Recommendations for rosacea diagnosis, classification and management: update from the global ROSacea COnsensus 2019 panel.

BACKGROUND: A transition from a subtyping to a phenotyping approach in rosacea is underway, allowing individual patient management according to presenting features instead of categorization by predefined subtypes. The ROSacea COnsensus (ROSCO) 2017 recommendations further support this transition and align with guidance from other working groups. OBJECTIVES: To update and extend previous global ROSCO recommendations in line with the latest research and continue supporting uptake of the phenotype approach in rosacea through clinical tool development. METHODS: Nineteen dermatologists and two ophthalmologists used a modified Delphi approach to reach consensus on statements pertaining to critical aspects of rosacea diagnosis, classification and management. Voting was electronic and blinded. RESULTS: Delphi statements on which the panel achieved consensus of ≥ 75% voting 'Agree' or 'Strongly agree' are presented. The panel recommends discussing disease burden with patients during consultations, using four questions to assist conversations. The primary treatment objective should be achievement of complete clearance, owing to previously established clinical benefits for patients. Cutaneous and ocular features are defined. Treatments have been reassessed in line with recent evidence and the prior treatment algorithm updated. Combination therapy is recommended to benefit patients with multiple features. Ongoing monitoring and dialogue should take place between physician and patients, covering defined factors to maximize outcomes. A prototype clinical tool (Rosacea Tracker) and patient case studies have been developed from consensus statements. CONCLUSIONS: The current survey updates previous recommendations as a basis for local guideline development and provides clinical tools to facilitate a phenotype approach in practice and improve rosacea patient management. What's already known about this topic? A transition to a phenotype approach in rosacea is underway and is being recommended by multiple working groups. New research has become available since the previous ROSCO consensus, necessitating an update and extension of recommendations. What does this study add? We offer updated global recommendations for clinical practice that account for recent research, to continue supporting the transition to a phenotype approach in rosacea. We present prototype clinical tools to facilitate use of the phenotype approach in practice and improve management of patients with rosacea.

Immunologic inhibition of ultraviolet radiation-induced tumor suppressor cell activity.

Long-term exposure of C3H mice to ultraviolet radiation resulted in the formation of suppressor T cells that recognize ultraviolet radiation-induced regressor skin cancers as a class before the appearance of overt tumors. Administration of monoclonal antibodies to the product of the I-Jk subregion of the major histocompatibility complex or low doses of cyclophosphamide in vivo inhibited the development or activity of these cells. This activity of the monoclonal antibody was eliminated by adsorption on B10.BR (I-Jk) but not B10.D2 (I-Jd) splenocytes. These findings provide evidence that elements expressing the I-J determinant are important in regulating the host response prior to the overt development of ultraviolet radiation-induced skin cancers and suggest novel therapeutic approaches to malignancies or other diseases involving suppressor T cells in their pathogenesis.

In vivo inflammatory activity of epidermal cell-derived thymocyte activating factor and recombinant interleukin 1 in the mouse.

Epidermal cell-derived thymocyte activating factor (ETAF), a cytokine produced by keratinocytes, has previously been shown to be biochemically and functionally very similar, if not identical, to interleukin 1 (IL-1). Both ETAF and IL-1 have been demonstrated to be chemotactic for neutrophils and mononuclear cells in vitro. In order to demonstrate that this activity has physiological relevance we have used a simple in vivo model. The present study demonstrates that injection of high-titer ETAF or purified recombinant murine IL-1 into the mouse footpad results in an influx of neutrophils into the site with peak accumulation at 4 h. Footpad swelling also occurs with a time course roughly paralleling that of the neutrophil accumulation. Injection of control proteins failed to reproduce this phenomenon. Margination of neutrophils within blood vessels was seen within 1 h of injection of ETAF or IL-1, followed by entry into the stroma by 4 h. This suggests that chemotactic activity and not merely increased adherence or inhibition of migration is occurring. 5-10 d of daily, subcutaneous injection of ETAF on the mouse flank resulted in an infiltrate of neutrophils, and to a lesser degree, mononuclear cells in association with epidermal hyperplasia, subcutaneous fibrosis, and focal muscle necrosis in the panniculus carnosus. These findings were not seen in control sites injected with media. These findings provide direct in vivo experimental evidence suggesting a physiologic role for ETAF/IL-1 in local inflammation.

