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Tumor-associated macrophages (TAMs) have been detected in most skin cancers. TAMs produce various chemokines and angiogenic factors that promote tumor development, along with other immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and tumor-associated neutrophils. TAMs generated from monocytes develop into functional, fully activated macrophages, and TAMs obtain various immunosuppressive functions to maintain the tumor microenvironment. Since TAMs express PD1 to maintain the immunosuppressive M2 phenotype by PD1/PD-L1 signaling from tumor cells, and the blockade of PD1/PD-L1 signaling by anti-PD1 antibodies (Abs) activate and re-polarize TAMs into immunoreactive M1 phenotypes, TAMs represent a potential target for anti-PD1 Abs. The main population of TAMs comprises CD163 + M2 macrophages, and CD163 + TAMs release soluble (s)CD163 and several proinflammatory chemokines (CXCL5, CXCL10, CCL19, etc.) as a result of TAM activation to induce an immunosuppressive tumor microenvironment together with other immunosuppressive cells. Since direct blockade of PD1/PD-L1 signaling between tumor cells and tumor-infiltrating T cells (both effector T cells and Tregs) is mandatory for inducing an anti-immune response by anti-PD1 Abs, anti-PD1 Abs need to reach the tumor microenvironment to induce anti-immune responses in the tumor-bearing host. Taken together, TAM-related factors could offer a biomarker for anti-PD1 Ab-based immunotherapy. Understanding the crosstalk between TAMs and immunosuppressive cells is important for optimizing PD1 Ab-based immunotherapy.

Minocycline/tetracycline is clinically used for the treatment of bullous pemphigoid (BP), and its clinical benefits are superior to those of prednisolone when considering adverse events. Although the clinical benefits of minocycline/tetracycline are well known, its immunosuppressive mechanisms are still unclear. In this study, we investigated the immunomodulatory effects of traditional anti-BP drugs (minocycline, nicotinic acid amide, dexamethasone and cyclosporine) on CD163 + M2 macrophages in vitro, with special focus on the production of CCL18 and CCL22, both of which are produced by CD163 + M2 macrophages in the lesional skin of BP and are increased in the serum of BP patients. Minocycline decreased the production of CCL22, CCL24 and CCL26 as well as CCL2 from M2 macrophages. CCL18 from M2 macrophages was decreased by dexamethasone and cyclosporine, but not decreased by minocycline. These data suggest that the clinical benefit of minocycline is partially explained by its suppressive effects against the production of specific Th2 chemokines from M2 macrophages, which should contribute to the recruitment of Th2 cells and eosinophils in the lesional skin of BP patients., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Primary cutaneous apocrine carcinoma (PCAC) is a rare and highly aggressive tumor entity. Since there is no conventional therapy for advanced PCAC, exploratory treatments are sometimes used. As we previously reported, receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL)/RANK signaling on M2 macrophages promotes the production of chemokines and proinflammatory cytokines to maintain the immunosuppressive tumor environment of extramammary Paget's disease (EMPD). Since EMPD is a skin adenocarcinoma of apocrine gland origin that expresses high levels of RANKL and matrix metalloproteinase (MMP) 7, and EMPD is associated with the presence of RANK + M2 macrophages, we hypothesized that tumor-associated macrophages (TAMs) in adenocarcinomas such as PCAC might also express RANKL and MMP7., Materials and Methods: We employed immunohistochemical staining of RANKL and MMP7 in the lesional skin from five patients with PCAC, and microarray analysis of MMPs using human monocyte-derived macrophages., Results: According to DNA microarray analysis, the expression of MMP1 and MMP25 was augmented. The DNA microarray results were verified by using real-time polymerase chain reaction (RT-PCR). Immunohistochemical staining of MMP1 and MMP25 as well as chemokine (C-C motif) ligand (CCL) 5 in the lesional skin from five patients with PCAC showed a substantial number of MMP1-bearing cells and MMP25-bearing cells, as well as CCL5-producing cells, that were distributed in the lesional skin., Conclusion: Our study suggests that the RANKL/RANK pathway contributes to the development and maintenance of the immunosuppressive tumor microenvironment and denosumab may be a promising adjuvant therapy targeting TAMs in cancer of apocrine origin., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Together with regulatory T cells (Tregs), tumor-associated macrophages (TAMs) play roles in maintaining the tumor microenvironment. Although cytotoxic antimelanoma drugs such as dacarbazine (DTIC), nimustine hydrochloride (ACNU) and vincristine (VCR) have been used for the treatment