Transforming growth factor beta 1 systemically modulates granuloid, erythroid, lymphoid, and thrombocytic cells in mice.

Transforming growth factor beta 1 (TGF-beta 1) has been shown to inhibit the development of most early hemopoietic progenitors in vitro. The present series of in vivo experiments show that TGF-beta 1 can simultaneously augment and suppress distinct cell lineages in peripheral and central hemopoietic compartments. Mice treated daily for 7-14 days with s.c. injections of TGF-beta 1 exhibited up to a 95% reduction in circulating platelets and a 50% reduction in red cell counts, whereas a 50%-400% increase occurred in circulating white cells with the morphology of small lymphocytes. Decreased erythrocytes were also evident in the splenic red pulp and bone marrow sinusoids. A dramatic increase in granulopoiesis occurred in the spleen and bone marrow, followed by a peripheral neutrophilia 1 week after treatments ceased. All effects were completely reversible, with normal histologic and hematologic profiles evident 2 weeks after cessation of treatments. Thus, TGF-beta 1 can differentially regulate multiple hemopoietic pathways in a systemic, reversible, and dose-dependent fashion. These actions may be mediated by the direct effects of TGF-beta 1 or through modulation of secondary cytokines and receptors.

Transforming growth factor-beta in leishmanial infection: a parasite escape mechanism.

The course of infection with the protozoan parasite Leishmania is determined in part by their early replication in macrophages, the exclusive host cells for these organisms. Although factors contributing to the survival of Leishmania are not well understood, cytokines influence the course of infection. Transforming growth factor-beta (TGF-beta) is a multipotential cytokine with diverse effects on cells of the immune system, including down-regulation of certain macrophage functions. Leishmanial infection induced the production of active TGF-beta, both in vitro and in vivo. TGF-beta was important for determining in vivo susceptibility to experimental leishmanial infection.

Differential modulation of transforming growth factor-beta 1 expression and mucin deposition by retinoic acid and sodium lauryl sulfate in human skin.

Immunohistochemical staining of skin sections with two polyclonal antibodies (anti-CC 1-30 and anti-LC 1-30), specific for transforming growth factor-beta 1, revealed increased extracellular and decreased intracellular expression of transforming growth factor-beta 1 in retinoic acid-treated, compared to vehicle-treated, skin. Transforming growth factor-beta 1 staining, with both antibodies, was most marked in the upper layers of the epidermis, although dermal staining was also evident. The modulation of transforming growth factor-beta 1 expression by retinoic acid occurred in the absence of any change in its mRNA level. Transforming growth factor-beta 1 protein, as detected by rabbit polyclonal antibody (anti-LC 50-75) and mRNA, were only minimally detected in either retinoic acid- or vehicle-treated skin. Similar changes in TGF-beta 1 and TGF-beta 2 immunoreactivity and mRNA levels, as observed in retinoic acid-treated skin, were observed in skin following topical application of the irritant sodium lauryl sulfate, indicating that the alterations induced by retinoic acid were not specific. In contrast, mucin deposition, which is induced by transforming growth factor-beta, was elevated in retinoic acid-treated but not sodium lauryl sulfate-treated skin. Cultured adult human keratinocytes also expressed predominantly transforming growth factor-beta 1 protein, as measured by ELISA, and mRNA. Treatment of keratinocytes with retinoic acid resulted in a 50% induction of transforming growth factor-beta 1 protein, without any detectable change in transforming growth factor-beta 2. These data demonstrate disassociation of modulation of transforming growth factor-beta 1 expression and mucin deposition by retinoic acid and sodium lauryl sulfate in human skin in vivo. Whereas alterations in transforming growth factor-beta 1 expression were observed in both retinoic acid- and sodium lauryl sulfate-treated skin, accumulation of mucin was specific to retinoic acid-treated skin.

Suppressive effects of monocytic cells and transforming growth factor-beta on natural killer cell differentiation in autoimmune viable motheaten mutant mice.

