Macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status.

BACKGROUND: Macrophages play a dual role in multiple sclerosis (MS) pathology. They can exert neuroprotective and growth promoting effects but also contribute to tissue damage by production of inflammatory mediators. The effector function of macrophages is determined by the way they are activated. Stimulation of monocyte-derived macrophages in vitro with interferon-γ and lipopolysaccharide results in classically activated (CA/M1) macrophages, and activation with interleukin 4 induces alternatively activated (AA/M2) macrophages. METHODS: For this study, the expression of a panel of typical M1 and M2 markers on human monocyte derived M1 and M2 macrophages was analyzed using flow cytometry. This revealed that CD40 and mannose receptor (MR) were the most distinctive markers for human M1 and M2 macrophages, respectively. Using a panel of M1 and M2 markers we next examined the activation status of macrophages/microglia in MS lesions, normal appearing white matter and healthy control samples. RESULTS: Our data show that M1 markers, including CD40, CD86, CD64 and CD32 were abundantly expressed by microglia in normal appearing white matter and by activated microglia and macrophages throughout active demyelinating MS lesions. M2 markers, such as MR and CD163 were expressed by myelin-laden macrophages in active lesions and perivascular macrophages. Double staining with anti-CD40 and anti-MR revealed that approximately 70% of the CD40-positive macrophages in MS lesions also expressed MR, indicating that the majority of infiltrating macrophages and activated microglial cells display an intermediate activation status. CONCLUSIONS: Our findings show that, although macrophages in active MS lesions predominantly display M1 characteristics, a major subset of macrophages have an intermediate activation status.

Detrimental dermal wound healing: what can we learn from the oral mucosa?

Wounds in adults are frequently accompanied by scar formation. This scar can become fibrotic due to an imbalance between extracellular matrix (ECM) synthesis and ECM degradation. Oral mucosal wounds, however, heal in an accelerated fashion, displaying minimal scar formation. The exact mechanisms of scarless oral healing are yet to be revealed. This review highlights possible mechanisms involved in the difference between scar-forming dermal vs. scarless oral mucosal wound healing. Differences were found in expression of ECM components, such as procollagen I and tenascin-C. Oral wounds contained fewer immune mediators, blood vessels, and profibrotic mediators but had more bone marrow-derived cells, a higher reepithelialization rate, and faster proliferation of fibroblasts compared with dermal wounds. These results form a basis for further research that should be focused on the relations among ECM, immune cells, growth factors, and fibroblast phenotypes, as understanding scarless oral mucosal healing may ultimately lead to novel therapeutic strategies to prevent fibrotic scars.

The number of immune cells is lower in healthy oral mucosa compared to skin and does not increase after scarring.

OBJECTIVE: Depending on the location of injury, wounds can heal with different outcomes. In addition foetal wounds heal fast without scar formation, while scars are a common feature of regular skin repair. Since inflammation is very limited in these wounds reduced numbers or even absence of immune cells might be responsible for scarless foetal wound healing. It is thought that various immune cells, such as macrophages, neutrophils and T-cells, play a role in aberrant wound healing and the fibrotic process seen in scar formation in the adult skin. Similar to the foetus, oral wounds show comparable healing properties by means of accelerated reepithelialization and negligible scar formation. It is possible that reduced inflammatory reaction as a result of lower numbers of immune cells are present in oral wounds compared to skin wounds. DESIGN: Here we investigated the presence of various immune cells in human skin and oral mucosa, with or without scars. The presence or absence of these cells may play a role in the different modes of healing observed between the two types of tissue. Mast cells, neutrophils, M1/M2 macrophages, T-cells and blood vessels were localized in healthy and scarred skin and oral mucosa (scars>1 year old). RESULTS: Oral mucosa had significantly fewer neutrophils, macrophages, mannose receptor-positive M2 macrophages, but more blood vessels. Scars contained similar numbers of immune cells compared to healthy tissues. CONCLUSIONS: Less immune cells in the healthy oral mucosa may induce a diminished immune reaction when wounding occurs, and could explain the better healing capacity of the oral mucosa.

Extracellular matrix components of oral mucosa differ from skin and resemble that of foetal skin.

