Vitam Kodelja

Alternative versus Classical Activation of Macrophages

In parallel to the Th1/Th2 paradigm, antigen-presenting cells (APC) are divided into classically activated APC (dendritic cells/effector macrophages) and alternatively activated APC (IL-4-induced, alternatively activated macrophages/IL-10-induced, immature dendritic cells). Alternatively activated APC share a special molecular repertoire including receptors of innate immunity with broad specificity for foreign antigen and anti-inflammatory cytokines such as IL-1Ra and alternative macrophage activation-associated CC-chemokine-1. Alternatively activated APC mediated tolerance and downregulated inflammation. Abuse of alternatively activated APC in support of infectious susceptibility or tumor immune escape is counteracted by the classical pathway. Thus, classically and alternatively activated APC secure the balance between proinflammatory and anti-inflammatory immune reactions.

DIFFERENCES IN ANGIOGENIC POTENTIAL OF CLASSICALLY VS ALTERNATIVELY ACTIVATED MACROPHAGES

Macrophages (M phi) are important for angiogenesis during inflammation, wound repair, and tumor growth. However, well-characterized M phi subsets such as IFN-gamma-induced, classically activated (ca) M phi or IL-4/glucocorticoid-induced, alternatively activated (aa) M phi have not been thoroughly examined for a positive or negative association with angiogenesis. While caM phi populate early inflammatory reactions and high-turnover granulomas, aaM phi occur in healing wounds and chronic inflammation. In contrast to caM phi-dominated lesions, aaM phi-rich lesions are highly vascularized. In order to determine their angiogenic potential in vitro, these M phi subsets as well as unstimulated control macrophages (coM phi) were analyzed by RT-PCR for mRNA expression of 10 angiogenic factors after 3 and 6 days of culture. Early during activation, caM phi and coM phi expressed equal levels of 8 of 10 angiogenic factors (PDGF-A, MK, TNF-alpha, TGF-beta 1, PDGF-B, HGF, TGF-alpha, IGF-1), while aaM phi showed expression of only 4 of these factors (TGF-beta 1, PDGF-B, HGF, GF-1). After maturation, TGF-alpha and IGF-1 showed a shift in mRNA expression from caM phi to aaM phi resulting in a considerably enhanced expression of these factors in day-6 aaM phi as compared to day-6 caM phi and coM phi while PDGF-A, MK, and TNF-alpha remained suppressed in day 6 aaM phi. In all M phi subsets including controls, mRNA expression of aFGF and bFGF was minimal or absent while TGFG-beta 1, HGF, and ODGF-B were constitutively expressed. In order to functionally integrate angiogenic factor mRNA expression profiles, mitogenic activity of M phi subsets towards microvascular endothelium was assessed by cocultivation. Coculture experiments revealed that endothelial proliferation induced by aaM phi was 3.0-3.5x higher than induced by caM phi. In conclusion, mature aaM phi are well equipped to play an important role in protracted M phi-associated angiogenic processes. Presumably due to expression of predominantly angio-inhibitory cytokines such as TNF-alpha by caM phi but much less by aaM phi, caM phi exhibit only a low angiogenic potential in vitro and in vivo despite considerable expression of angiogenic factor mRNA.

Immunohistochemical identification of type II alternatively activated dendritic macrophages (RM 3/1+++, MS-1± 25F9−) in psoriatic dermis

Immunological mechanisms play an important role in the pathogenesis of psoriasis. Lesional psoriatic skin-derived T-cell clones have been shown to stimulate keratinocyte proliferation and to predominantly express a T-helper type 1 cytokine pattern. However, T-helper type 2-like cytokines have also been identified in some psoriatic T-cell clones. In parallel to the T-helper type 1/type 2 dichotomy, a distinction between interferon-γ-induced (classically activated) macrophages and interleukin-4/glucocorticoid-induced (alternatively activated) macrophages has been put forward as a conceptual framework for a better understanding of immunopathological processes. In the present study, the phenotype of mononuclear phagocytes in psoriatic skin lesions (n=21), allergic contact dermatitis (n=4) and normal skin (n=2) was investigated using a panel of monoclonal antibodies (mAb) against monocytes/macrophages and dendritic cells (mAb MS-1, RM 3/1 and 25F9 against subsets of in vitro alternatively activated macrophages, and mAb against myeloid antigens CD1a, CD11b, CD11c, CD34, CD36, and CD68). With regard to mononuclear phagocytes, psoriatic skin was found to be compart-mentalized into epidermis, subepidermal space, and upper and lower dermis. RM 3/1+++, MS-1± 25F9− dendritic macrophages previously classified as type II alternatively activated macrophages were the dominant dermal macrophage population in psoriatic skin, while intraepidermal and epithelium-lining macrophages expressed a different, presumably classically activated, macrophage phenotype (RM 3/1−, MS-1−, 25F9−, CD68++, CD11b++). In allergic contact dermatitis a classical T-helper type 1 disease, RM 3/1+++ macrophages were less prominent. Since MS-1 high molecular weight protein is much more sensitive to interferon-γ-induced suppression than RM 3/1 antigen a predominance of T-helper type 1 cytokines in psoriasis could explain why dermal dendritic macrophages do not express the fully induced MS-1+++, RM 3/1+++, 25F9± phenotype of type I alternatively activated macrophages.