Viable motheaten (mev) mice are homozygous for a recessive single gene mutation at chromosome 6. These mice develop numerous inflammatory and arthritic syndromes and exhibit abnormal B cell functions as well as lower T and NK cell activity. In this study, the differentiation of NK cells in mev mice was examined to elucidate the underlying basis for decreased NK activity. Although NK cells appear to be present in mev mice, their activity was demonstrable only when the spleen cells were enriched by nylon wool passage. Similarly bone marrow cells from these mice could be shown to contain precursors of NK cells when they were passed over nylon wool and transplanted into irradiated recipients. The adherent cells from both the spleen and bone marrow of mev mice suppressed the differentiation of NK cells from normal splenic populations. These suppressive adherent cells were F4/80(+), AsGm-1(+), Qa-5(+), and NK-1.1(+). They were not cytolytic when cultured in IL-2. Antibodies to a number of cytokines, such as IFN-alpha, -beta, and gamma, or TNF-alpha, could not reverse the suppressive effect of the adherent cells. Addition of anti-TGF-beta antibody could, however, overcome the suppression, suggesting that TGF-beta was partly responsible for the defective NK differentiation in the mev mice.

Stimulation of granulopoiesis by transforming growth factor beta: synergy with granulocyte/macrophage-colony-stimulating factor.

Transforming growth factor beta 1 (TGF-beta 1) is known to inhibit the growth of immature hematopoietic progenitor cells, whereas more mature, lineage-restricted progenitors are not inhibited. In contrast, in the presence of saturating concentrations of granulocyte/macrophage-colony-stimulating factor (GM-CSF), TGF-beta promoted a 3- to 5-fold increase in the number and size (greater than 0.5 mm) of bone marrow colonies in a dose-dependent manner with an ED50 of 10-20 pM; TGF-beta 1 alone had no effect. Morphological examination showed an increase in granulocyte colonies. In suspension cultures, TGF-beta 1 and GM-CSF stimulated an increase in total viable cells with markedly enhanced neutrophilic differentiation and a concomitant decrease in the number of monocytes/macrophages by day 6 in culture. Limiting dilution analysis demonstrated a 2- to 5-fold increase in the frequency of progenitor cells that responded to GM-CSF plus TGF-beta 1 vs. GM-CSF alone. Bone marrow progenitors obtained from mice 3 days after treatment with 5-fluorouracil responded to a combination of GM-CSF and TGF-beta 1, whereas either factor alone had no effect. A single-cell assay identified a progenitor cell that directly responded to TGF-beta and GM-CSF. TGF-beta increased the number of GM-CSF receptors on bone marrow cells. Thus, TGF-beta 1 can act as a bifunctional mediator of hematopoietic cell growth, and TGF-beta 1 and GM-CSF act together to stimulate granulopoiesis as measured by large granulocyte colony formation; the progenitor cell is tentatively designated granulocyte burst-forming unit.

Macrophage- and astrocyte-derived transforming growth factor beta as a mediator of central nervous system dysfunction in acquired immune deficiency syndrome.

The multifunctional cytokine, transforming growth factor beta (TGF-beta), was identified by immunocytochemistry in the brain tissues of four patients with acquired immune deficiency syndrome (AIDS), but not in control brain tissue. The TGF-beta staining was localized to cells of monocytic lineage as well as astrocytes, especially in areas of brain pathology. In addition, the brain tissues from the AIDS patients contained transcripts for human immunodeficiency virus 1 (HIV-1) by in situ hybridization, suggesting a correlation between the presence of HIV-1 in the brain and the expression of TGF-beta. However, the expression of TGF-beta was not limited to HIV-1-positive cells, raising the possibility of alternative mechanisms for the induction of TGF-beta in these AIDS patients' brains. To investigate these mechanisms, purified human monocytes were infected in vitro with HIV-1 and were shown to secrete increased levels of TGF-beta. In addition, HIV-1-infected monocytes released a factor(s) capable of triggering cultured astrocytes that are not infected with HIV-1 to secrete TGF-beta. The release of TGF-beta, which is an extremely potent chemotactic factor, may contribute to the recruitment of HIV-1-infected monocytic cells, enabling viral spread to and within the central nervous system (CNS). Moreover, TGF-beta augments cytokine production, including cytokines known to be neurotoxic. The identification of TGF-beta within the CNS implicates this cytokine in the immunopathologic processes responsible for AIDS-related CNS dysfunction.

