Regulation of murine macrophage function by IL-4. I. Activation of macrophages by a T-T cell hybridoma is due to IL-4

A T-cell hybridoma produced by fusion of concanavalin A-stimulated murine splenocytes produced a factor (MAFH) capable of activating tumoricidal capacity by responsive murine peritoneal macrophages. Macrophages treated with the MAFH required an additional trigger signal of bacterial lipopolysaccharide (LPS) for maximal activity. In contrast to interferon-gamma (IFN gamma), which induced tumoricidal activity against all tumor cells tested, MAFH only induced macrophage-mediated kill of the BI6P51 and 168 lines, and not of the P815 or B16BL6 lines. An identical pattern of tumoricidal activity was obtained by treating macrophages with recombinant interleukin-4 (IL-4). The active moiety of MAFH appeared to be IL-4 as (i) monoclonal antibody against IL-4 blocked MAFH, but not IFN gamma, activity, and (ii) the T-cell hybridoma contained large amounts of mRNA for IL-4 and no detectable mRNA for IFN gamma (as determined by Northern dot analysis). The pattern of tumoricidal activity observed may be due to an IL-4 mediated enhancement of tumor necrosis factor production by LPS-triggered macrophages.     

Renaturation of Escherichia coli-derived recombinant human macrophage colony-stimulating factor.

Recombinant human macrophage colony-stimulating factor (rhM-CSF), a homodimeric, disulfide bonded protein, was expressed in Escherichia coli in the form of inclusion bodies. Reduced and denatured rhM-CSF monomers were refolded in the presence of a thiol mixture (reduced and oxidized glutathione) and a low concentration of denaturing agent (urea or guanidinium chloride). Refolding was monitored by nonreducing gel electrophoresis and recovery of bioactivity. The effects of denaturant type and concentration, protein concentration, concentration of thiol/disulfide reagents, temperature, and presence of impurities on the kinetics of rhM-CSF renaturation were investigated. Low denaturant concentrations (<0.5 M urea) and high protein concentrations (>0.4 mg/ml) in the refolding mixture resulted in increased formation of aggregates, although aggregation was never significant even when refolding was carried out at room temperature. Higher protein concentration resulted in higher rates but did not lead to increased yields, due to the formation of unwanted aggregates. Experiments conducted at room temperature resulted in slightly higher rates than those conducted at 4 degrees C. Although the initial renaturation rate for solubilized inclusion body protein without purification was higher than that of the reversed-phase purified reduced denatured rhM-CSF, the final renaturation yield was much higher for the purified material. A maximum refolding yield of 95% was obtained for the purified material at the following refolding conditions: 0.5 M urea, 50 mM Tris, 1.25 mM DTT, 2 mM GSH, 2 mM GSSG, 22 degrees C, pH 8, [protein] = 0.13 mg/ml.     

Immunoreactive macrophage colony-stimulating factor is increased in atherosclerotic lesions of watanabe heritable hyperlipidemic rabbits after recombinant human macrophage colony-stimulating factor therapy

Infiltration of monocytes into arteries is an early event in the pathogenesis of atherosclerosis. This recruitment is interpreted as enhancing lesion development, but it could also be a host response limiting lipid accumulation. The ability of macrophages to limit cholesterol uptake, however, can be reduced by the impaired mobility and metabolic activity associated with foam cell development. As lesions enlarge, foam cells die and become the nidus for the necrotic core. Treatments to improve viability might improve foam cell function and promote regression. Macrophage colony-stimulating factor (M-CSF) is vital to monocyte/macrophage differentiation, proliferation, and activation. We found that foam cells of Watanabe heritable hyperlipidemic (WHHL) rabbits had faint staining for M-CSF. Treatment of rabbits with recombinant human M-CSF (rhM-CSF) increased M-CSF staining, which correlated with reduced cholesterol content of these foam cells. Mol Reprod Dev 46:92–95, 1997. © 1997 Wiley-Liss, Inc.     

Stimulation of macrophage plasminogen activator activity by colony-stimulating factors

Colony-stimulating factors (CSFs) stimulate granulocyte-macrophage production from single hemopoietic progenitor cells. Various preparations of purified CSFs of two different subclasses have been shown here to stimulate a plasminogen-dependent fibrinolytic (plasminogen activator) activity from resident and starch-induced mouse peritoneal macrophages. Lymphocyte supernatants also stimulate macrophage plasminogen activator (PA) activitty. Since they contain colony stimulating activity, it is possible that one or more sublcasses of CSF in these supernatants is responsible for this effect. Since both colony-stimulating and macrophage growth activities have been detected at inflammatory sites, these findings could reflect a role for CSF in inflammatory processes.     

