokine. Treatment of normal murine stromal macrophages in culture with hydroquinone (HQ) also showed an inhibition in processing of pre-IL-1 alpha. Hydroquinone is oxidized by a peroxidase-mediated reaction in the stromal macrophage to p-benzoquinone, which interacts with the sulfhydryl (SH) groups of proteins and was shown to completely inhibit the activity of calpain, the SH-dependent protease that cleaves pre-IL-1 alpha. In a similar manner, HQ, via peroxidase oxidation to p-benzoquinone, was capable of preventing the IL-1 beta autocrine stimulation of growth of human B1 myeloid tumor cells by preventing the processing of pre-IL-1 beta to mature cytokine. Benzoquinone was also shown to completely inhibit the ability of the SH-dependent IL-1 beta converting enzyme. Thus benzene-induced bone marrow hypocellularity may result from apoptosis of hematopoietic progenitor cells brought about by lack of essential cytokines and deficient IL-1 alpha production subsequent to the inhibition of calpain by p-benzoquinone and the prevention of pre-IL-1 processing.

P-Benzoquinone, a reactive metabolite of benzene, prevents the processing of pre-interleukins-1 alpha and -1 beta to active cytokines by inhibition of the processing enzymes, calpain, and interleukin-1 beta converting enzyme.

Chronic exposure of humans of benzene affects hematopoietic stem and progenitor cells and leads to aplastic anemia. The stromal macrophage, a target of benzene toxicity, secretes interleukin-1 (IL-1), which induces the stromal fibroblast to synthesize hematopoietic colony-stimulating factors. In a mouse model, benzene causes an acute marrow hypocellularity that can be prevented by the concomitant administration of IL-1 alpha. The ability of benzene to interfere with the production and secretion of IL-1 alpha was tested. Stromal macrophages from benzene-treated mice were capable of the transcription to the IL-1 alpha gene and the translation of the message but showed an inability to process the 34-kDa pre-IL-1 alpha precursor to the 17-kDa biologically active cytokine. Treatment of normal murine stromal macrophages in culture with hydroquinone (HQ) also showed an inhibition in processing of pre-IL-1 alpha. Hydroquinone is oxidized by a peroxidase-mediated reaction in the stromal macrophage to p-benzoquinone, which interacts with the sulfhydryl (SH) groups of proteins and was shown to completely inhibit the activity of calpain, the SH-dependent protease that cleaves pre-IL-1 alpha. In a similar manner, HQ, via peroxidase oxidation to p-benzoquinone, was capable of preventing the IL-1 beta autocrine stimulation of growth of human B1 myeloid tumor cells by preventing the processing of pre-IL-1 beta to mature cytokine. Benzoquinone was also shown to completely inhibit the ability of the SH-dependent IL-1 beta converting enzyme. Thus benzene-induced bone marrow hypocellularity may result from apoptosis of hematopoietic progenitor cells brought about by lack of essential cytokines and deficient IL-1 alpha production subsequent to the inhibition of calpain by p-benzoquinone and the prevention of pre-IL-1 processing.

A novel bone marrow frozen section assay for studying hematopoietic interactions in situ: the role of stromal bone marrow macrophages in erythroblast binding.

Bone marrow macrophages are found in intimate contact with erythroblasts. Exact mechanisms and functions of this interaction are unclear. New insights into erythroblast binding were obtained using a newly developed bone marrow frozen section assay. This modified Woodruff and Stamper assay has some important advantages compared to other adhesion assays. Erythroblasts specifically adhered to bone marrow macrophages forming clusters, as appear in vivo. Selective depletion of bone marrow macrophages by intravenous injection of dichloromethyl-enediphosphonate resulted in a release of immature erythroid cells to the peripheral blood. Furthermore no erythroblasts adhered to bone marrow sections without macrophages. Evaluating the binding of erythroblasts to bone marrow macrophages we found that this binding is temperature and cation dependent. The receptor for erythroblasts present on macrophages recognizes a sialyated protein as ligand on erythroblasts, since neuraminidase treatment of erythroblasts resulted in a decrease in binding. A possible candidate for the erythroblast receptor on macrophages is the ED2 antigen. ED2 is a differentiation antigen present on resident macrophages that has some biochemical features characteristic of an adhesion molecule. Erythroblast binding to bone marrow was inhibited using a monoclonal antibody directed against ED2.