Induction of transforming growth factor-beta 1 (TGF-beta 1), receptor expression and TGF-beta 1 protein production in retinoic acid-treated HL-60 cells: possible TGF-beta 1-mediated autocrine inhibition.

Treatment of HL-60 cells, a human promyelocytic leukemia cell line, with the vitamin A derivative retinoic acid (RA) for 7 days resulted in a dose-dependent decrease in proliferation and increase in granulocytic differentiation. The role of transforming growth factor-beta 1 (TGF-beta 1), a protein with pleiotropic effects on the proliferation and differentiation of various cell types, was examined during RA-induced differentiation of HL-60 cells. Although TGF-beta 1 alone had little effect on proliferation or differentiation of HL-60 cells, addition of TGF-beta 1 to HL-60 cells treated with a suboptimum concentration of RA (1.0 nmol/L) resulted in a marked decrease in proliferation with no effect on granulocytic differentiation. Studies of the mechanism of RA-induced TGF-beta sensitivity showed that although untreated HL-60 cells expressed low levels of TGF-beta 1 binding proteins on the cell surface, the levels were increased in a dose-dependent manner after RA treatment. Maximum induction was achieved after treatment with 10 nmol/L RA and consisted predominantly of the 65-Kd TGF-beta 1 receptor type. Moreover, RA treatment also resulted in a dose-dependent increase in both TGF-beta 1 steady-state mRNA expression and production of active TGF-beta with maximum induction at 10 nmol/LRA. RA treatment of HL-60 cells had no effect on TGF-beta 2 and TGF-beta 3 mRNA expression. These data suggest that the effects of RA may be mediated by a TGF-beta 1-mediated autocrine antiproliferative loop during differentiation of HL-60 cells.

A novel polyclonal antibody (CL-B1/29) for immunolocalization of transforming growth factor-beta 2 (TGF-beta 2) in adult mouse.

A polyclonal antibody (CL-B1/29) raised against a synthetic peptide with an amino acid sequence identical to the first 29 N-terminal residues of bovine bone-derived transforming growth factor-beta 2 (TGF-beta 2) was characterized and used for immunolocalization of TGF-beta 2 in adult mice. Reduced staining of immunoblots and tissue after absorption of the antiserum with the immunizing peptide or with TGF-beta 2 but not with purified TGF-beta 1 demonstrated that the reagent is specific for TGF-beta 2, with little or no crossreactivity with TGF-beta 1. The immunolocalization of TGF-beta 2 was investigated in formalin-fixed, paraffin-embedded cultured cells and murine tissue. Specimens pre-digested with testicular hyaluronidase demonstrated immunostaining predominantly of extracellular connective tissue matrix, whereas specimens pre-digested with pronase E demonstrated primarily cytoplasmic staining. Immunoreactivity was widely distributed in connective tissue, muscle, adsorptive and secretory epithelia, especially of endocrine tissue, and neural tissue of adult mice.

Immune responses to allogeneic and xenogeneic implants of collagen and collagen derivatives.

Whereas xenogeneic collagen has provided a safe and effective biomaterial for numerous medical applications, there are few instances in which data permit the correlation of the immunologic profile of well-defined devices with their clinical sequelae. A major exception is the use of injectable bovine dermal collagen for soft-tissue contour correction. The low incidence of hypersensitivity has been studied in the context of clinical efficacy and safety with several devices. The findings indicate that such immunity usually results in the manifestation of local symptoms of dermal inflammation at sites of treatment that resolve as the implant is resorbed by the host. In contrast, more immunogenic hemostatic agents may elicit a more frequent or vigorous immune response that is not clinically visible or relevant in that application. Recent experiences with collagen-based devices for the repair and regeneration of bone have also demonstrated that the presence of immunity to their collagenous or non-collagenous components does not necessarily predict adverse clinical sequelae. Indeed, numerous specific data indicate that this immunity can exist as an epiphenomenon with no effect on osteogenesis. To get a true composite picture of biocompatibility, significant steps must be taken to characterize biomaterials properly and to ensure that immunologic, clinical, histologic, and other pertinent laboratory data are viewed in relation to one another and not in isolation.

