John A. Hamilton

Colony-stimulating factors in inflammation and autoimmunity

Although they were originally defined as haematopoietic-cell growth factors, colony-stimulating factors (CSFs) have been shown to have additional functions by acting directly on mature myeloid cells. Recent data from animal models indicate that the depletion of CSFs has therapeutic benefit in many inflammatory and/or autoimmune conditions and as a result, early-phase clinical trials targeting granulocyte/macrophage colony-stimulating factor and macrophage colony-stimulating factor have now commenced. The distinct biological features of CSFs offer opportunities for specific targeting, but with some associated risks. Here, I describe these biological features, discuss the probable specific outcomes of targeting CSFs in vivo and highlight outstanding questions that need to be addressed.

Macrophage plasminogen activator: Modulation of enzyme production by anti-inflammatory steroids, mitotic inhibitors, and cyclic nucleotides

Plasminogen activator production by cultured mouse peritoneal macrophages can be modulated in vitro by low concentrations of various pharmacologically active molecules. Glucocorticoid hormones and their synthetic derivatives, as well as cholera toxin, colchicine, and vinblastine markedly inhibit production of this enzyme without affecting other important macrophage functions. The effect of glucocorticoids is of particular interest, both because their relative in vivo anti-inflammatory potencies correlate exactly with their effect on plasminogen activator production in culture and because this effect occurs at near physiological concentrations. In view of the correlations established in other systems between plasminogen activator production and cell migration, we have also examined the age of the macrophages in thioglycollate-induced exudates. Confirming the results of Van Furth and Cohn (1968), we have found that the majority of these cells are young, having recently replicated and arrived in the peritoneal cavity. Using a fibrinagar overlay technique which allowed us to determine the production of plasminogen activator by individual cells. we have found that the majority of these cells produce the enzyme. The potential roles of plasminogen activator in monocyte migration and the relationship of this enzyme to the anti-inflammatory effect of gluccorticoids are correlated and emphasized.

Granulocyte-Macrophage Colony-Stimulating Factor (CSF) and Macrophage CSF-Dependent Macrophage Phenotypes Display Differences in Cytokine Profiles and Transcription Factor Activities: Implications for CSF Blockade in Inflammation

GM-CSF and M-CSF (CSF-1) can enhance macrophage lineage numbers as well as modulate their differentiation and function. Of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. What the critical actions are of these CSFs on macrophages during inflammatory reactions are unknown. To address this issue, adherent macrophages (GM-BMM and BMM) were first derived from murine bone marrow precursors by GM-CSF and M-CSF, respectively, and stimulated in vitro with LPS to measure secreted cytokine production, as well as NF-κB and AP-1 activities. GM-BMM preferentially produced TNF-α, IL-6, IL-12p70, and IL-23 whereas, conversely, BMM generated more IL-10 and CCL2; strikingly the latter population could not produce detectable IL-12p70 and IL-23. Following LPS stimulation, GM-BMM displayed rapid IκBα degradation, RelA nuclear translocation, and NF-κB DNA binding relative to BMM, as well as a faster and enhanced AP-1 activation. Each macrophage population was also pretreated with the other CSF before LPS stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and NF-κB activity. Thus, GM-CSF and M-CSF demonstrate, at the level of macrophage cytokine production, different and even competing responses with implications for their respective roles in inflammation, including a possible dampening or suppressive role for M-CSF in certain circumstances.

GM-CSF in inflammation and autoimmunity.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is now best viewed as a major regulator governing the functions of granulocyte and macrophage lineage populations at all stages of maturation. There is recent evidence for a key role for GM-CSF in inflammatory and autoimmune diseases, therefore making it worthy of consideration for targetting. Such evidence includes disease exacerbation following its administration and amelioration of disease in animal models by GM-CSF gene targetting or by anti-GM-CSF antibody blockade. The interdependence of GM-CSF formation and that of the important proinflammatory cytokines, interleukin-1 and tumour necrosis factor-alpha (TNF-alpha), is discussed, as is the greater disease suppression found in arthritis models following GM-CSF depletion compared with that observed in the absence of TNF-alpha.