The outcome of the parasitic process initiated by Leishmania infantum in laboratory mice: a tissue-dependent pattern controlled by the Lsh and MHC loci.

Human visceral leishmaniasis is mainly due to intracellular protozoan parasites of the Leishmania donovani complex, i.e., L. donovani and L. infantum (or L. chagasi). A mouse model has been established to monitor 1) the parasitic process initiated by L. infantum in three tissues they invade, and 2) parameters of the acquired immune response they trigger. Mice congenic at the Lsh locus and mice of inbred strains differing at the MHC locus have been inoculated by the i.v. route with L. infantum. The parasitic process has been evaluated by the follow-up of the parasitic load in the liver, the spleen, and, for the first time, in the bone marrow using a very sensitive limiting dilution assay. As previously established for L. donovani, the early outcome of L. infantum is also under the control of the Lsh locus in the liver; genes of the MHC complex are involved in the development of the subsequent acquired immune response. "Cure" or "noncure" haplotypes are the same for the two species of Leishmania; as far as the cure haplotype is concerned, whatever the tissues being screened, the parasites are never totally cleared, although the liver is the tissue in which the best parasite load reduction is achieved. Through immunostaining, it was established that sialoadhesin-positive stromal bone marrow macrophages contain parasites; such long-lived mononuclear phagocytes could be the host cells where the parasite can find "safe targets" unreactive to the dominant effector immune mechanism triggered by the replicative stage of the parasites.

The expression of fetuin in the development and maturation of the hemopoietic and immune systems.

The distribution and expression of fetuin, a fetal plasma protein that has been shown to have a wide-spread intracellular presence in many developing tissues including the central nervous system, has been studied in the developing immune and hemopoietic organs of fetal and adult sheep. The presence of fetuin was demonstrated using immuno-cytochemistry and expression of fetuin was studied using northern blot analysis and in situ hybridization. In the developing sheep fetus, fetuin was shown to be expressed first in the hemopoietic cells of the fetal liver and subsequently in the forming spleen. The very first stromal, bone marrow-forming cells, also expressed fetuin mRNA. These cells became more numerous during gestation and by embryonic day (E)115 (term is 150 days), fetuin-expressing cells were identified morphologically to be monocytes/macrophages. Fetuin protein, on the other hand, was present in all hemopoietic and immune organs from the earliest age studied (E30) but was confined initially to matrix, mesenchymal tissue. Fetuin-positive cells could be identified in the spleen at E60 as early hemopoietic cells, in the lymph nodes at E60 as stromal cells and macrophages, and at E115 in the thymus as macrophages and squamous cells. In the adult, fetuin mRNA was only detectable by northern blot in the liver and the bone marrow. Using in situ hybridization in adult tissue, fetuin mRNA-positive cells were identified in the bone marrow to be monocytes/macrophages. Additionally, in the spleen germinal centres, fetuin mRNA was identified in cells with the morphology of dendritic cells. Using three separate cellular markers: lysozyme, S-100, and alpha 1-antitrypsin, the cellular identification of fetuin-positive cells was confirmed to be in the monocyte/macrophage lineage.

Depletion of stromal and intraepithelial antigen-presenting cells in cervical neoplasia in human immunodeficiency virus infection

Human immunodeficiency virus-positive (HIV+) women have an increased risk of lower genital tract dysplasia and neoplasia, and studies of the central lymphoid system suggest that impaired immunosurveillance plays a role in the development of their cervical tumors. Intraepithelial and stromal immunocompetent cell counts were compared in cervical specimens from 50 HIV+ and 50 appropriately matched HIV-women (controls) with low and high grade squamous intraepithelial lesions (SIL), or carcinoma. Each histological class of HIV+ women displayed fewer intraepithelial Langerhans' (S100+) cells (LC) (as already known), and also fewer stromal LC and both intraepithelial and stromal (CD68+) macrophages. LC and macrophages were reduced in all HIV+ patients, whereas reduction of cervical T lymphocytes was found in only immunocompromised subjects (peripheral blood CD4+ T-cell count <500/μL). A mucosal quantitative deficiency of antigen-presenting cells (APC) thus precedes that of T cells. HIV infection appears to lead to early impairment of mucosal immunoreactivity mainly because of defective antigen presentation. This impairment may be one mechanism underlying the increased frequency of cervical dysplasia/neoplasia, and the enhanced aggressiveness of invasive cancers in HIV+ women.