Clinical applications of recombinant human colony-stimulating factors.

The differentiation and maturation of hematopoietic progenitor cells are regulated by certain growth factors. Several of these glycoproteins have been characterized, and their amino acid sequences have been delineated. Modern DNA technology provides sufficient quantities of these hormones for testing in clinical trials. Erythropoietin (EPO) has been shown to increase the hemoglobin level and hematocrit in patients with end-stage renal disease. Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) can increase the numbers of neutrophils and monocytes, in a dose-dependent fashion. The function of granulocytes and monocytes is also enhanced. Clinical studies of the toxicity and activity of G-CSF and GM-CSF have been conducted in patients with acquired immune deficiency syndrome, aplastic anemia, myelodysplastic syndromes, and neutropenia due to cancer and chemotherapy. In almost all patients the neutrophil count increased within 24 hours after the start of treatment. Side effects of G-CSF and GM-CSF are infrequent and usually mild. Combinations of CSFs may be even more effective.     

The adaptor protein Tom1L1 is a negative regulator of Src mitogenic signaling induced by growth factors

The Src family of protein-tyrosine kinases (SFK) play important roles in mitogenesis and morphological changes induced by growth factors. The involved substrates are, however, ill defined. Using an antiphosphotyrosine antibody to screen tyrosine-phosphorylated cDNA expression library, we have identified Tom1L1, an adaptor protein of the Tom1 family and a novel substrate and activator of the SFK. Surprisingly, we found that Tom1L1 does not promote DNA synthesis induced by Src. Furthermore, we report that Tom1L1 negatively regulates SFK mitogenic signaling induced by platelet-derived growth factor (PDGF) through modulation of SFK-receptor association: (i) Tom1L1 inhibits DNA synthesis induced by PDGF; (ii) inhibition is overcome by c-myc expression or p53 inactivation, two regulators of SFK mitogenic function; (iii) Src or Fyn coexpression overrides Tom1L1 mitogenic activity; (iv) overexpression of the adaptor reduces Src association with the receptor; and (v) protein inactivation potentiates receptor complex formation, allowing increased SFK activation and DNA synthesis. However, Tom1L1 affects neither DNA synthesis induced by the constitutively active allele SrcY527F nor SFK-regulated actin assembly induced by PDGF. Finally, overexpressed Tom1 and Tom1L2 also associate with Src and affected mitogenic signaling in agreement with some redundancy among members of the Tom1 family. We concluded that Tom1L1 defines a novel mechanism for regulation of SFK mitogenic signaling induced by growth factors.     

Regulation of macrophage activation markers by IL-4 and IFN-gamma is subpopulation-specific

We have addressed the differential regulatory properties that IFN-gamma and IL-4 exert on macrophage (M phi) subpopulations. For this purpose, Thyoglicolate-, Peptone-, and Con A-elicited M phi, as well as bone marrow-derived M phi and P388D1 cells, were cultured in the presence of either IFN-gamma or IL-4. The expression of LFA-1, Mac-1, and Mac-2 after this treatment was studied by FACS analysis. We have found that these surface molecules are differentially modulated by the two lymphokines, depending on the M phi subpopulation studied. Mac-1 is upregulated only in Thyoglicolate-elicited cells after treatment with IFN-gamma, while no change in the expression of Mac-2 was observed in any of the groups. LFA-1 is upregulated by IFN-gamma in Thyoglicolate- and bone marrow-derived M phi and P388D1 cells, while IL-4 does not induce LFA-1 on these cells. Interestingly, however, we have observed the reverse situation on Con A-elicited M phi, where a strong induction of LFA-1 is achieved by treatment of the cells with IL-4, while IFN-gamma does not modify the expression of this antigen. Our results obtained with the lymphokine-stimulated M phi are interpreted in the context of functionally induced M phi subpopulations, which might be regulated by either Th1 or Th2 CD4+ T cells. Thyoglicolate-elicited M phi may represent the in vitro equivalent of a M phi subpopulation regulated in vivo by Th1 cells while Con A-elicited M phi could be the equivalent of a subpopulation regulated by Th2 cells.     

Human recombinant IL-3 is a growth factor for normal B cells.