Growth inhibition of a human lymphoma cell line: induction of a transforming growth factor-beta-mediated autocrine negative loop by phorbol myristate acetate.

Transforming growth factor-beta (TGF-beta) exerts profound inhibitory effects on a number of cell types, including normal B- and T-lymphocytes. In contrast, we have found a number of lymphoid tumor cell lines to be insensitive to the antiproliferative effects of TGF-beta 1 or TGF-beta 2. Binding and cross-linking with radioiodinated TGF-beta 1 demonstrated either low or absent expression of all three TGF-beta receptor species on three B-cell tumor lines, but T-cell and non-T, non-B tumors expressed large numbers of receptors. Treatment of the B-cell lines with phorbol 12-myristate 13-acetate (PMA) induced the expression of TGF-beta receptors and inhibited proliferation in all three lines in a dose- and time-dependent manner. The cell lines constitutively produced TGF-beta mRNA and released small amounts of latent TGF-beta; however, PMA induced the release of active TGF-beta. A neutralizing antibody to TGF-beta was able to reverse the PMA-induced growth inhibition of the malignant lymphoma cell line, RL, and addition of exogenous TGF-beta reversed the effect of the neutralizing antibody. Thus, TGF-beta can inhibit human lymphoma cell growth in vitro through an autocrine mechanism. Some lymphoma cells appear to have escaped from TGF-beta negative regulation by failing to express functional TGF-beta receptors and/or by failing to secrete active TGF-beta receptors and/or by failing to acts to inhibit lymphoma cell growth is by inducing the expression of TGF-beta receptors and the secretion of active TGF-beta, thereby reestablishing an autocrine growth-inhibitory loop.

Antagonistic and agonistic effects of transforming growth factor-beta and IL-1 in rheumatoid synovium.

We investigated potential mechanisms by which lymphocytes infiltrating rheumatoid synovium become immunosuppressed. In 20 of 22 synovial fluids and 12 of 13 synovial tissue culture supernatants, no IL-1 bioactivity could be detected in the thymocyte proliferation assay. These same preparations could, however, support proliferation of fibroblast monolayers, consistent with the presence of IL-1 and/or other fibroblast growth factors. Addition of either rheumatoid synovial fluids or synovial culture supernatants to exogenous IL-1 in the IL-1 bioassay caused marked inhibition of the assay indicative of an IL-1 inhibitor. This inhibition of IL-1 could be reversed by treating the effusions or supernatants with a neutralizing antibody to transforming growth factor-beta (TGF-beta). Furthermore, monocyte-macrophages isolated from rheumatoid synovial fluid constitutively released both latent and active TGF-beta in culture at levels sufficient to completely block the biologic activity of 100 U/ml IL-1. The production of substantial levels of TGF-beta by synovial macrophages, as well as the apparent ability of these inflammatory macrophages to activate latent TGF-beta, implicates TGF-beta not only as an important inhibitor of IL-1-induced lymphocyte proliferation, but also as a key cytokine in promoting synovial fibroblast hyperplasia and pathology.

Immunohistochemical evidence of a role for transforming growth factor beta in the pathogenesis of nodular sclerosing Hodgkin's disease.