Gamma-interferon inhibits collagen synthesis in vivo in the mouse.

Subcutaneous implantation of osmotic pumps into CAF1 mice resulted in the formation of thick fibrous capsules around the pumps. When pumps were loaded with recombinant murine gamma-interferon (rMuIFN-gamma) to deliver 2 X 10(3) U/h for 14 d, there was a marked decrease in thickness and collagen content of the capsules from rMuIFN-gamma-treated animals compared with capsules from animals receiving diluent alone. The collagen content of the capsules was estimated by hydroxyproline analysis of the tissue and by quantitative electron microscopy of collagen bundles. Heat-inactivated rMuIFN-gamma failed to reduce the fibrotic response in this assay. These results provide compelling evidence that gamma-interferon can down-regulate collagen synthesis in vivo and suggest the possibility that this lymphokine may be useful in the treatment of disease states characterized by excessive fibrosis.

Suppressor T cells and the immune response to tumors.

In this paper we have attempted to define the role of suppressor T cells in many well-defined murine tumor systems. We have knowingly omitted a blocking antibodies, suppressor B cells as mediators of tumor immunosuppression in various murine tumor systems; these have been well reviewed elsewhere. Also, we have focused on the importance of two different types of antigen-presenting cells in the induction and suppression of cell-mediated immunity and on some of the different modalities employed to inhibit Ts function. Finally, we have discussed the acquired immunodeficiency syndrome and the possible role of a defective helper pathway and enhanced suppressor pathway in its pathogenesis. We and others believe that the suppressor pathway is preferentially activated by tumor antigen(s) in the cases of many immunogenic murine tumors--possibly due to the release of tumor antigen(s) from tumor cells, their subsequent trafficking to specific areas of the spleen and other organs, and, ultimately, their presentation by certain APC to Ts. Ts may then act directly upon helper Lyt 1+2- T cells as these cells interact with tumor antigen(s) on I-A+ APC. Alternatively, if the effector pathway were somehow impaired--e.g., by ultraviolet radiation or a virus--then the suppressor pathway may be activated in an unregulated manner, often to the detriment of the host. Biochemical characterization of the tumor antigens that stimulate Ts generation and, presumably, tumor growth and definitive documentation of a role of APC in the processing and presentation of these tumor antigens to Ts need to be done. Then selective stimulation of the effector immune response, along with inhibition of the suppressor response, to tumor antigens with drugs, monoclonal antibodies, and soluble mediators or their analogues may be possible in the near future.

Interferon-gamma inhibits tumor antigen presentation by epidermal antigen-presenting cells.

Murine I-A+ epidermal antigen-presenting cells (APCs) have been shown to be capable of presenting soluble tumor fragments (TFs), as a source of tumor-associated antigens (TAAs), for primary and secondary tumor immune responses. In this study we investigated whether incubation of epidermal APCs in interferon-gamma (IFN-gamma) modulates their ability to present TAA and whether the effects of IFN-gamma on the presentation of tumor antigen correspond to its effects on alloantigen presentation in both primed and unprimed systems. Our results show that three weekly subcutaneous injections of naive mice with GM-CSF-cultured but not with fresh TAA-pulsed epidermal APCs induce protective tumor immunity in naive mice and that the immunostimulatory effect of GM-CSF in this system is abrogated by coculture of epidermal cells in IFN-gamma. Furthermore, epidermal APCs are able to present TAA to primed, tumor-immune mice, as assessed by the elicitation of tumor-specific delayed-type hypersensitivity after injection of TAA-pulsed epidermal APCs. IFN-gamma was found to inhibit tumor antigen presentation by freshly prepared epidermal APCs in this system. The effects of IFN-gamma on the presentation of tumor antigen correlated well with its effects on the primary and secondary mixed epidermal cell-lymphocyte reaction, indicating that IFN-gamma differentially modulates the function of epidermal APCs with regard to induction versus elicitation of immunity.