of malignant melanoma as adjuvant therapy in Japan, the detailed mechanisms of their immunomodulatory effects are not fully understood. As the majority of TAMs are alternatively activated M2 macrophages that favour tumor development, the aim of this study was to elucidate the immunomodulatory effects of these reagents on human monocyte-derived M2 macrophages. First, mRNA expressions and protein production of immune checkpoint molecules, PD-L1 and chemokines by CD163 + CD206 + M2 macrophages derived from peripheral blood mononuclear cells were investigated to determine the immunomodulatory effects of DTIC, ACNU, and VCR. DTIC and VCR significantly decreased PD-L1 mRNA expression, which was confirmed by flow cytometry. Moreover, the mRNA expression and production of CCL22 were significantly decreased by DTIC, which suggested that DTIC might suppress the recruitment of Tregs in the tumor site. Furthermore, the decreased expression of PD-L1 and production of CCL22 were validated in vivo, using the B16F10 mouse melanoma model, leading to abrogation of the suppressive function of T-cell proliferation. The present report suggests one of the possible antimelanoma mechanisms of DAV combination chemotherapy for melanoma patients., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pemphigus vulgaris (PV) and bullous pemphigoid (BP) are autoimmune blistering diseases, and substantial numbers of CD163 + tissue-associated macrophages (TAMs) are detected in both diseases. PV and BP possess different subsets of helper T cells, suggesting that the cytokine profiles of PV and BP might be different. The purpose of this study was to investigate the microenvironment of lesional skin and serum of PV and BP patients, focusing on the immunomodulatory factors related to TAMs, such as periostin (POSTN), chemokines, cytokines and matrix metalloproteinases (MMPs). We first performed immunohistological staining of POSTN in PV and BP lesions. POSTN was prominent in the superficial dermis in both PV and BP lesions. Next, to validate the activation of CD163 + TAMs in PV and BP patients, we examined the serum levels of soluble (s)CD163. The serum sCD163 levels in PV and BP patients are significantly higher than in healthy controls. To further elucidate the molecular mechanisms of the effects of POSTN on CD163 + TAMs in PV and BP, we examined chemokines, MMPs and cytokines selected by DNA microarray database. The serum CXCL5 levels from PV patients are significantly higher than those in BP patients and healthy controls. The IL-36γ expression on infiltrating macrophages was prominent only in the lesional skin of PV, while the MMP12 deposition was detected in both PV and BP lesions. Our results shed light on the novel pathogenesis of PV through CD163 + TAMs., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: Flavonoids are converted to inactive metabolites like glucuronides in the gut, and circulate mainly as glucuronides in blood stream, resulting in low concentrations of active aglycones in plasma. It is therefore unclear how oral flavonoids exert their effects in tissues. We recently reported the plasma pharmacokinetics of some flavonoids and suggested the possibility that the absorbed flavonoids modified macrophage functions leading to enhance bacterial clearance. We aimed to confirm their pharmacological profiles focusing on tissue macrophages., Methods: Pseudoinfection was induced by intradermal injection of FITC-conjugated and killed Staphylococcus aureus into the ears of mice treated with or without genistein 7-O-glucuronide (GEN7G, 1 mg/kg, i.v.). FACS analysis was performed on single cell suspensions dispersed enzymatically from the skin lesions at 6 h post pseudoinfection to evaluate phagocytic activities of monocytes/macrophages (CD11b + Ly6G - ) and neutrophils (CD11b + Ly6G + ). Phagocytosis of the FITC-conjugated bacteria by four glucuronides including GEN7G was evaluated in cultures of mouse macrophages., Results: After GEN7G injection, genistein was identified in the inflamed ears as well as GEN7G, and the phagocytic activity of CD11b + Ly6G - cells was increased. GEN7G was converted to genistein by incubation with macrophage-related β-glucuronidase. Macrophage culture assays revealed that GEN7G increased phagocytosis, and the action was dampened by a β-glucuronidase inhibitor. Binding of aglycones to estrogen receptors (ERs), putative receptors of flavonoid aglycones, correlated to biological activities, and glucuronidation reduced the binding to ERs. An ER antagonist suppressed the increase of macrophage function by GEN7G, whereas estradiol enhanced phagocytosis as well., Conclusions: This study suggests a molecular mechanism by which oral flavonoids are carried as glucuronides and activated to aglycones by β-glucuronidase in tissue macrophages, and contributes to the pharmacological study of glucuronides., (© 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.)

