Production of multilineage growth factors by hematopoietic stromal cells: an intercellular regulatory network involving mononuclear phagocytes and interleukin-1

In the past 8 years, our group has carried out a series of in-vitro studies designed to characterize the role of mononuclear phagocytes as regulators of human hematopoiesis. The results of this program of investigation, some of which are reviewed below, led to the discovery that mononuclear phagocytes are more efficient recruitors of growth factor release by other cells than they are direct stimulators of progenitor cell growth. Specifically, mononuclear phagocytes release soluble factors (MRA) that stimulate other cells, including vascular endothelial cells, skin fibroblasts, and marrow fibroblasts, to release multilineage hematopoietic growth factors. Experiments designed to purify and characterize these monokines indicated unambiguously that the MRA that stimulates granulocyte/macrophage colony stimulating factor (GM-CSF) release is interleukin-1 (IL-1). Based on these observations and recent observations by other groups on the hematopoietic effects of other monokines including tumor necrosis factor alpha, we argue that mononuclear phagocytes serve as important regulators of hematopoiesis by producing monokines that, in turn, induce the expression of multiple hematopoietic growth factor genes in stromal cells of the hematopoietic microenvironment. Because IL-1 molecules and the mononuclear phagocytes producing them are evolutionarily conserved, and in view of the heterogeneous nonhematopoietic effects of these monokines, studies on their role in hematopoiesis may also provide new understanding of the molecular evolution of multicellular organisms.     

Human mononuclear phagocyte activation antigens

Activation of mononuclear phagocytes causes changes in plasma membrane composition that include the expression of surface antigens and receptors. Monoclonal antibody technology has made it possible to identify and characterize newly expressed surface antigens. Among these "activation antigens" is a glycoprotein, Mo3, which (among hematopoietic cells) is selectively expressed by human mononuclear phagocytes that have been exposed to inflammatory factors in vitro and in vivo. Progress toward a functional and structural analysis of Mo3 is described.     

Interleukin-1 and tumor necrosis factor alpha can be induced from mononuclear phagocytes by human immunodeficiency virus type 1 binding to the CD4 receptor.

Cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) are important in normal immune processes. In this study, we demonstrate that human immunodeficiency virus type 1 (HIV-1) virions induce normal peripheral blood mononuclear phagocytes to produce both IL-1 and TNF within a few hours after their exposure to virus. The induction of these cytokines by HIV-1 does not require a productive infection. Blocking studies with soluble CD4 indicate that the effect is mediated through the CD4 molecule. In addition, the treatment of mononuclear phagocytes with OKT4A monoclonal antibody mimics the effects of HIV-1. Thus, these results indicate that induction of IL-1 and TNF alpha can occur via signals mediated through the CD4 molecule on mononuclear phagocytes. TNF has been shown by other investigators to induce HIV-1 expression. Therefore, TNF alpha may play a role in autocrine and paracrine regulation of HIV-1 expression. In addition, the induction of IL-1 and TNF by HIV-1 may also contribute to some of the neurologic and physiologic disorders associated with acquired immunodeficiency syndrome.     

CX3CR1+ lung mononuclear phagocytes spatially confined to the interstitium produce TNF-α and IL-6 and promote cigarette smoke-induced emphysema

Increased numbers of macrophages are found in the lungs of smokers and those with chronic obstructive pulmonary disease. Experimental evidence shows the central role of macrophages in elaboration of inflammatory mediators such as TNF-α and the progression toward cigarette smoke-induced emphysema. We investigated the role of CX3CR1 in recruitment of mononuclear phagocytes, inflammatory cytokine responses, and tissue destruction in the lungs after cigarette smoke exposure. Using mice in which egfp is expressed at the locus of the cx3cr1 gene, we show that alveolar macrophages increased transmembrane ligand CX3CL1 expression and soluble CX3CL1 was detectable in the airspaces, but cx3cr1(GFP/GFP) and cx3cr1(GFP/+) mice failed to show recruitment of CX3CR1(+) cells into the airspaces with cigarette smoke. In contrast, cigarette smoke increased the accumulation of CX3CR1(+)CD11b(+) mononuclear phagocytes that were spatially confined to the lung interstitium and heterogenous in their expression of CD11c, MHC class II, and autofluorescent property. Although an intact CX3CL1-CX3CR1 pathway amplified the percentage of CX3CR1(+)CD11b(+) mononuclear phagocytes in the lungs, it was not essential for recruitment. Rather, functional CX3CR1 was required for a subset of tissue-bound mononuclear phagocytes to produce TNF-α and IL-6 in response to cigarette smoke, and the absence of functional CX3CR1 protected mice from developing tissue-destructive emphysema. Thus, CX3CR1(+) "tissue resident" mononuclear phagocytes initiate an innate immune response to cigarette smoke by producing TNF-α and IL-6 and are capable of promoting emphysema.     