OBJECTIVE: Wounds of both the oral mucosa and early-to-mid gestation foetuses have a propensity to heal scarless. Repair of skin wounds in adults, however, regularly results in scar formation. The extracellular matrix (ECM) plays an important role in the process of healing. The fate of scarless or scar forming healing may already be defined by the ECM composition, prior to wounding. In this study, the presence of several ECM components in oral mucosa (palatum) and skin was investigated. DESIGN: Immunohistochemical stainings of different ECM components were performed on skin, obtained from abdominal dermolipectomy surgery, and oral mucosa, derived after pharynx reconstruction. RESULTS: Expression of fibronectin, its splice variant ED-A, and chondroitin sulphate was elevated in oral tissue, whereas elastin expression was higher in skin. Tenascin-C, hyaluronic acid, biglycan, decorin, and syndecan-1 were expressed at similar levels in both tissues. Oral mucosa contained more blood vessels than skin samples. Finally, oral keratinocytes proliferated more, while dermal keratinocytes demonstrated higher differentiation. CONCLUSIONS: Comparing ECM components of the skin and oral mucosa coincides with differences earlier observed between foetal and adult skin, and this might indicate that some ECM components are involved in the mode of repair.

Human macrophage polarization in vitro: maturation and activation methods compared.

Macrophages form a heterogeneous cell population displaying multiple functions, and can be polarized into pro- (M1) or anti-inflammatory (M2) macrophages, by environmental factors. Their activation status reflects a beneficial or detrimental role in various diseases. Currently several in vitro maturation and activation protocols are used to induce an M1 or M2 phenotype. Here, the impact of different maturation factors (NHS, M-CSF, or GM-CSF) and activation methods (IFN-γ/LPS, IL-4, dexamethason, IL-10) on the macrophage phenotype was determined. Regarding macrophage morphology, pro-inflammatory (M1) activation stimulated cell elongation, and anti-inflammatory (M2) activation induced a circular appearance. Activation with pro-inflammatory mediators led to increased CD40 and CD64 expression, whereas activation with anti-inflammatory factors resulted in increased levels of MR and CD163. Production of pro-inflammatory cytokines was induced by activation with IFN-γ/LPS, and TGF-ß production was enhanced by the maturation factors M-CSF and GM-CSF. Our data demonstrate that macrophage marker expression and cytokine production in vitro is highly dependent on both maturation and activation methods. In vivo macrophage activation is far more complex, since a plethora of stimuli are present. Hence, defining the macrophage activation status ex vivo on a limited number of markers could be indecisive. From this study we conclude that maturation with M-CSF or GM-CSF induces a moderate anti- or pro-inflammatory state respectively, compared to maturation with NHS. CD40 and CD64 are the most distinctive makers for human M1 and CD163 and MR for M2 macrophage activation and therefore can be helpful in determining the activation status of human macrophages ex vivo.

Platelet derived growth factor-CC secreted by M2 macrophages induces alpha-smooth muscle actin expression by dermal and gingival fibroblasts.

Dermal wounds can heal detrimentally by formation of excess fibrosis or hypertrophic scarring. These phenomena are normally absent in the oral mucosa. Macrophages play an important role in wound repair, have a marked heterogeneity and are thought to contribute to fibrosis. To investigate to what extend macrophages are involved in the occurrence of fibrosis, the effect of differently activated macrophages on dermal and gingival fibroblasts was studied in vitro. Macrophages were differentiated into a classical (M1) or alternative (M2) phenotype, which was assessed by receptor expression (CD40/mannose receptor) and cytokine secretion (interleukin-4 and -12). Fibroblasts were exposed to these macrophages and/or conditioned medium (cm), and differentiation into α-SMA-expressing myofibroblasts was quantified. M2, but not M1 macrophages induced α-SMA expression in both dermal and gingival fibroblasts. Blocking of transforming growth factor-ß1 did not decrease the α-SMA expression mediated by M2 macrophages. It appeared that this induction was mediated by platelet derived growth factor-CC (PDGF-CC), produced by M2 macrophages. The expression and role of this growth factor was confirmed by ELISA, RT-PCR, and blocking experiments. Our results indicate that M2 macrophages are able to induce myofibroblast differentiation via production of PDGF-CC. Based on our findings we conclude that PDGF-CC may play a hitherto unknown role in the differentiation of myofibroblasts.