Background/aim: Tumor-associated macrophages (TAMs), together with splenic CD11b + cells, help maintain the tumor microenvironment. The immunomodulatory compound imiquimod (IQM) stimulates innate immune cells, including macrophages, to induce antitumor effects. In order to elucidate the effects of IQM on the tumor microenvironment, we investigated the immunomodulatory effect of IQM during melanoma growth by using the B16F10 melanoma model., Materials and Methods: To elucidate the immunomodulatory effects of IQM on the tumor microenvironment, we isolated CD11b + TAMs and splenic CD11b + cells and evaluated the immunomodulatory effects of IQM, using the B16F10 melanoma model., Results: IQM suppressed B16F10 melanoma growth in parallel with reduction of Foxp3 + regulatory T cells (Tregs) at the tumor site, caused by the down-regulation of CCL22 production by tumor-derived and splenic CD11b + cells. Subsequently, we investigated the antitumor or tumor-loading effects of splenic CD11b + cells on B16F10 melanoma growth in vivo. B16F10 melanoma growth was accelerated by splenic CD11b + cells from untreated mice, but was inhibited by splenic CD11b + cells from IQM-treated mice. Consistent with these results, Foxp3 + Tregs were significantly decreased in tumors of mice implanted with both melanoma and splenic CD11b + cells from topical IQM-treated mice. Furthermore, intratumoral administration of anti-CCL22 antibody inhibited B16F10 melanoma growth by decreasing Treg recruitment at the tumor site., Conclusion: Our results suggest a possible mechanism for the antitumor immune response induced by IQM through tumor-associated macrophages., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Dermatofibrosarcoma protuberance (DFSP) is a fibrohistiocytic tumor of intermediate malignancy characterized by slow infiltrative growth and a high tendency to recur locally. Periostin is involved in modulating cell function and inducing the production of proinflammatory cytokines, chemokines, and matrix metalloproteinases (MMPs) from tumor-associated macrophages (TAMs) to promote fibrosis and tumor growth. This study aimed to examine the cancer stroma of DFSP, focusing on TAMs-related proteins and MMPs., Patients and Methods: Using immunohistochemical staining and DNA microarray database, we evaluated periostin, CD163, CD206, MMP1 and MMP12 in 10 cases of DFSP and dermatofibroma., Results: Dense deposits of periostin as well as a substantial number of CD163 + TAMs were detected at the peripheral areas of DFSP. Moreover, MMP1 and MMP12, that were selected by using a DNA microarray database of monocyte-derived macrophages, were observed in the TAMs-detected area., Conclusion: Increased levels of MMP1 and MMP12 on TAMs in the peripheral areas of DFSP might contribute to local invasion., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Immunosuppressive tumor-associated macrophages (TAMs) promote an immunosuppressive environment in the tumor-bearing host, together with regulatory T cells (Tregs). TAMs compose cancer stroma in skin cancers including melanomas and non-melanomas. The majority of tumor-associated macrophages (TAMs) are alternatively activated M2 macrophages that favor tumor development, and they comprise one of the main populations of inflammatory cells in skin cancers. On the other hand, TAMs could be modulated into M1-type macrophages that suppress tumor growth by stimulating and recruiting Th1 and effector cells in the tumor sites. Therefore, TAMs are a target for immunotherapy in various cancers. In this review, we discuss the definition and suppressive mechanisms of TAMs, as well as their biological activities in tumor-bearing hosts to assess potential therapeutic strategies., (Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: Tumor-associated M2 macrophages (TAMs) produce chemokines that affect the formation of cutaneous T-cell lymphoma (CTCL) by stromal factors. Since IFNs are an effective treatment for advanced-stage mycosis fungoides (MF), we hypothesized that IFNs might modulate M2 macrophages., Objective: To prove our hypothesis, we stimulated monocyte-derived M2 macrophages with IFN-α2a or IFN-γ and examined the mRNA expression of chemokines., Methods: By using a microarray, we selected