Supernatants from UVB radiation-exposed keratinocytes inhibit Langerhans cell presentation of tumor-associated antigens via IL-10 content.

Exposure of mice to midrange UV radiation (UVB) (280-320 mm) in vivo leads to suppression of the ability to induce delayed-type hypersensitivity (DTH). Systemic administration of supernatants from UVB-exposed keratinocytes (KC) similarly inhibits the ability to induce DTH and the presence of interleukin-10 (IL-10) in the supernatants has been shown to be responsible for this effect. It has been hypothesized that release of IL-10 by KC after exposure to UVB radiation in vivo may be responsible for UVB-induced inhibition of DTH and also for the inability of chronically UVB-irradiated mice to immunologically reject immunogenic UVB-induced skin tumors. To test directly whether supernatants from UVB-irradiated KC can inhibit presentation of tumor-associated antigens (TAA) by epidermal Langerhans cells (LC), cultures of the transformed murine KC line PAM 212 were exposed to 200 J/m2 of UVB radiation and 24 h supernatants obtained. CAF1 (H-2a/d) epidermal cells (EC) enriched for LC content were exposed to supernatants from irradiated (UV-SN) or mock-irradiated (MI-SN) PAM 212 cells for 3 h followed by culture for 16 h in granulocyte-macrophage colony-stimulating factor and then were pulsed with soluble TAA derived from the murine spindle cell tumor S1509a (H-2a). ECs were then washed and injected subcutaneously into naive CAF1 mice three times at weekly intervals for priming. One week after the final immunization these mice were challenged subcutaneously with live S1509a cells and tumor growth scored over time. Pretreatment of EC with UV-SN but not MI-SN inhibited the induction of effective immunity by this immunization scheme. ECs were also treated with UV-SN or MI-SN for 3 h then pulsed with TAA and injected into a hind footpad of previously immunized mice for elicitation of a DTH response. Pretreatment of EC with UV-SN but not MI-SN inhibited the ability of EC to elicit DTH. Neutralization studies with specific neutralizing antibodies to IL-10 demonstrated that the presence of IL-10 in UV-SN was responsible for the inhibition of antigen presentation both for induction and elicitation of immunity. UV-SN inhibits tumor antigen presentation by epidermal LC through the action of IL-10.

Tumor antigen presentation by epidermal antigen-presenting cells in the mouse: modulation by granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha, and ultraviolet radiation.

I-A+ epidermal antigen-presenting cells (APCs, Langerhans cells) have been shown to present tumor-associated antigens (TAAs) and to induce tumor immunity in vivo. This study examined the effects of ultraviolet radiation (UVR) and the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha) on the ability of epidermal cells (ECs) to induce or to elicit immunity against the murine spindle cell tumor S1509a. Naive syngeneic mice were immunized three times at weekly intervals with ECs that had been cultured in GM-CSF for 18 h and then pulsed with TAA derived from S1509a. This resulted in protective immunity against subsequent tumor challenge, providing a model to study the conditions required for sensitization against TAAs by epidermal APCs. Culture of ECs in GM-CSF was required for induction of significant protective tumor immunity, and UV irradiation or incubation in TNF-alpha for 2 h after GM-CSF incubation abrogated the immunostimulatory effect of GM-CSF. However, unlike UVR, TNF-alpha did not significantly inhibit the induction of immunity when ECs were exposed to TNF-alpha before overnight incubation in GM-CSF, together with GM-CSF, or after pulsing with TAA, and anti-TNF-alpha antibody treatment did not abrogate the effects of UVR on this system. Furthermore, TNF-alpha incubation of ECs augmented their ability to elicit delayed-type hypersensitivity (DTH) and also enhanced elicitation of DTH by GM-CSF-cultured ECs, whereas UV-irradiation reduced it in a dose-dependent fashion. Taken together, these results demonstrate that GM-CSF, TNF-alpha, and UVR are significant regulators of tumor antigen presentation by epidermal APCs and that the effects of the cytokines examined differ with regard to induction or elicitation of immunity.