Killing of Escherichia coli by mononuclear phagocytes and neutrophils stimulated in vitro with beta-1,3-D-polyglucose derivatives.

Human monocytes, human peritoneal macrophages, mouse peritoneal macrophages and human peripheral neutrophils pretreated with beta-1,3-D-polyglucose derivatives showed pronounced bactericidal capacity to Escherichia coli compared to control cells. The increased bactericidal capacity was detectable in mononuclear phagocytes over a wide range of concentrations of bacteria. Granulocytes, however, showed bactericidal capacity only at low concentrations of bacteria. The pretreated mononuclear phagocytes released significant amounts of IL-1 and PGE2. However, there was no significant release of tumor necrosis factor (TNF). By incubating unstimulated cells with purified IL-1 and TNF, the bactericidal activity of neutrophils and mononuclear phagocytes was enhanced. Our data indicate that the inability of neutrophils stimulated with beta-1,3-D-polyglucose derivatives to kill large numbers of bacteria could be overcome by a combined treatment with purified IL-1 or TNF in addition to beta-1,3-D-polyglucose derivatives. By incubating unstimulated cells with medium from beta-1,3-D-polyglucose-treated human peritoneal macrophages, the bactericidal activity of the cells was enhanced to the same extent as cells pretreated with purified TNF and IL-1. Cells incubated with IL-1-depleted medium from beta-1,3-D-polyglucose-treated human peritoneal macrophages, showed reduced bactericidal activity compared to cells incubated with undepleted medium. These studies demonstrate that beta-1,3-D-polyglucose-treated mononuclear phagocytes and neutrophils show enhanced bactericidal activity. The enhanced activity is partly caused by stimulation of the cells with IL-1 released from mononuclear phagocytes and partly by other unknown effects of beta-1,3-D-polyglucose derivatives on both mononuclear phagocytes and neutrophils.     

Inhibition of hBD-3, but not hBD-1 and hBD-2, mRNA expression by corticosteroids

Intensive use of corticosteroids may be accompanied by increased susceptibility to infections; hence, we investigated the effects of dexamethasone on the expression of antimicrobial peptides, termed human beta-defensins (hBD), by cultured bronchial epithelial cells and mononuclear phagocytes. The results revealed that dexamethasone inhibited the (stimulated) expression of mRNA for hBD-3, but not hBD-1 and hBD-2 by these epithelial cells. Dexamethasone did not affect the (stimulated) mRNA expression of hBD-1 and hBD-2 by mononuclear phagocytes, whereas these cells did not express hBD-3 mRNA.     

Negative trans-acting factors extinguish Ia expression in B cell-L 929 somatic cell hybrids

Numerous studies have implicated trans-acting factors in the regulation of MHC class II gene expression. Some of these factors have been shown to act by inducing the expression of class II genes while others have been demonstrated to downregulate such expression. These reports have dealt almost exclusively with the role of trans-acting factors in the regulation of class II gene expression in hematopoietic-derived cells. We decided to extend these studies to the role trans-acting factors play in nonhematopoietic-derived (NHD) cells. In order to address this question we made somatic cell hybrids between the NHD Ltk- cell line and normal B cells to determine if the existence of positive trans-acting factors from the B cell would lead to the expression of Ltk- class II genes in the resultant hybrid. Our results clearly indicate that not only was there no induction of Ltk- class II gene expression in the hybrids, but there was a loss of B cell class II gene expression as well. These results suggest that Ltk- cells possess negative trans-acting factors that appear to predominate over the positive trans-acting factors possessed by B cells. We have further extended these studies to test the MHC-inducing activity of IFN-gamma and IL-4 on these hybrids. Our results indicate that the hybrids responded to IFN-gamma with an increase in class I but not class II expression for both fusion partners. Furthermore, neither B cell nor L cell class II genes were induced by IL-4. Taken together, these results indicate that Ltk- cells possess negative trans-acting factors that not only maintain the Ia- phenotype of these cells, but also block the action of positive trans-acting factors from B cells.     