Molecular Interactions between Human B-Cell Progenitors and the Bone Marrow Microenvironment

Apoptosis of normal and leukemic immature B-cellsin vitrois suppressed when either cell type is grown in direct contact with a feeder layer of bone marrow-derived stromal cells, including fibroblasts, macrophages, endothelial cells, and adipocytes. In this study, our objective was to identify a stromal cell type which is essential for lymphoblast survival and to characterize the molecules involved in lymphoblast adhesion to these cells. In experiments with B-lineage acute lymphoblastic leukemia (ALL) cells (n= 28) and purified CD19+cells from normal bone marrow (n= 6) we found that homogeneous populations of bone marrow fibroblasts could sustain survival of normal and leukemic immature B-cells as efficiently as composite bone marrow stromal layers. Electron microscopic studies showed that leukemic lymphoblasts associate with fibroblasts and with the extracellular matrix (ECM) primarily via their specialized cell surface structures. Immunogold labeling/electron microscopy analysis revealed that the areas of contact between lymphoblasts and fibroblasts contained β1 integrins (VLA-4 and VLA-5), fibronectin, vascular cell adhesion molecule (VCAM-1), and a cartilage-link protein, CD44. Double immunogold labeling studies disclosed a directin siturelationship between fibronectin and VLA-4, VLA-5, and CD44. We hypothesize that these molecular interactions either bring lymphoblasts into close physical proximity with other fibroblast-bound or ECM-bound survival factors or provide survival signals themselves.

Interferon-y Inhibits the Synthesis and Release of Renin from Human Decidual Cells1

Experiments were performed to examine the effect of interferon-gamma (IFN gamma) on the expression of renin by human uterine decidual cells and decidual macrophages. Exposure of a mixed population of decidual cells consisting of 80% decidualized stromal cells and 20% macrophages to IFN gamma for 4 days caused a dose-dependent inhibition of renin release beginning 2 days after exposure. Renin release on Day 4 was inhibited by a maximum of 83.9%, and the half-maximal effective dose of IFN gamma was 5 ng/ml (290 pM). The inhibition of renin release in response to IFN gamma was accompanied by a comparable inhibition of renin mRNA levels. In addition to inhibiting basal renin expression, IFN gamma potentiated the inhibitory effect of tumor necrosis factor alpha (TNF alpha) on renin expression. IFN gamma also inhibited basal renin release and potentiated the inhibitory effect of TNF alpha by highly purified populations of decidual stromal cells and decidual macrophages prepared by immunomagnetic separation with beads coupled to an anti-human leukocyte antigen (HLA-DR) antibody that binds macrophages but not stromal cells. Reverse transcription-polymerase chain reaction analysis showed that HLA-DR(+) cells express IFN gamma mRNA, and that both HLA-DR(+) and HLA-DR(-) cells express IFN gamma receptors. Since IFN gamma is expressed only by decidual macrophages, the results of this study strongly suggest that IFN gamma inhibits the expression of decidual renin by a paracrine action.

Immunophenotyping of human bone marrow-derived macrophages.

In vitro cultured cells derived from human bone marrow stimulated with macrophage-colony stimulating factor (M-CSF) or granulocyte-macrophage-colony stimulating factor (GM-CSF) were investigated for their immunophenotypic surface characteristics by flow cytometry. Approximately 80-90% of the cells showed morphological and histochemical features of macrophages and bore CD11a, CD11b, CD11c, CD14, CD29, CD32, CD33, CD44, CD45, CD54, CD64, CD71, HLA-DR antigen. The expression of CD4, CD25 and CD45RO were detected only in low density. Bone marrow-derived macrophages were negative for CD8, CD45RA, CD16 and CD23. Functionally, macrophages were able to phagocytose opsonized Escherichia coli, but no oxidative bursts were induced either by fmlp or by opsonized bacteria. Cell surface phenotyping revealed a CD pattern very similar to monocyte-derived phagocytes, and was partially distinct from resident stromal macrophages.

Preparation of a population of macrophages from human third trimester decidua.