Regulation of cytokine expression in macrophages and the Langerhans cell-like line XS52 by calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP) inhibits antigen presentation by Langerhans cells (LC) and macrophages, and LC are anatomically associated with CGRP-containing epidermal nerves. To determine whether CGRP may produce some of its functional effects through regulation of cytokine expression, we utilized enzyme-linked immunosorbent assay (ELISA) of conditioned supernatants to examine production of interleukin (IL)-10 and IL-1 beta protein in the LC-like cell line XS52 as well as the reverse transcriptase-polymerase chain reaction (RT-PCR) to examine levels of mRNA for IL-10, IL-1 beta, and the 40-kDa subunit (p40) of IL-12. CGRP augmented the lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF) -induced release of IL-10 protein and the induced expression of IL-10 mRNA in these cells. However, it suppressed the induction of release of IL-1 beta protein and the induction of mRNA for IL-12 p40 and IL-1 beta by LPS and GM-CSF. Regulation of cytokine expression in peritoneal macrophages was also examined. By ELISA, the LPS-induced expression of IL-10 was augmented by CGRP, whereas the induction of IL-1 beta was suppressed. Northern analysis demonstrated augmentation of LPS-induced IL-10 mRNA levels and inhibition of LPS-induced IL-1 beta mRNA by CGRP. CGRP inhibited the LPS-induced induction of IL-12 mRNA as assessed by RT-PCR. Up-regulation of B7-2 expression by LPS and GM-CSF was suppressed by CGRP in both XS52 cells and macrophages, as previously reported. This suppression, however, could be abrogated by co-culture with neutralizing antibodies to IL-10. Furthermore, the presence of neutralizing antibodies to IL-10 during exposure of epidermal cells (EC) to CGRP prevented the CGRP-mediated suppression of EC presentation of tumor-associated antigens (from the S1509a spindle cell carcinoma) for elicitation of delayed-type hypersensitivity in S1509a-immune mice. These data suggest that suppression of antigen-presenting function by CGRP is mediated, at least in part, by changes in cytokine expression that favor less robust antigen presentation for cell-mediated immunity.

Calcitonin gene-related peptide: key regulator of cutaneous immunity.

Calcitonin gene-related peptide (CGRP) has been viewed as a neuropeptide and vasodilator. However, CGRP is more appropriately thought of as a pleiotropic signalling molecule. Indeed, CGRP has key regulatory functions on immune and inflammatory processes within the skin. CGRP-containing nerves are intimately associated with epidermal Langerhans cells (LCs), and CGRP has profound regulatory effects on Langerhans cell antigen-presenting capability. When LCs are exposed to CGRP in vitro, their ability to present antigen for in vivo priming of naïve mice or elicitation of delayed-type hypersensitivity is inhibited in at least some situations. Administration of CGRP intradermally inhibits acquisition of immunity to Th1-dominant haptens applied to the injected site while augmenting immunity to Th2-dominant haptens, although the cellular targets of activity in these experiments remain unclear. Although CGRP can be a pro-inflammatory agent, several studies have demonstrated that administration of CGRP can inhibit the elicitation of inflammation by inflammatory stimuli in vivo. In this regard, CGRP inhibits the release of certain chemokines by stimulated endothelial cells. This is likely to be physiologically relevant as cutaneous blood vessels are innervated by sensory nerves. Exciting new studies suggest a significant role for CGRP in the pathogenesis of psoriasis and, most strikingly, that CGRP inhibits the ability of LCs to transmit the human immunodeficiency virus 1 to T lymphocytes. A more complete understanding of the role of CGRP in the skin immune system may lead to new and novel approaches for the therapy of immune-mediated skin disorders.

Epidermal I-J-bearing cells are responsible for transferable suppressor cell generation after immunization of mice with ultraviolet radiation-treated epidermal cells.