Transforming growth factor beta (TFG-beta) is a multifunctional growth factor that promotes the growth of fibroblasts, collagen synthesis and angiogenesis, and stimulates monocyte migration and activation, but suppresses the growth and differentiation of immune lymphocytes and killer cells. Previously we demonstrated biologic activity for TGF-beta in supernatants of fresh Hodgkin's disease (HD) cell cultures and the cell line L428 derived from nodular sclerosing HD. This study was undertaken to find evidence of TGF-beta activity directly in tissues affected by HD. Formalin-fixed tissue from 14 patients with HD, including 8 nodular sclerosis, 4 mixed cellularity, 1 lymphocyte predominance, and 1 lymphocyte depletion type were studied by immunoperoxidase technique with antibody CC (1-30) raised against a synthetic polypeptide with the same N-terminal amino acid sequence as TGF-beta 1. Transforming growth factor beta activity was demonstrated in six cases of nodular sclerosis but not in other histologic types of HD. Staining for TGF-beta was found in the cytoplasm of Reed-Sternberg (RS) cells in one case and on the surface of RS cells and their lacunar variants in five cases. Transforming growth factor beta activity associated with the extracellular matrix was localized mainly around blood vessels, zones of necrosis, at the margins of bands of collagen sclerosis, and in areas containing syncytia of RS cells. In two cases TGF-beta was associated with collections of epithelioid histiocytes or granulomas. Small lymphocytes, granulocytes, and germinal center cells were unreactive. These results suggest that TGF-beta is a growth factor of biologic importance in HD and may be responsible for many of the histologic features, such as nodular sclerosis and granulomas, that may have prognostic significance.

Synergy between transforming growth factor-beta and tumor necrosis factor-alpha in the induction of monocytic differentiation of human leukemic cell lines.

We examined the effect of transforming growth factor-beta (TGF-beta) alone and in combinations with other factors on the growth and differentiation of the human promyelocytic cell line HL60 and the human monoblastic cell line U937. Treatment with TGF-beta alone did not significantly affect growth or differentiation of HL60 cells, while it significantly inhibited proliferation and induced monocytic differentiation of a small percentage of U937 cells. Combinations of TGF-beta and tumor necrosis factor-alpha (TNF-alpha) acted in synergy to inhibit cell proliferation and to induce monocytic differentiation of both HL60 and U937 cells. In contrast, no synergy was observed when HL60 cells were treated with TGF-beta in various combinations with interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), and retinoic acid. Examination of TNF-alpha receptor expression on HL60 and U937 cells showed that these cell lines expressed comparable levels of high-affinity TNF-alpha binding sites. Treatment of HL60 and U937 cells with TGF-beta did not induce significant changes in TNF-alpha receptor expression in either cell line. In contrast, HL60 cells expressed much lower levels of TGF-beta receptors than did U937 cells. Treatment of both HL60 and U937 cells with TNF-alpha induced a dose-dependent increase in expression of TGF-beta receptors, suggesting that the synergy between TNF-alpha and TGF-beta may result, at least in part, from upregulation of TGF-beta receptor expression by TNF-alpha.

Transforming growth factor beta directly regulates primitive murine hematopoietic cell proliferation.

We previously reported that transforming growth factor beta (TGF-beta) selectively inhibits colony-stimulating factor-driven hematopoietic progenitor cell growth. We report here that TGF-beta 1 can act directly on hematopoietic progenitors to inhibit the growth of the most primitive progenitors measurable in vitro. Highly enriched populations of hematopoietic progenitor cells were obtained by isolating lineage negative (Lin-), Thy-1-positive (Thy-1+) fresh bone marrow cells, or by isolating cells from interleukin-3 (IL-3) supplemented bone marrow cultures expressing Thy-1 antigen with the fluorescent activated cell sorter. TGF-beta 1 inhibited IL-3-induced Thy-1 expression on Thy-1-negative (Thy-1-) bone marrow cells in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. In addition, TGF-beta 1 inhibited the formation of multipotent and mixed colonies by isolated Thy-1+ cells, while single lineage granulocyte and macrophage colonies were not affected. The growth of Thy-1+ Lin- cells incubated as single cells in Terasaki plates in medium supplemented with IL-3 were inhibited by TGF-beta, demonstrating a direct inhibitory effect. Hematopoietic stem cells, which have a high proliferative potential (HPP) when responding to combinations of growth factors in vitro, have been detected in the bone marrow of normal mice and mice surviving a single injection of 5-fluorouracil. TGF-beta 1 inhibited the growth of all subpopulations of HPP colony forming cells (CFC) in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. Thus, TGF-beta directly inhibits the growth of the most immature hematopoietic cells measurable in vitro.