Interleukin-4. A regulatory protein

Since its discovery in 1982, numerous biological activities of interleukin-4 (IL-4) have been described. Like other cytokines, IL-4 is highly pleiotropic, both with respect to the number of different target cells that are responsive to it and with respect to the number of different biological responses it elicits. Interleukin-4 was initially described as a costimulant for the proliferation of B lymphocytes stimulated with anti-IgM antibody. Synonyms for this cytokine are B cell growth factor-1 (BCGF-1) and B cell stimulatory factor-1 (BSF-1). After cloning of both the murine and human IL-4, the use of recombinant IL-4 enabled detailed studies of its biological functions. Many cell types, mainly of hematological origin, express receptors for IL-4. Accordingly, effects of IL-4 have been described on B lymphocytes, T lymphocytes, NK cells, mononuclear phagocytes, mast cells, fibroblasts and hematopoietic progenitor cells. Currently, there are three major areas in which IL-4 appears to play an important role: 1) regulation of B cell growth and of antibody isotype expression. In this context, a possible role for IL-4 in allergic reactions is of special interest. 2) Stimulation of T cell growth and the generation of cytotoxic T lymphocytes. In addition to the suppressive effects on the induction of non HLA-restricted cellular cytotoxicity by natural killer- (NK) and lymphokine-activated killer (LAK) cells, this suggests a role for IL-4 in the regulation of cellular immune responses. 3) Regulation of the growth and differentiation of hematopoietic bone marrow stem cells. IL-4 itself does not induce proliferation of hematological progenitor cells but it can modulate the growth-factor dependent proliferation of these cells. In this review the biological functions of IL-4, reported until present, are discussed.     

Morphine Up-regulates expression of substance P and its receptor in human blood mononuclear phagocytes and lymphocytes

In vitro and in vivo studies have indicated that there is an important relationship between morphine and neuropeptide substance P (SP). We therefore investigated the interaction of morphine and cultured human immune cells on the expression of SP, a neuropeptide which we have recently demonstrated to be produced by human monocytes and lymphocytes. Morphine up-regulated SP production in human mononuclear phagocytes and lymphocytes at both the mRNA and the protein level. In addition, morphine induced SP receptor (NK-1R) expression in human lymphocytes. The specific morphine receptor antagonist (naltrexone) blocked morphine-induced SP expression in human mononuclear phagocytes, supporting the concept of authentic morphine receptor-mediated regulation. Since SP modulates neurogenic inflammation and immunologic events, these data suggest that morphine-induced SP expression in cells of the immune system may be of importance in the pathogenesis of immune-mediated diseases, including neuroimmunologic diseases and AIDS.     

Role of cytokines in alveolar macrophage accessory cell function in HIV-infected individuals.

Mononuclear phagocytes, including alveolar macrophages (AM), can be chronically infected with HIV and thus serve as a reservoir for the virus. Acting as AC during the generation of an immune response, HIV-infected mononuclear phagocytes can facilitate viral T cell infection by several mechanisms, including direct contact of T cells with HIV-infected macrophages as well as cytokine-induced up-regulation of latent T cell infection. Our laboratory has shown that AM from HIV-infected individuals have enhanced AC function compared to normal AM. In this study we explored AM production and secretion of IL-1 beta and IL-6, two cytokines critical for optimal AC function, in normal volunteers and HIV-infected patients. Cultured AM supernatants and lysates were generated in the presence and absence of LPS and standard mitogens. In initial mixing experiments HIV AM supernatants enhanced mitogen-induced T cell proliferation using normal AM as AC significantly more than normal AM supernatants, suggesting that HIV AM secreted more T cell stimulatory factors than normal AM. Neither group could enhance T cell proliferation induced by HIV AM suggesting these cells already secreted optimal levels of these factors. AM from HIV+ individuals produced and secreted more IL-1 beta (measured by ELISA) and IL-6 (measured in a B9 bioassay and by immunoprecipitation) than normal AM both spontaneously and in the presence of low LPS concentrations and mitogens. In some cases depleting HIV AM supernatants of IL-1 beta and IL-6 on immunoaffinity columns abrogated their enhancement properties indicating that these cytokines were important in the observed enhancement. However, in other patients different factors must also be involved as depletion of IL-1 beta and IL-6 in their AM supernatants had no effect on enhancement function. These results show that HIV AM secretory products are important in the enhanced AC function demonstrated by these cells. However, although augmented IL-1 beta and IL-6 secretion likely contribute significantly to this enhancement, other AC secretory factors and/or functions must also be involved.     