Subcutaneous immunization of mice with hapten-coupled, ultraviolet radiation (UVR)-treated epidermal cells (EC) results in a hyporesponsive delayed-type hypersensitivity (DTH) response associated with the appearance of afferent-acting, hapten-specific T suppressor (Ts) cells. Depletion of I-J-bearing cells from the EC population prior to UVR-exposure and hapten coupling prevents the appearance of these Ts cells. However, non-UVR-treated EC depleted of I-J-bearing cells and hapten-coupled are capable of immunizing mice for a DTH response. Therefore, the set of I-J-bearing EC appears to be distinct from classic Langerhans cells. A novel set of I-J-bearing EC appears to be responsible for Ts activation after subcutaneous immunization with hapten-coupled UVR-treated EC.

Experimental photoaging in C3H/HeN, C3H/HeJ, and Balb/c mice: comparison of changes in extracellular matrix components and mast cell numbers.

Chronic exposure of human or murine skin to ultraviolet B (UVB) radiation alters dermal extracellular matrix composition and increases the number of mast cells and inflammatory cells. Experiments were designed to test the possible role of UVB-induced tumor necrosis factor-alpha in these photoaging changes based on reports that C3H/HeN, but not C3H/HeJ or Balb/c mice, produce excess TNF-alpha in response to UVB exposure. Pigmented C3H/HeN and C3H/HeJ strains were exposed to a total of 75 J/cm2 of UVB radiation, and unpigmented Balb/c mice were exposed to 19 J/cm2. The UVB-induced increases in collagen, glycosaminoglycans, and neutrophil number were similar or the same in all three strains. The elastin increase was greater in C3H/HeJ than in C3H/HeN mice. The most striking difference between the strains was a 7.7-fold UVB-induced increase in mast cells in C3H/HeN mice compared to no increase in irradiated C3H/HeJ mice and a 2.3-fold increase in Balb/c mice. These results suggest that excess TNF-alpha (or other mediator) produced in C3H/HeN skin (but not C3H/HeJ skin) in response to UVB exposure is involved in the mast cell increase and partial inhibition of elastin increase, but that neither these mediators nor mast cell products are important mediators for the chronic UVB-induced increases in neutrophils, glycosaminoglycans, and collagen. When a possible source of the excess TNF-alpha was investigated, it was found that isolated epidermal cells from all three strains produced increases in TNF-alpha in response to UVB radiation. These results, as well as the previous results showing differences between these strains in UVB-induced effects on cutaneous immune function, are consistent with a model in which UVB-induced mediators from the epidermis stimulate another cell type to produce excess TNF-alpha (and other mediators) in the C3H/HeN but not C3H/HeJ or Balb/c mice.

Prolongation of murine skin allograft survival by the systemic effects of 8-methoxypsoralen and long-wave ultraviolet radiation (PUVA).

Systemic administration of the photoactive drug 8-methoxypsoralen to a group of mice bearing cutaneous allografts, followed by exposure to long-wave ultraviolet radiation (UVA, 320-400 nm) (PUVA) daily for 14 days at a site distant from the allograft, significantly increased the survival time of the allografts. This effect was seen both in donor-recipient combinations that differ at the major histocompatibility complex and in those differing only at minor histocompatibility loci. Treatment with 8-methoxypsoralen or long-wave UV radiation alone was ineffective in prolonging allograft survival, as were doses of mid-wave UV radiation (UVB, 280-320 nm) that produced greater inflammation than the PUVA protocol. Allografted, PUVA-treated animals also demonstrated decreased alloantigen reactivity against donor-strain spleen cells during the period of treatment by cytotoxicity assays. Allografts of skin in the murine system are highly immunogenic and are generally rejected faster than organ allografts; thus PUVA treatment appears to exert a potent effect on prolonging allograft survival. The systemic nature of the effect and the fact that adverse side effects from PUVA are largely limited to the skin suggest that PUVA might have a role in clinical organ transplantation management.

Effects of immunomodulatory cytokines on the presentation of tumor-associated antigens by epidermal Langerhans cells.