Expression of vascular endothelial growth factor (VEGF) in various compartments of the human hair follicle

Abstract Hair follicle vascularization appears to be closely related to the processes involved in hair cycle regulation, in which growth factors, cytokines and other bioactive molecules are involved. In particular, vascular endothelial growth factor (VEGF), essential for angiogenesis and vascular permeability, may be responsible for maintaining proper vasculature around the hair follicle during the anagen growth phase. The aim of our study was to compare the in vitro angiogenic capacity, i.e. the steady-state expression of the VEGF gene, of different cultured cell types derived from normal human hair follicles, corresponding to different follicular compartments. Human dermal papilla cells (DPC), fibrous sheath fibroblasts, dermal fibroblasts, and follicular and interfollicular keratinocytes were cultured and studied in vitro for VEGF expression at the mRNA level using RT-PCR, and for VEGF protein synthesis by radioimmunoprecipitation and immunocytochemistry. In vivo examination for VEGF expression in human terminal hair follicles was performed using immunohistochemical methods. In the present report the expression of four different VEGF molecular isoforms, differing in their angiogenic capacity, are described in different cultured follicular cell types for the first time. Cultured follicular cells strongly expressed mRNA of four VEGF molecular species identified as the 121-, 145-, 165- and 189-amino acid splice variants, the most prominent being the 121-amino acid molecule. DPC, and also other mesenchymal cells such as fibrous sheath fibroblasts and dermal fibroblasts, in vivo and in vitro strongly expressed VEGF mRNA and synthesized a 46-kDa VEGF protein, whereas follicular and interfollicular keratinocytes in vitro expressed lower levels of VEGF mRNA and proteins than mesenchymal cells. As the highest expression of VEGF was found in DPC, we suggest that DPC are mainly responsible for angiogenic processes possibly related to the human hair cycle.

Dissection of macrophage differentiation pathways in cutaneous macrophage disorders and in vitro

Abstract Macrophages play important roles in immunity and inflammation, and in allergic, granulomatous and neoplastic diseases. Here, we present the indepth results of an ongoing study of macrophage differentiation pathways in cutaneous macrophage disorders and in vitro. Up to now, a total of 40 cases of cutaneous macrophage disorders (histiocytoses and granulomas) and related diseases were examined using a panel of monoclonal and polyclonal antibodies to macrophage differentiation antigens (mAb MS‐1, mAb αCDla, mAb αCD34, mAb RM 3/1, mAb αCD11c, mAb αCD36, mAb MAC 387, mAb 27E10, polyclonal antibodies ccMRP‐8 and ‐14, mAb aCD68. mAb 25F9, mAb DRC1‐R4/23, and mAb 1F10). Of these, MS‐1 high molecular weight protein, synthesized by non‐continuous sinusoidal endothelial cells and highly dendritic perivascular macrophages in normal human organs, is the most specific macrophage differentiation marker. MS‐1 high molecular weight protein is selectively expressed by cutaneous non‐Langerhans cell histiocytoses, and proves to be a valuable diagnostic tool for these diseases. MS‐1 high molecular weight protein is not found in Langerhans cell histiocytosis cells, epithelioid cells in sarcoidosis, and palisading histio‐cytes in granuloma annulare. MS‐1+ macrophages may be found intermingled in cellular type dermatofibroma and in foreign body granulomas; they differ from MS‐1+ non‐Langerhans cell histiocytosis cells by their highly dendritic morphology, and thus rather resemble the MS‐1+ macrophages in normal skin. RM 3/1 antigen shows a similar, but broader expression pattern including non‐Langerhans cell histiocytoses, xanthel‐asmata palpebrarum, foreign body granulomas, granuloma annulare, and cellular type dermatofibroma. Moreover, xanthelasmata palpebrarum para‐digmatically represent a class of macrophage lesions with strong RM 3/1, but little MS‐1 antigen expression. In sarcoidosis, RM 3/1 + macrophages are only found at the very periphery of epithelioid cell granulomas. In contrast, 25F9 antigen is strongly and consistently expressed in epithelioid cells of sarcoidosis, and in foreign body granulomas. In cultured human monocytes/macrophages, RM 3/1 antigen is expressed early on, while MS‐1 high molecular weight protein and 25F9 antigen are late and very late macrophage differentiation antigens, respectively. Expression of RM 3/1 antigen and MS‐1 high molecular weight protein is inducible by glucocorticoid and interleukin‐4, and less so by interleukin‐13 and interlcukin‐10, and combinations thereof, while 25F9 antigen seems to be less influenced by these agents. Interferon‐y (and less so tumor necrosis factor‐ex) inhibit expression of all three antigens in cultured human monocytes/macrophages. In monocytic leukemia cell line THP‐1. RM 3/1 antigen is only induced by a combined treatment of phorbolester and glucocorticoid and peaks at 3‐7 days. In different subclones of the cell line, 25F9 antigen is either inducible by phorbolester alone or by combined treatment with glucocorticoid or it is constitutively expressed and hardly modulated. In contrast, MS‐1 high molecular weight protein cannot be induced in THP‐1 cells by the agents tested. Inhibition of MS‐1 high molecular weight protein, and RM 3/1 and 25F9 antigens by interferon‐y suggests that macrophages characterized by these phenotypic traits must by counted among the alternatively activated macrophage populations. As a result of our study, alternative macrophage activation should be viewed as a multifacetted process resulting in various, partially overlapping, partially complementary macrophage phenolypes both in vivo and in vitro.