IL-3 is a well known hemopoietic cell growth and differentiation factor. However, its functional role in normal B cell differentiation has not been established. We have investigated the effect of IL-3 on the growth and differentiation of human B cells. IL-3 enhanced the proliferation of Staphylococcus aureus Cowan 1 strain-stimulated B cells. The optimal time of IL-3 to stimulate B cell growth was on day 2 to day 3, suggesting that IL-3 was a B cell growth factor acting in the late stage. IL-3 synergized with IL-2 to enhance B cell proliferation and differentiation. Pretreatment of B cells with IL-3 for more than 3 days increased the expression of IL-2R on B cells. However, pretreatment of B cells with IL-2 did not alter the subsequent response to IL-3, suggesting that the synergy between IL-2 and IL-3 may be attributed to the up-regulation of IL-2 response by IL-3. In addition, pretreatment of B cells with IL-4 decreased subsequent response of B cells to IL-3 as well as IL-2, suggesting that IL-3- and IL-2-responding cells passed a similar way during the early stage of B cell activation. It appears that IL-3 and IL-6 mediate normal B cell differentiation via separate mechanisms. IL-3-induced B cell differentiation was mainly mediated by increasing cell growth, whereas IL-6 induced B cell differentiation without affecting proliferation.     

Properties of primary murine stroma induced by macrophage colony-stimulating factor

The ability of purified human macrophage colony-stimulating factor (M-CSF) to accelerate the formation of stromal cells from murine bone marrow cells was investigated. The liquid culture of the marrow cells with M-CSF resulted in the formation of monolayers of macrophages on day 7. When the M-CSF was removed on that day and the residual adherent cells were cultured in the absence of M-CSF for an additional 7 days, many colonies appeared with cells that were morphologically distinguishable from M-CSF-derived macrophages. The appearance of the colonies was dependent on the concentration of M-CSF used at the beginning of the culture. Each colony was isolated as a single clone and analyzed. All clones were negative for esterase staining. These cells did not express M-CSF receptor mRNA and did not show a mitogenic response to M-CSF. On the contrary, these cells could be stimulated to proliferate by fibroblast growth factor and platelet-derived growth factor. The polymerase chain reaction analysis of these cells demonstrated constitutive expression of mRNA for M-CSF, stem cell factor, and interleukin (IL)-1, but not IL-3. Some clones expressed mRNA for granulocyte/M-CSF and IL-6. We also examined the ability of the cells to maintain murine bone marrow high proliferative potential colony-forming cells (HPP-CFC) in a coculture system. Most of the clones showed a significant increase in total HPP-CFC numbers after 2 weeks of coculture, although the extent of stimulation differed among clones. These results suggested that the colonies established by M-CSF were composed of functional stromal cells that were phenotypically different from macrophages. J. Cell. Physiol. 173:1–9, 1997. © 1997 Wiley-Liss, Inc.     

Expression and processing of a recombinant human macrophage colony-stimulating factor in mouse cells.

A human macrophage colony-stimulating factor encoded by a 4-kilobase cDNA was expressed with bovine papillomavirus vectors in mouse cells. Pulse-chase analyses revealed that the 62-kilodalton (kDa) translation product was glycosylated, cleaved, and efficiently secreted within 1 h of synthesis. The secreted product contained both N-linked and O-linked oligosaccharide chains. Macrophage colony-stimulating factor was present extracellularly as an 80-kDa homodimer and as a multimeric species of greater than 200 kDa that may be associated with other glycoproteins.     

Murine eotaxin-2: a constitutive eosinophil chemokine induced by allergen challenge and IL-4 overexpression

The generation of tissue eosinophilia is governed in part by chemokines; initial investigation has identified three chemokines in the human genome with eosinophil selectivity, referred to as eotaxin-1, -2, and -3. Elucidation of the role of these chemokines is dependent in part upon analysis of murine homologues; however, only one murine homologue, eotaxin-1, has been identified. We now report the characterization of the murine eotaxin-2 cDNA, gene and protein. The eotaxin-2 cDNA contains an open reading frame that encodes for a 119-amino acid protein. The mature protein, which is predicted to contain 93 amino acids, is most homologous to human eotaxin-2 (59.1% identity), but is only 38.9% identical with murine eotaxin-1. Northern blot analysis reveals three predominant mRNA species and highest constitutive expression in the jejunum and spleen. Additionally, allergen challenge in the lung with Aspergillus fumigatus or OVA revealed marked induction of eotaxin-2 mRNA. Furthermore, eotaxin-2 mRNA was strongly induced by both transgenic over-expression of IL-4 in the lung and administration of intranasal IL-4. Analysis of eotaxin-2 mRNA expression in mice transgenic for IL-4 but genetically deficient in STAT-6 revealed that the IL-4-induced expression was STAT-6 dependent. Recombinant eotaxin-2 protein induced dose-dependent chemotactic responses on murine eosinophils at concentrations between 1-1000 ng/ml, whereas no activity was displayed on murine macrophages or neutrophils. Functional analysis of recombinant protein variants revealed a critical role for the amino terminus. Thus, murine eotaxin-2 is a constitutively expressed eosinophil chemokine likely to be involved in homeostatic, allergen-induced, and IL-4-associated immune responses.     