Human decidua in the third trimester of pregnancy contains at least four different cell types (stromal cells, macrophages, T-lymphocytes and granulocytes). The contributions of these cells to the overall function of the tissue are not known, although it is anticipated that both stromal cells and macrophages will produce the prostaglandins involved in labour. In addition, the decidua produces cytokines, but the cellular source has not been identified. As a first stage to addressing these questions, we have developed magnetic separation methods to obtain enriched populations of decidual macrophages which contain at least 5 x 10(6) cells. We found that a preparation of up to 10(7) cells which are up to 80% pure (as assessed by a fluorescence-activated cell sorting procedure) and approximately 90% pure (as assessed by immunocytochemistry) can be obtained reliably with this method. These cells are viable for at least 48 h in culture after the enrichment procedure, and produce prostaglandins after stimulation with interleukin-1 beta.

Benzene-induced bone marrow cell depression caused by inhibition of the conversion of pre-interleukins-1alpha and -1beta to active cytokines by hydroquinone, a biological reactive metabolite of benzene.

Benzene (BZ), a widely used industrial chemical and a ubiquitous environmental pollutant, is a hematotoxin that causes bone marrow (BM) cell depression in experimental animals and aplastic anemia in humans that are chronically exposed1-3. Marrow stromal macrophage (SO) dysfunction and deficient interleukin-1 (IL-1) production have been demonstrated in aplastic anemia. Markedly depressed IL-1 production was observed in adherence-separated, LPS-stimulated monocytes from 75 to 80 percent of patients with severe aplastic anemia compared with normal control subjects4,5. The SO is involved in hematopoietic regulation6–8 through the synthesis of several cytokines including IL-1, which synergizes with IL-3 to promote the development of the pluripotent stem cell to myeloid and lymphoid stem cells9. IL-1 is also involved in lymphocyte development and in the induction of cytokine production by stromal fibroblasts8,10,11. Inhibition of the production of active IL-1 in SO could result in a lack of cytokines, increased apoptosis of hematopoietic progenitor cells and thus aplastic anemia. Two IL-1 cytokines, IL-1 α and IL-1 β, are the products of distinct genes located on chromosome 212. The transcript of each gene is translated as a precursor protein of approximately 34 kDa that is converted by a specific protease to a biologically active cytokine of 17 kDa13. The processing of pre-IL-1α to mature cytokine is catalyzed by the calcium-activated, cysteine protease, calpain14,15. The release of IL-1β from the cell is also associated with the cleavage of its precursor form by a sulfhydryl-dependent protease referred to as IL-1β convertase (ICE)16–18.

Inhibition of the conversion of pre-interleukins-1α and 1β to mature cytokines by p-benzoquinone, a metabolite of benzene

Chronic exposure of humans to benzene causes severe bone marrow cell depression leading to aplastic anemia. Marrow stromal macrophage dysfunction and deficient interleukin-1 production has been reported for patients with severe aplastic anemia. The stromal macrophage, a target of benzene toxicity, is involved in hematopoietic regulation through the synthesis of several cytokines including interleukin-1, which is required for production by stromal fibroblasts of a number of cytokines required for the survival of hematopoietic progenitor cells. We have previously demonstrated that hydroquinone, a major toxic metabolite of benzene in marrow, prevents the proteolytic conversion of 31 kDa pre-interleukin-1α to the 17 kDa cytokine by calpain in purified murine stromal macrophages. Furthermore, stromal macrophages from benzene-treated mice produce the 31 kDa pre-interleukin-1α when stimulated in culture with endotoxin, but cannot convert the precursor to interleukin-1α. In this report, we show that 1,4-benzoquinone, the oxidation product of hydroquinone in the cell, causes a concentration-dependent inhibition of highly purified human platelet calpain with an IC 50 of 3 μM. Hydroquinone also inhibits the processing of pre-interleukin-1β by interleukin-1β convertase. The addition of 2 μM hydroquinone to B1 cells that undergo autocrine stimulation by interleukin-1β resulted in the cessation of autocrine cell growth and interleukin-1β secretion into the culture medium, as determined by Western immunoblots of the culture supernatants. Purified converting enzyme treated with 3 μM benzoquinone was incapable of converting 31 kDa recombinant pre-interleukin-1β to the 17 kDa mature cytokine as analyzed by polyacrylamide gel electrophoresis and Western immunoblotting. These findings support our observations in a mouse model that benzene-induced bone marrow cell depression results from a lack of interleukin-1α subsequent to an inhibition by benzoquinone of calpain, the protease required for converting pre-interleukin-1α to active cytokine. The results may provide a basis for studying benzene-induced aplastic anemia in a mouse model.