The recognition and presentation of tumor-associated antigens by cutaneous antigen-presenting cells (APC) may play an important role in the establishment of effective defense mechanisms against newly emerging tumors in the skin. Recent data demonstrate the ability of I-A+ epidermal cells (Langerhans cells) to present tumor-associated antigens for the induction of protective tumor immunity and elicitation of delayed-type hypersensitivity against the murine spindle cell tumor, S1509a. Furthermore, the local cytokine microenvironment in the vicinity of a cutaneous neoplasm may regulate the ability of resident epidermal APC to initiate and/or to elicit protective immunity against incipient cutaneous neoplasms. This article summarizes the effects of granulocyte-macrophage/colony-stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha (TNF alpha), transforming growth factor-beta (TGF beta), and interferon-gamma (IFN gamma) on the modulation of antigen presentation by epidermal APC. Our data indicate that these cytokines significantly and differentially modify the ability of epidermal cells to present tumor-associated antigens and that their effects differ with regard to induction of primary immunity (sensitization) or elicitation of secondary immune responses.

Interleukin 1 alpha but not transforming growth factor beta inhibits tumor antigen presentation by epidermal antigen-presenting cells.

Cutaneous I-A+ Langerhans cells are the principal antigen-presenting cells within the epidermis, capable of both initiating and eliciting CD4-dependent immune reactions. We recently demonstrated that epidermal Langerhans cells can present tumor-associated antigens and thus may be important in cutaneous tumor immunity. Despite the ability of Langerhans cells to present tumor antigens, they generally fail to induce protective tumor immunity against growing tumors in situ. We therefore investigated whether locally produced cytokines may be able to down-regulate the presentation of tumor-associated antigens and alloantigen by epidermal antigen-presenting cells in primed as well as in unprimed systems in vivo and in vitro. Naive syngeneic mice could be successfully immunized against the spindle cell tumor S1509a by injecting them with granulocyte-macrophage colony-stimulating factor-exposed and tumor-associated antigen-pulsed epidermal cells three times at weekly intervals. Co-incubation of epidermal cells in granulocyte-macrophage colony-stimulating factor and interleukin-1 alpha inhibited tumor-antigen presentation by epidermal antigen-presenting cells in this system and also inhibited alloantigen presentation in the primary mixed epidermal cell-lymphocyte reaction. Tumor necrosis factor-alpha appeared to be a significant mediator of the inhibitory effect of interleukin-1 alpha on the ability of epidermal antigen-presenting cells to induce protective tumor immunity, because addition of anti-tumor necrosis factor-alpha antibody abrogated the observed effect of interleukin-1 alpha. However, the effects of interleukin-1 alpha and tumor necrosis factor-alpha differed with regard to presentation of tumor-associated antigens by epidermal antigen-presenting cells in a primed system. Whereas incubation of epidermal cells in interleukin-1 alpha before or after tumor antigen pulse inhibited their ability to elicit a delayed-type hypersensitivity response against S1509a tumor-associated antigens in tumor-immune mice, culture in tumor necrosis factor-alpha significantly enhanced delayed-type hypersensitivity. Again, these in vivo data corresponded well to similar results obtained in vitro using the secondary mixed epidermal cell-lymphocyte reaction. Incubation of epidermal cells in transforming growth factor-beta, which has been shown to down-regulate T-cell-mediated immune responses in other systems, did not suppress tumor immunity in our assays. Thus, interleukin-1 alpha may be an important regulator of Langerhans cell antigen-presenting function, having effects that are partially mediated via interleukin-1 alpha-induced up-regulation of tumor necrosis factor-alpha secretion within the skin.

Induction of anti-tumor immunity with epidermal cells pulsed with tumor-derived RNA or intradermal administration of RNA.