Defining GM-CSF– and Macrophage-CSF–Dependent Macrophage Responses by In Vitro Models

GM-CSF and M-CSF (CSF-1) induce different phenotypic changes in macrophage lineage populations. The nature, extent, and generality of these differences were assessed by comparing the responses to these CSFs, either alone or in combination, in various human and murine macrophage lineage populations. The differences between the respective global gene expression profiles of macrophages, derived from human monocytes by GM-CSF or M-CSF, were compared with the differences between the respective profiles for macrophages, derived from murine bone marrow cells by each CSF. Only 17% of genes regulated differently by these CSFs were common across the species. Whether a particular change in relative gene expression is by direct action of a CSF can be confounded by endogenous mediators, such as type I IFN, IL-10, and activin A. Time-dependent differences in cytokine gene expression were noted in human monocytes treated with the CSFs; in this system, GM-CSF induced a more dramatic expression of IFN-regulated factor 4 (IRF4) than of IRF5, whereas M-CSF induced IRF5 but not IRF4. In the presence of both CSFs, some evidence of “competition” at the level of gene expression was observed. Care needs to be exercised when drawing definitive conclusions from a particular in vitro system about the roles of GM-CSF and M-CSF in macrophage lineage biology.

The colony‐stimulating factors and collagen‐induced arthritis: exacerbation of disease by M‐CSF and G‐CSF and requirement for endogenous M‐CSF

There is increasing evidence that the colony‐stimulating factors (CSFs) may play a part in chronic inflammatory autoimmune diseases, such as rheumatoid arthritis (RA). We examined the involvement of macrophage CSF (M‐CSF or CSF‐1) and granulocyte CSF (G‐CSF) in collagen‐induced arthritis (CIA), a murine model of RA. Daily injections of M‐CSF or G‐CSF, 20–24 days postprimary immunization with type II collagen, exacerbated disease symptoms in suboptimally immunized DBA/1 mice. Support for the involvement of endogenous M‐CSF in CIA was obtained by studies in which neutralizing monoclonal antibody reduced the severity of established CIA and also by studies showing the resistance of M‐CSF‐deficient op/op mice to CIA induction. These studies show that M‐CSF and G‐CSF can be proinflammatory in CIA and provide evidence that macrophage‐ and granulocyte‐lineage cells can exacerbate CIA. Our results also show that M‐CSF‐dependent cells are essential for CIA development, suggesting M‐CSF may be a suitable target for therapeutic intervention in RA.

Macrophage plasminogen activator: induction by concanavalin A and phorbol myristate acetate

The synthesis and secretion of plasminogen activator by cultured macrophages can be induced and stimulated by concanavalin A and by phorbol myristate acetate, and inhibited by such agents as glucocorticoids, mitotic inhibitors and compounds affecting cAMP metabolism. By the manipulation of stimulatory and inhibitory influences, enzyme production can be modulated continuously over a 200 fold range. In the same way, the proportion of cells that secrete detectable levels of enzyme can be varied from 1-90%. No comparable modulation of lysozyme or acid hydrolase production is observed under the same conditions. These results suggest that the physiological control of macrophage plasminogen activator production is achieved by the interacting effects of mutually antagonistic stimuli; this emphasizes the utility of this enzyme for the study of regulatory phenomena, including those relating to inflammation.