a series of chemokines and MMPs that were strongly connected with the IL-4 stimulation. Then, we investigated the effects of IFN-α2a and IFN-γ on these chemokines., Results: IFN-α2a and IFN-γ decreased the expression and production of CCL17 and CCL18 and increased those of CXCL10 and CXCL11. Moreover, the subcutaneous administration of IFN-α2a increased the CXCL11-producing cells in the lesional skin of patients with advanced MF., Conclusion: Our data suggest one possible mechanism of the therapeutic effects of IFNs through TAMs for the treatment of advanced-stage MF., (Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Mycosis fungoides (MF) starts as an indolent disease, progresses from a patch stage to confluent plaques and ultimately develops skin tumors. Tumor-associated macrophages (TAMs) play roles in maintaining the tumor microenvironment in MF. The purpose of this study was to elucidate the involvement of TAMs in the lesional skin of different stages of MF. First, we immunohistologically examined the percentage of CD163+ macrophages and CD206+ cells, as well as the levels of periostin and IL-4 in cancer stroma. The percentage of CD206+ cells increased in parallel with tumor progression, while there was no significant difference in the percentage of CD163+ cells. Periostin was prominent in the stromal area at the patch and plaque stages but decreased at the tumor stage. In contrast, IL-4 was prominently stained at both plaque and tumor stages. To further elucidate the molecular mechanisms of the effects of these stromal factors on TAMs, we examined their effects on mRNA expression in monocyte-derived macrophages in vitro. Based on microarray analysis and gene ontology, we examined a series of chemokines and MMPs whose expression was strongly connected with periostin stimulation. The DNA microarray results were verified in M2 macrophages using real-time PCR. We further examined the mRNA expression of these chemokines and MMPs in the presence of periostin and IL-4 to simulate the advanced stages of MF and validated their protein expression by ELISA. Our present report suggests possible roles of periostin on TAMs in establishing the tumor microenvironment in MF., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: M2 macrophages play a critical role in the recruitment of T helper 2 (Th2) regulatory T cells (Treg)., Objectives: To study the role of M2 macrophages and Treg cells in eosinophilic celulitis., Material and Methods: We employed immunohistochemical staining for CD163( )and CD206 (macrophages) as well as FoxP3 (Treg), in lesional skin of four cases of eosinophilic cellulitis., Results: CD163(+) CD206(+) M2 macrophages, which were previously reported to produce CCL17 to induce Th2 cells and Treg cells, were predominantly infiltrating the subcutaneous tissues and interstitial area of the dermis. M2 macrophages derived from PBMC showed significantly increased expression of CCL11, CCL17, CCL24 and CCL26 mRNA and production of CCL17 and CCL24, when stimulated by IL-4 or IL- 13. In addition, CCL17-producing cells and CCL24-producing cells were prominent in the lesional skin of EC., Conclusion: Our study sheds light on one of the possible immunological mechanisms of eosinophilic cellulitis.

Background: M2 macrophages play a critical role in the induction of T helper 2 (Th2) polarization., Methods: To investigate the contribution of M2 macrophages to the pathogenesis of bullous pemphigoid (BP) and pemphigus vulgaris (PV), we employed immunohistochemical staining for CD163 and CD206, as well as pSTAT1, pSTAT6, interleukin (IL)-4, IL-13, CCL17, CCL18 and Foxp3 in the lesional skin of 10 cases of BP and PV., Results: The numbers of CD163+ CD206+ M2 macrophages were higher in BP than in PV. Moreover, pSTAT6+ cells, CCL17+ cells, CCL18+ cells and Foxp3+ regulatory T cells were prominent only in the lesional skin of BP. To further investigate the function of M2 macrophages, we examined the mRNA expression and production of Th2-related chemokines from M2 macrophages in vitro, which showed a significant increase in the mRNA expression and production of CCL18 when stimulated by IL-4 or IL-13., Conclusion: Our study sheds light onto one of the possible immunological mechanisms of BP and PV.