The mononuclear phagocyte–dendritic cell dichotomy: myths, facts, and a revised concept

Since Aschoff s reticuloendothelial system was abandoned a few decades ago, classification and characterization of the mononuclear phagocyte and dendritic cell systems have evolved separately or even in competition with one another. New information has now become available indicating that monocytes/macrophages and dendritic cells have a common origin in the bone marrow, and may even transdifferentiate. Morphological and functional distinctions—although valid under certain conditions—have been blurred by revelation of the versatility of monocytes/macrophages and dendritic cells in response to different contextual needs in inflammation and immunity. Monocytes/macrophages and dendritic cells share a sentinel, receptor/effector, and presentation mode, and may either activate or silence specific immune reactions. In keeping with the view of monocytes/macrophages and dendritic cells as interactive sentinels, we suggest that the mononuclear phagocyte and dendritic cell systems be replaced by the custocyte system (custos, Lat = sentinel, guard) as a unifying concept. Within the custocyte system, we recognize type I, type II, and type III custocytes. Type I and II custocytes exhibit predominance of presentation or effector/presenter interdependency, respectively, while type III custocytes are bipolar, passing through type I‐ and type II‐like phases during their development and in inflammatory responses. The custocyte system brings into view monocytes/macrophages and dendritic cells as dynamic players in immunity and inflammation with a high degree of derivational, phenotypic, functional, and molecular plasticity.

Balanced Macrophage Activation Hypothesis: A Biological Model for Development of Drugs Targeted at Macrophage Functional States

Macrophages play a central role in the immune response and are major targets for chronic infection with viruses such as HIV. Recent studies on macrophage differentiation have shown the existence of classical activation and the counter-balancing anti-inflammatory alternative activation states. In the ‘balanced macrophage activation hypothesis’ we propose that macrophage activation is a cyclic process that balances these two states to achieve proper immunologic function. Dysregulation of this cycle would, therefore, be associated with various forms of chronic disease. This model has been utilized in the drug development of WF10, a novel macrophage-targeted drug, currently in advanced clinical testing for the treatment of HIV disease.

Langerhans cells do not express alternative macrophage activation-associated CC chemokine (AMAC)-1

We have cloned a novel human CC chemokine, alternative macrophage activation-associated CC chemokine (AMAC)-1 that is highly homologous to macrophage inflammatory protein (MIP)-1alpha. In contrast to MIP-1alpha, AMAC-1 is induced in macrophages by Th2-associated cytokines IL4, IL13, and IL10 in vitro; in addition, AMAC-1 is expressed by Th1-suppressive alveolar macrophages in vivo. Surprisingly, however, AMAC-1 is also expressed by GM-CSF-induced, in vitro monocyte-derived dendritic cells when treated by IL4. Here, we present a detailed analysis of AMAC-1 expression in monocyte-derived dendritic cells in vitro and show that the prime dendritic cells in vivo, i.e. epidermal Langerhans cells, do not express AMAC-1 mRNA. In conclusion, AMAC-1 is a novel CC chemokine whose Th2-associated expression pattern in alternatively activated suppressor macrophages in vivo and in vitro and its absence from epidermal Langerhans cells in vivo suggest that it may be involved in inhibition of Th1 reactions and in tolerance induction.