The skin is well-suited to serve as a substrate for vaccination strategies. In this regard, epidermal cells exposed to granulocyte-macrophage colony-stimulating factor can, upon subcutaneous injection into naïve mice, present a soluble extract of tumor as a source of tumor-associated antigens for the induction of in vivo anti-tumor immunity. Use of RNA for immunization has a potential advantage over this technique. Because RNA can be amplified, only a small amount of tumor is needed for antigen preparation and, as with a soluble extract, it is not necessary to know the molecular nature of the antigen(s) relevant to immunity. To test the hypothesis that RNA-pulsed epidermal cells can induce anti-tumor immunity, total cellular RNA was isolated from the S1509a spindle cell tumor and used to pulse CAF1 epidermal cells enriched for Langerhans cell content and pre-exposed to granulocyte-macrophage colony-stimulating factor. These cells were then injected subcutaneously into naïve CAF1 mice three times at weekly intervals followed by challenge with living S1509a cells. Tumor growth was significantly less than in control animals immunized in an identical fashion but with irrelevant RNA. Digestion of S1509a RNA with RNase prior to pulsing of epidermal cells prevented the development of immunity. In separate experiments, intradermal injection of S1509a RNA into naïve mice three times at weekly intervals also induced immunity to challenge with the tumor. Digestion of S1509a RNA with RNase also prevented development of immunity in this system. Effective anti-tumor immunity can be induced in mice utilizing RNA-pulsed epidermal cells for in vivo immunization or by injecting RNA intradermally into naïve mice.

Interferons and collagen production.

The immunoregulatory, antiviral, and antiproliferative agents known as the interferons have profound effects on collagen synthesis. Interferons alpha, beta, and gamma suppress collagen synthesis by dermal fibroblasts. In addition, interferon gamma (IFN-gamma) inhibits the constitutively increased collagen synthesis characteristic of fibroblasts derived from lesions of patients with scleroderma. IFN-gamma also inhibits collagen synthesis by myofibroblasts and synovial fibroblast-like cells. Inhibition of collagen synthesis by IFN-gamma is associated with a coordinate inhibition of transcription for types I and III collagen. In addition, IFN-gamma suppresses levels of procollagen mRNA and type II collagen synthesis in human articular chondrocytes. In vivo studies in mice have demonstrated that IFN-gamma inhibits the collagen synthesis associated with the fibrotic response to an implanted foreign body, bleomycin-induced pulmonary fibrosis, and the healing response to cutaneous thermal burns. In the latter case, while collagen content of the wound scar was decreased, hyaluronic acid was increased in mice receiving IFN-gamma compared to controls. This is in accord with in vitro studies showing that, while interferons alpha and beta decrease production of glycosaminoglycans, IFN-gamma increases production of glycosaminoglycans. Of interest, acute inflammation at sites of thermal injury, or when elicited by proinflammatory agents in separate experiments, also was suppressed in mice treated with IFN-gamma. The means by which IFN-gamma inhibits collagen synthesis involves transcriptional regulation. There is a single report that interferon alpha can decrease the size of a keloid of recent onset in a human patient. Because the interferons can inhibit collagen synthesis in vivo, further studies may be warranted to evaluate the usefulness of these agents in the treatment of disease states characterized by abnormal fibrotic responses as well as their potential for altering the healing response associated with particular therapeutic interventions.

Expression of neurotrophic factors and neuropeptide receptors by Langerhans cells and the Langerhans cell-like cell line XS52: further support for a functional relationship between Langerhans cells and epidermal nerves.

Epidermal Langerhans cells are frequently anatomically associated with calcitonin gene-related peptide-containing nerves. Furthermore, calcitonin gene-related peptide inhibits Langerhans cells antigen-presenting function in several assays. Studies were performed to further explore the hypothesis that Langerhans cells and nerves have a functional relationship. To examine whether Langerhans cells may produce factors that influence nerve cell differentiation, we utilized the Langerhans cell-like cell line XS52 as a surrogate for Langerhans cells and compared it with Langerhans cells enriched to 90%. Supernatants conditioned by lipopolysaccharide-stimulated XS52 cells were able to induce the differentiation of the pheochromocytoma line PC12 into sympathetic neuron-like cells. This was also the case with enriched Langerhans cells stimulated by lipopolysaccharide. Pretreatment of conditioned supernatants with specific neutralizing anti-sera indicated that most of the differentiation-inducing activity was due to interleukin-6 and a small amount was due to nerve growth factor and basic fibroblast growth factor. By reverse transcriptase polymerase chain reaction, three clones of the XS52 cell line, XS52-4D, XS52-11D, and XS52-8B, were found to express mRNA for interleukin-6 and expression was markedly augmented by lipopolysaccharide. mRNA for nerve growth factor and basic fibroblast growth factor was detected in XS52-4D and XS52-11D, but not in XS52-8B. The expression of these neurotrophic factors by enriched Langerhans cells was quite similar to that of XS52-4D. In order to examine whether Langerhans cells may express receptors for nerve-derived peptides, reverse transcriptase polymerase chain reaction was employed to look for pituitary adenylate cyclase activating polypeptide type I, type II, and type III, and gastrin-releasing peptide receptors. All clones examined, as well as enriched Langerhans cells, expressed pituitary adenylate cyclase activating polypeptide type II and type III, and gastrin-releasing peptide receptors. These results suggest bi-directional signalling between Langerhans cells and nerves; nerve cells may regulate Langerhans cell function by elaboration of certain neuropeptides whereas Langerhans cells may promote the differentation of nerves by elaboration of interleukin-6 and, possibly, other factors.