An imbalance of immunosuppressive and immunomodulatory cells plays an important role in inhibiting the anti-tumour immune response in a tumour-bearing host. Among such cells, regulatory T cells (Tregs), together with immunosuppressive macrophages, such as CD163+ M2 macrophages, play roles in maintaining the tumour microenvironment. In contrast, interleukin-27 (IL-27) induces STAT1 and STAT3 activation, thus resulting in the enhancement of naive CD4 T-cell proliferation, the promotion of early Th1 differentiation, and the induction of the anti-tumour immune response. The purpose of this study was to investigate the involvement of immunosuppressive cells, such as Tregs and CD163+ macrophages, as well as immunomodulatory cells (i.e. IL-27-producing cells) in keratoacanthoma (KA) and invasive squamous cell carcinoma (SCC). We also examined the presence of CD3+ Foxp3+ Tregs cells in lesional skin from 10 patients with KA and 18 patients with SCC. Increased numbers of CD3+ Foxp3+ Tregs were observed in SCC compared with KA. In parallel with Tregs, higher numbers of CD163+ macrophages and MMP-9+ cells were detected only in SCC. In contrast, IL-27-producing cells were increased only in KA. In addition, the expression of pSTAT1 on tumour cells was observed only in KA. These findings suggest that the induction of immunosuppressive and immunomodulatory cells differs between KA and SCC.

An imbalance of immunosuppressive cells and cytotoxic cells plays an important role in inhibiting the antitumor immune response of the tumor-bearing host. We previously reported the profiles of tumor infiltrating leukocytes in cutaneous angiosarcoma (AS) and suggested that a combination of docetaxel (DTX) with bisphosphonate risedronate sodium (RS) might be effective for MMP9-expressing AS by targeting immunosuppressive cells such as M2 macrophages. To further confirm the effect of this combination therapy, in this report we investigated the immunomodulatory effect of DTX and RS on CD163(+) arginase 1 (Arg1)(+) M2 macrophages in vitro. Interestingly, our present study demonstrated that DTX in combination with RS significantly upregulated the mRNA expression of CXCL10 on M2 macrophages and significantly decreased the mRNA expression of CCL17 and Arg1. Moreover, the production of CXCL10 and CXCL11 from M2 macrophages was significantly increased by DTX with RS though there was no effect of DTX with RS on the production of CCL5 and CCL17. Furthermore, DTX with RS significantly decreased the production of CCL18, which was previously reported to correlate with the severity and prognosis in cancer patients. Our present report suggests one of the possible mechanisms of DTX with RS in the supportive therapy for angiosarcoma.

Regulatory T cells (Tregs), identified by the expression of CD4, CD25 and Foxp3, together with immunosuppressive macrophages, such as CD163+ M2 macrophages, are involved in maintaining peripheral tolerance. The aim of this study was to elucidate the involvement of Tregs and CD163+ macrophages in invasive and non-invasive extramammary Paget's disease. The presence of CD4+CD25+Foxp3+ Tregs, CD163+ M2 macrophages and matrix metalloproteinase-9+ cells was examined immunohistologically in fixed sections of lesional skin from 10 patients with non-invasive extramammary Paget's disease and 7 patients with invasive extramammary Paget's disease. Fewer CD4+CD25+Foxp3+ Tregs were observed in non-invasive extramammary Paget's disease than in invasive extramammary Paget's disease. In contrast, higher numbers of CD163+ macrophages and metalloproteinase-9+ cells were detected only in invasive extramammary Paget's disease. These findings suggest that the induction of immunosuppressive cells in extramammary Paget's disease differs according to the tumour stage.

Several studies have suggested that interleukin (IL)-8 can serve as a biomarker for discrimination of skin sensitizers from nonsensitizers. We established a stable THP-1-derived IL-8 reporter cell line, THP-G8, which harbors SLO and SLR luciferase genes under the control of IL-8 and glyceraldehyde 3-phosphate dehydrogenase promoters, respectively. After 6 h treatment with chemicals, normalized SLO luciferase activity (nSLO-LA) was calculated by dividing SLO-LA by SLR-LA, and the fold induction of nSLO-LA (FInSLO-LA) was calculated by dividing nSLO-LA of chemically treated cells by that of nontreated cells. The nSLO-LA of THP-G8 cells increased in response to lipopolysaccharide (LPS) and several sensitizers. The FInSLO-LA in THP-G8 cells induced by LPS or sensitizers positively correlated with their induction of IL-8 messenger RNA in THP-1 cells. The nSLO-LA value of THP-G8 cells was significantly increased (FInSLO-LA ≥ 1.4) by 13 of the 15 sensitizers as well as by 5 of the 7 nonsensitizers. Interestingly, pretreatment with N-acetylcysteine suppressed the increase in FInSLO-LA induced by all sensitizers (inhibition index (II) ≤ 0.8) but did not suppress that induced by most of the nonsensitizers. We then evaluated the performance of this assay using values of FInSLO-LA ≥ 1.4 and II ≤ 0.8 in at least two of three independent experiments as the criteria of a sensitizer, which resulted in test accuracies of 82% for the 22 chemicals used and of 88% for the chemicals proposed by European Center for the Validation of Alternative Methods. This newly developed assay is a candidate replacement for animal tests of skin sensitization because of its accuracy, convenience, and high throughput performance.