Effects of endotoxin on proliferation of human hematopoietic cell precursors

In examining the effects of corticosteroids on hematopoiesis in vitro, we observed that results were highly dependent on the lot of commercial fetal calf serum (FCS) utilized. We hypothesized that this variability correlated with the picogram (pg) level of endotoxin contaminating the FCS. Randomly obtained commercial lots of FCS contained 0.39 to 187 pg/ml of lipopolysaccharide (LPS). Standard FCS concentrations in hematopoietic precursor proliferation assays (granulocyte-marcrophage colony forming units [CFU-GM]) resulted in final LPS levels as high as 40 pg/ml. LPS (2–5 pg/ml) added to essentially endotoxin-free cultures, induced human mononuclear cell release of interleukin (IL)-1, IL-6 and granulocyte colony stimulating factor (G-CSF). Lots of FCS induced the release of IL-1, IL-6, and G-CSF from human mononuclear cells and the release of these factors correlated with the level of contaminating LPS. Human bone marrow CFU-GM proliferation, in response to granulocyte-macrophage colony stimulating factor (GM-CSF), positively correlated with the level of LPS contaminating the FCS and the FCS-induced release of IL-6 from mononuclear cells. CFU-GM proliferation of human bone marrow cluster of differentiation (CD) 34+CD14-cells were not affected by the presence of endotoxin. These data suggest that LPS at 2–5 pg/ml may induce bone marrow accessory cell release of hematopoietic growth factors, thus altering proliferative response of hematopoietic precursors and confounding the study of exogenously added cytokines to culture systems. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of cytokines by immature erythroid cells derived from human embryonic liver

It is well known that regulatory interactions between hematopoietic and lymphoid cells are mediated by different mediators. The cells of erythroid lineage are not an exception and have a regulatory effect on hemato- and immunopoiesis that can be mediated through the production of cytokines i.e. by soluble factors - a universal mechanism for cell regulation in hematopoietic and immune systems. It has been previously shown that erythroid progenitor cells from mice express mRNA of cytokines such as IL-1 alpha and beta, IL-4, IL-6, IFN-gamma, GM-CSF and TGF-beta. In this report we present the results of the production of the main immunoregulatory cytokines by erythroid cells derived from human embryonic liver. It was revealed that the cell population enriched with erythroid progenitors, isolated from human fetal liver, can produce IL-1 beta, IL-2, IL-4, IL-6. The levels of production of cytokines by immature erythroid progenitor cells is compared to the levels of corresponding cytokines produced by mitogen-stimulated peripheral blood mononuclear cells. The production of these cytokines changed quantitatively under the effect of erythropoietin, and are correlated with the expression of differentiation markers of erythroid cells such as AG-EB and Glycophorin A. The role of cytokine production by erythroid cells in hemato- and immunopoiesis and the mechanisms of self-regulation of proliferation and differentiation of erythroid progenitor cells is discussed.     

Regulation of cell surface receptors for different hematopoietic growth factors on myeloid leukemic cells.

There are clones of myeloid leukemic cells which are different from normal myeloid cells in that they have become independent of hematopoietic growth factor for cell viability and growth. The ability of these clones to bind three types of hematopoietic growth factors (MGI-1GM = GM-CSF, IL-3 = multi-CSF and MGI-1M = M-CSF = CSF-1) was measured using the method of quantitative absorption at 1 degree C and low pH elution of cell-bound biological activity. Results of binding to normal myeloid and lymphoid cells were similar to those obtained by radioreceptor assays. The results indicate that the number of receptors on different clones of these leukemic cells varied from 0 to 1,300 per cell. The receptors have a high binding affinity. Receptors for different growth factors can be independently expressed in different clones. There was no relationship between expression of receptors for these growth factors and the phenotype of the leukemic cells regarding their ability to be induced to differentiate. The number of receptors on the leukemic cells was lower than on normal mature macrophages. Myeloid leukemic cells induced to differentiate by normal myeloid cell differentiation factor MGI-2 (= DF), or by low doses of actinomycin D or cytosine arabinoside, showed an up-regulation of the number of MGI-1GM and IL-3 receptors. Induction of differentiation of leukemic cells by MGI-2 also induced production and secretion of the growth factor MGI-1GM, and this induced MGI-1GM saturated the up-regulated MGI-1GM receptors. It is suggested that up-regulation of these receptors during differentiation is required for the functioning of differentiated cells.