Structure-function relations in the inhibition of murine contact hypersensitivity by amiloride.

Topical application of amiloride, a potent inhibitor of the Na+/H+ antiport, inhibits cutaneous inflammation induced by ultraviolet radiation or contact hypersensitivity in mice. Amiloride analogues with greater and lesser inhibition of the Na+/H+ exchange were tested to determine whether anti-inflammatory effects correlate with this activity. Structural analogues of amiloride without significant activity at the Na+/H+ antiport (pyrazine, pyrazinamide, and chloropyrazine) failed to inhibit contact hypersensitivity. N-amidino-3-amino-5-dimethyl amino-6-chloropyrazinecarboxamide (DMA) has a 23-fold greater affinity for the Na+/H+ antiport compared to amiloride, but failed to inhibit contact hypersensitivity in this assay. 3,5-diamino-6-chloropyrazine-amido-guanidine (DCG), which has only 7% of the affinity of amiloride for the antiport, suppressed contact hypersensitivity as well as amiloride. Experiments examining the ability of these agents to diffuse through mouse skin revealed amiloride to be superior to both DCG and DMA, which were approximately equal. DMA, with greater inhibition of the Na+/H+ antiport but lesser ability to inhibit contact hypersensitivity, inhibited protein synthesis and induced cell death more than amiloride or DCG. Amiloride and DCG hold promise as topical anti-inflammatory agents. Their anti-inflammatory properties do not correlate with affinity for the Na+/H+ antiport, ability to penetrate murine skin, or inhibition of protein synthesis.

Inhibition of neutrophil elastase suppresses the development of skin tumors in hairless mice.

In this study we investigated whether a reduction in neutrophil elastase activity in mice would alter the development of ultraviolet B or chemically induced skin tumors. A mutant strain of neutrophil elastase-deficient mice was developed by crossing beige mice with SKH 1 hairless mice. Ultraviolet irradiation three times per week for 20 wk developed an average of 10 tumors per normal mouse, whereas elastase-deficient hairless mice receiving the same treatment developed only 0.4 tumors per mouse. Benzopyrene administered topically for 20 wk resulted in an average of seven tumors per control mouse, while similar treatment to elastase-deficient hairless mice reduced the tumor count to 0.2 per mouse. Two small molecular weight elastase inhibitors, which were shown to inhibit mouse neutrophil elastase, were administered subcutaneously to normal SKH-1 mice during 16 wk of ultraviolet B exposure. Both inhibitors significantly reduced the incidence of ultraviolet B-induced tumors. When control and elastase-deficient mice were immunized with 2,4,6-trinitrochlorobenzene and oxazolone, both molecules elicited a significant contact hypersensitivity response. Ultraviolet B irradiation prior to immunization at a nonirradiated site completely suppressed the induction of contact hypersensitivity in both the normal and the deficient mice, suggesting that prevention of systemic immunosuppression was not the reason for the resistance to skin tumors observed in the elastase-deficient mice. The results suggest that neutrophil elastase can be an important factor in squamous cell tumor development.