Although cadexomer iodine is widely used for the treatment of skin ulcers such as decubitus ulcers, the mechanism by which it enhances wound healing is not clear. Recently, it has been demonstrated that macrophages play an important role in wound healing by inducing inflammation and angiogenesis. We examined the effects of cadexomer and cadexomer iodine on tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-6, IL-8, IL-10, IL-12 p 40, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) production by macrophages. Human macrophages were obtained by culturing CD14+ monocytes with macrophage colony stimulating factor (M-CSF) in the presence or absence of cadexomer or cadexomer iodine. The production of cytokines and the expression of mRNA were evaluated by enzyme linked immunosorbent assay (ELISA) of the culture supernatants and by reverse transcriptase polymerase chain reaction (RT-PCR) analysis, respectively. In addition, we examined the tissue concentration of VEGF in the wounds treated with or without cadexomer iodine. Cadexomer and cadexomer iodine significantly increased the expression of IL-1 beta, IL-8, TNF-alpha and VEGF mRNA, while they did not affect that of IL-6, IL-10, IL-12 p 40 or bFGF mRNA. In addition, they significantly increased the production of IL-1 beta and TNF-alpha. Although we could not detect increased production of VEGF in the culture supernatants, the western blot analysis of macrophages demonstrated the increased production of VEGF by the treatment with either cadexomer or cadexomer iodine. The treatment with cadexomer iodine increased the tissue concentration of VEGF in the skin wounds. These data suggest that cadexomer and cadexomer iodine have beneficial effects on wound healing in addition to those related to their antibacterial activity.

In this study of the hematopoietic functions of the cytokines released by the epidermal cells, we examined the effects of culture supernatants of epidermal cells and dermal cells from new born mice on uncommitted spleen cells. The epidermal cell culture supernatants (ECCS) stimulated the proliferation of nylon wool-passed I-A-spleen mononuclear cells (SC) much more vigorously than did the dermal cell culture supernatants (DCCS). The main responding cells were I-A-Thy-1-Lyt-1-Lyt-2-cells. Giemsa staining of cytocentrifuge preparations disclosed that SC cultured with ECCS or DCCS were composed of about 50% lymphoid cells, 30-40% granulocytes, 10% macrophages, and a few mast cells and megakaryocytes. Immunoperoxidase staining of the cytocentrifuge preparations showed that SC cultured with ECCS consisted of less than 10% I-A-, about 40% Thy-1+, 10-20% Lyt-1+, less than 6% Lyt-2+, 30-40% Mac-1+, 10-30% Mac-2+, 40-55% Mac-3+, and 10-20% Asialo-GM1+ cells. A double immunofluorescence study showed that 11% of Mac-1+ cells, 34% of Mac-2+ cells and 14% of Mac-3+ cells were I-A+, whereas all the I-A+ cells were either Mac-1+, Mac-2+, or Mac-3+, and that the percentage of Thy-1+ Asialo-GM1+ was less than 5%. In a panel of 3 available purified cytokines known to be secreted by keratinocytes which were used as a positive control, a similar stimulatory effect was recognized with GM-CSF and IL-3 but no such effect was found with recombinant IL-1. These data suggest that ECCS can stimulate the development of uncommitted SC into lymphocytes, different subsets of macrophages probably including Langerhans cells, granulocytes, mast cells and megakaryocytes. They constitute direct and convincing evidence indicating the combined effect of various cytokines released by epidermal cells on the proliferation and maturation of hematopoietic precursor cells into different immunocompetent cells in the skin.