The development of bone changes induced in rats by recombinant human granulocyte colony-stimulating factor is suppressed by bisphosphonate.

We have previously demonstrated that high doses of recombinant human granulocyte colony-stimulating factor (rhG-CSF) induce bone changes characterized by osteoclastic bone resorption and osteogenesis due to intramembranous ossification in rats. In this communication we examined the effects of a pretreatment with 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP), which is a powerful inhibitor of osteoclastic bone resorption, on bone changes induced by rhG-CSF in order to investigate the relation between osteoclastic bone resorption and osteogenesis. AHPrBP (5 mg/kg/day) was subcutaneously given to 6-week-old rats for 2 days. From the following day of the final injection of AHPrBP, rats received a subcutaneous injection of rhG-CSF (1,000 micrograms/kg/day) for 14 days, and the femur and tibia were evaluated histopathologically. By the analysis of peripheral blood leukocyte counts, spleen weights and bone marrow findings, the pretreatment with AHPrBP had no effect on the activation of hematopoiesis related to the major pharmacological activity of rhG-CSF. In the rats treated with rhG-CSF alone, accelerated osteoclastic bone resorption and osteogenesis due to intramembranous ossification were observed in the trabeculae of metaphyseal spongiosa. The accelerated osteoclastic bone resorption induced by rhG-CSF was suppressed by the pharmacological activity of AHPrBP. Furthermore, the osteogenesis induced by rhG-CSF was also suppressed by AHPrBP. These results suggest that the osteogenesis induced by rhG-CSF is a sequential reaction of accelerated osteoclastic bone resorption, and moreover that the main action of rhG-CSF on bone is an acceleration of osteoclastic bone resorption.     

Dok-1 is a positive regulator of IL-4 signalling and IgE response

Interleukin-4 (IL-4) plays an essential role in the control of humoral immunity by regulating lymphocyte proliferation and differentiation, including the T helper type 2 lineage commitment of CD4(+) T cells as well as the isotype switching to IgE in B cells. The adaptor protein Dok-1 is known to have an essential role in the negative regulation of a variety of cytokine signalling events. However, here we have found that the loss of Dok-1 impaired the proliferative response of CD4(+) T cells and B cells to IL-4. Conversely, the forced expression of Dok-1 in the myeloid cell line 32D augmented the IL-4-induced proliferation, indicating a positive role for Dok-1. Tyrosine phosphorylation, and thereby the activation of Stat6 and IRS-2, is critical for IL-4 signalling; however, only the activation of Stat6, not the IRS-2-dependent phosphorylation of Akt, was perturbed in Dok-1-deficient cells stimulated with IL-4. Furthermore, mice lacking Dok-1 showed an impaired IgE response to thymus-dependent antigen. Thus, Dok-1 is a positive regulator of IL-4 signalling and IgE response.     

IL-10 synergizes with IL-4 and transforming growth factor-beta to inhibit macrophage cytotoxic activity.

After activation with IFN-gamma, thioglycollate-elicited murine peritoneal macrophages kill schistosomula of Schistosoma mansoni in vitro by an L-arginine-dependent mechanism which involves the production of reactive nitrogen oxides (NO). In the present study we demonstrate that the regulatory cytokines IL-10, IL-4, and transforming growth factor-beta (TGF-beta) are potent inhibitors of this extracellular killing function of activated macrophages. Each cytokine was found to suppress killing of schistosomula in a dose-dependent fashion. The activity of IL-10 was not permanent, because subsequent treatment with additional IFN-gamma 2 to 6 h later reversed the inhibition of macrophage larval killing. More importantly, the combination of suboptimal levels of any two of these three cytokines was found to give a potent synergistic suppression of schistosomulum killing by IFN-gamma-treated macrophages. Similarly, IL-10, IL-4, or TGF-beta alone blocked the production of NO, and when used in combination these cytokines exhibited an enhanced inhibitory effect on nitrite production. Macrophage-mediated killing of schistosomula through the generation of NO has been shown previously to be a major effector mechanism of schistosome immunity. The results presented here suggest that the suppression of this mechanism by induction of the regulatory cytokines IL-10, IL-4, and TGF-beta, which are known to be produced during schistosome infection, may be an important strategy used by the parasite to evade macrophage-mediated immune destruction.     