Rapid onset synovial inflammation and hyperplasia induced by transforming growth factor beta.

After intraarticular injection of TGF-beta 1 or TGF-beta 2, marked swelling and erythema of the injected joints were apparent within 12-24 h. On a scale of 0 to 4, by day 3, the TGF-beta-treated joints had articular indices (AI) of 3.6 +/- 0.5 to 4.0 +/- 0.0 compared with no response for the vehicle-injected contralateral joints. Histopathologic evaluation revealed a predominantly mononuclear phagocyte infiltrate with some neutrophils and T lymphocytes, consistent with active inflammation. The monocytic pattern of leukocyte infiltration at 2-3 d was comparable to that seen in animals with antigen-induced arthritis after 2-3 wk. Extensive synovial fibroblast hyperplasia became apparent within 48 h, likely as a result of TGF-beta induction of growth factor synthesis by the accumulating monocytes. TGF-beta 2, a homologue of TGF-beta 1, was found to induce a similar level of synovitis and synovial hyperplasia consistent with its parallel monocyte and fibroblast chemotactic properties and ability to induce transcription and translation of monocyte/macrophage-derived growth factors. These data suggest that TGF-beta, released by platelets and activated inflammatory cells, may play a direct role in leukocyte recruitment and activation in arthritic and other chronic inflammatory lesions.

Transforming growth factor-beta 1 specifically localizes in elastin during synovial inflammation: an immunoelectron microscopic study.

We report here that extracellular TGF-beta 1 is associated exclusively with microfibrils of elastin which are present in the extracellular matrix of the inflamed articular joint of the rat. Inflammation was initiated by bacterial cell walls localized in the synovium following intraperitoneal injection of the bacterial components. This synovitis is associated with both destruction of connective tissue components and matrix deposition. The growth factor was localized by using a polyclonal antibody raised to a synthetic peptide corresponding to amino terminal 30 amino acids of TGF-beta 1 in conjunction with a gold-labeled secondary antibody. The results suggest a close association of TGF-beta 1 with proteoglycans which are known to be a major component of the microfibrils in elastin. Proteoglycan-mediated binding and concentration of TGF-beta 1 in specific areas of the extracellular matrix may constitute a mechanism whereby the growth factor could be targeted to specific sites of action.

In situ expression of transforming growth factor beta in streptococcal cell wall-induced granulomatous inflammation and hepatic fibrosis.

The expression of transforming growth factor beta (TGF-beta) was examined during the evolution of streptococcal cell wall (SCW)-induced hepatic granulomas in rats to evaluate the role of TGF-beta in chronic inflammation progressing to fibrosis. As determined by immunocytochemistry, Kupffer cells rapidly expressed TGF-beta 1 following intraperitoneal (i.p.) injection of SCW, and TGF-beta was expressed by mononuclear phagocytes in the earliest cell aggregates as well as by mononuclear phagocytes within the capsule of mature lesions. Interestingly, apparent extracellular TGF-beta was observed in mature lesions at the interface of the capsule and the cellular core, a region of active fibrogenesis. Granulomas isolated 3, 6, and 12 weeks post-SCW injection elaborated nanogram (ng) quantities of latent and active TGF-beta into culture supernatants, and expressed high levels of 2.4 and 1.9 kb TGF-beta 1 transcripts. Expression of procollagen type I and III mRNAs were observed in parallel with the expression of the TGF-beta 1 transcripts. Thus, TGF-beta is expressed throughout SCW-granuloma development, and, based on known bioactivities, it appears that TGF-beta mediates, in part, the recruitment and activation of monocytes and fibroblasts and deposition of collagen in SCW-granulomas and likely other chronic inflammatory lesions progressing to fibrosis.