Collagen‐induced arthritis in C57BL/6 (H‐2b) mice: new insights into an important disease model of rheumatoid arthritis

Collagen‐induced arthritis (CIA) is a widely used model of rheumatoid arthritis (RA) and has been important for understanding autoimmunity. CIA is purportedly restricted to mice bearing the MHC class II H‐2q or H‐2r haplotypes. In this study, we re‐examined established concepts regarding susceptibility to CIA. We found mice derived from the C57BL/6 (B6) (H‐2b) background can develop CIA with high incidence (60–70%), and sustained severity by using an immunization procedure modified for optimum response in DBA/1 (D1) (H‐2q) mice. Clinically and histologically the B6 disease resembles that of D1 mice and is dependent on immunization with type II collagen, as well as on B and CD4+ T cells. In contrast, 129/Sv mice, which share H‐2b, are resistant to CIA. We conclude that susceptibility to CIA may reflect immunization conditions and/or important contributions from non‐MHC genes, revealed by different immunization protocols. A practical outcome is that CIA can be directly applied to gene knockout mice generated from B6 embryonic stem cells without need for backcross onto the D1 background. This model may lead to improved understanding of autoimmunity in CIA and RA and may provide a platform for analysis of the contribution of non‐MHC genes to CIA.

GM-CSF- and M-CSF-dependent macrophage phenotypes display differential dependence on Type I interferon signaling

M‐CSF and GM‐CSF are mediators involved in regulating the numbers and function of macrophage lineage populations and have been shown to contribute to macrophage heterogeneity. Type I IFN is an important mediator produced by macrophages and can have profound regulatory effects on their properties. In this study, we compared bone marrow‐derived macrophages (BMM) and GM‐CSF‐induced BMM (GM‐BMM) from wild‐type and IFNAR1−/− mice to assess the contribution of endogenous type I IFN to the phenotypic differences between BMM and GM‐BMM. BMM were capable of higher constitutive IFN‐β production, which contributed significantly to their basal transcriptome. Microarray analysis found that of the endogenous type I IFN‐regulated genes specific to either BMM or GM‐BMM, 488 of these gene alterations were unique to BMM, while only 50 were unique to GM‐BMM. Moreover, BMM displayed enhanced basal mRNA levels, relative to GM‐BMM, of a number of genes identified as being dependent on type I IFN signaling, including Stat1, Stat2, Irf7, Ccl5, Ccl12, and Cxcl10. As a result of prior type I IFN “priming,” upon LPS stimulation BMM displayed increased activation of the MyD88‐independent IRF‐3/STAT1 pathways compared with GM‐BMM, which correlated with the distinct cytokine/chemokine profiles of the two macrophage subsets. Furthermore, the autocrine type I IFN signaling loop regulated the production of the M1 and M2 signature cytokines, IL‐12p70 and IL‐10. Collectively, these findings demonstrate that constitutive and LPS‐induced type I IFN play significant roles in regulating the differences in phenotype and function between BMM and GM‐BMM.

CSF-1 signal transduction.

Colony‐stimulating factor‐1 (CSF‐1) or macrophage‐CSF (M‐CSF) is a growth factor involved in the proliferation, differentiation, and activation of cells of the monocyte/macrophage lineage. Its receptor is the homodimeric, tyrosine kinase product of the c‐fms proto‐oncogene, which contains a so‐called kinase insert domain. This review focuses mainly on recent studies of signal transduction events that are initiated on interaction of CSF‐1 and its receptor. A summary is given of the tyrosine autophosphorylation sites on c‐Fms identified to date, including their interaction with various substrates and their possible significance for signal transduction and cellular function. In addition, the signal transduction pathways that have been identified to lie downstream of activated c‐Fms are reviewed. Although it is apparent that there have been many recent significant developments in our understanding of CSF‐1 signaling, a number of examples are mentioned of significant discrepancies in the literature, some possible reasons for which can sometimes be offered. It is also apparent that any particular biochemical response or signal transduction pathway, even though widespread in other ligand receptor/cellular systems, including those with similar receptor structures to c‐Fms, may not be relevant to CSF‐1 signaling. The relevance of any potentially important molecular signaling pathway activated by CSF‐1 in cells in vitro will ultimately have to be related to the functions of monocytes/macrophages in vivo. J. Leukoc. Biol. 62: 145–155; 1997.