T-cell protein tyrosine phosphatase (Tcptp) is a negative regulator of colony-stimulating factor 1 signaling and macrophage differentiation

Mice null for the T-cell protein tyrosine phosphatase (Tcptp-/-) die shortly after birth due to complications arising from the development of a systemic inflammatory disease. It was originally reported that Tcptp-/- mice have increased numbers of macrophages in the spleen; however, the mechanism underlying the aberrant growth and differentiation of macrophages in Tcptp-/- mice is not known. We have identified Tcptp as an important regulator of colony-stimulating factor 1 (CSF-1) signaling and mononuclear phagocyte development. The number of CSF-1-dependent CFU is increased in Tcptp-/- bone marrow. Tcptp-/- mice also have increased numbers of granulocyte-macrophage precursors (GMP), and these Tcptp-/- GMP yield more macrophage colonies in response to CSF-1 relative to wild-type cells. Furthermore, we have identified the CSF-1 receptor (CSF-1R) as a physiological target of Tcptp through substrate-trapping experiments and its hyperphosphorylation in Tcptp-/- macrophages. Tcptp-/- macrophages also have increased tyrosine phosphorylation and recruitment of a Grb2/Gab2/Shp2 complex to the CSF-1R and enhanced activation of Erk after CSF-1 stimulation, which are important molecular events in CSF-1-induced differentiation. These data implicate Tcptp as a critical regulator of CSF-1 signaling and mononuclear phagocyte development in hematopoiesis.     

Effects of recombinant human granulocyte and macrophage colony-stimulating factors on signal transduction pathways in human granulocytes

We studied the ability of the recombinant human-active hemopoietic growth factors granulocyte-macrophage colony-stimulating factor (GM-CSFrh) and granulocyte colony-stimulating factor (G-CSFrh) to activate receptor-mediated transduction pathways which have been implicated in the stimulation of cytotoxic functions in granulocytes. With the use of a panel of fluorescent probes, we found that these two growth factors exerted no detectable immediate effect on the resting transmembrane electrical potential, the intracellular concentration of free calcium ions, or the cytosolic pH of isolated, mature granulocytes. However, when granulocytes were "primed" by preincubation for 90 min with GM-CSFrh or G-CSFrh, the rate of membrane depolarization induced by 10(-7) M N-formyl-methionyl-leucyl-phenylalanine, but not the rate of rise in free calcium ions, was greatly accelerated. In examining potential mechanisms to account for the priming effect of these growth factors, we found that although they did not induce translocation of protein kinase C or stimulate significant degranulation, they each directly caused prompt release of arachidonic acid from plasma membrane phospholipids. Our data indicate that although GM-CSFrh and G-CSFrh do not activate the transduction signals that have most clearly been implicated in receptor-mediated activation of cytotoxic functions in granulocytes--namely, those coupled to membrane depolarization or release of intracellular calcium ions--they appear directly to induce the release of arachidonic acid esterified to membrane phospholipids, an event which may represent the receptor-mediated activation of membrane phospholipases and which may contribute to the "priming" of the cells for enhancement of their functional responsiveness.     

Murine B-cell stimulatory factor-1 (BSF-1)/interleukin-4 (IL-4) is a multilineage colony-stimulating factor that acts directly on primitive hemopoietic progenitors

Interleukin-4 (IL-4), which was originally identified as a B-cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL-4 on early hemopoetic progenitors in methylcellulose culture. IL-4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5-fluorouracil (5-FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI-3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL-4. To test whether or not IL-4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL-3 and IL-4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL-4 was significantly smaller than that supported by IL-3. When colony-supporting abilities of IL-4 and IL-3 were compared using day-4 post-5-FU spleen and day-2 post-5-FU marrow cells, IL-4 supported the formation of fewer blast cell colonies than did IL-3. IL-4 and IL-6 revealed synergy in support of colony formation from day 2 post-5-FU marrow cells. These results indicate that murine IL-4 is another direct-acting multilineage colony-stimulating factor (multi-CSF), similar to IL-3, that acts on primitive hemopoietic progenitors.