European Guidelines (S1) on the use of high-dose intravenous immunoglobulin in dermatology.

BACKGROUND: The treatment of severe dermatological autoimmune diseases and toxic epidermal necrolysis (TEN) with high-dose intravenous immunoglobulin (IVIg) is a well-established procedure in dermatology. As treatment with IVIg is usually considered for rare clinical entities or severe clinical cases, the use of immunoglobulin is not generally based on data from randomized controlled trials that are usually required for the practice of evidence-based medicine. Owing to the rarity of the indications for the use of IVIg, it is also unlikely that such studies will be available in the foreseeable future. Because the high costs of IVIg treatment also limit its first-line use, the first clinical guidelines on its use in dermatological conditions were established in 2008 and renewed in 2011. MATERIALS AND METHODS: The European guidelines presented here were prepared by a panel of experts nominated by the EDF and the EADV. The guidelines were developed to update the indications for treatment currently considered as effective and to summarize the evidence base for the use of IVIg in dermatological autoimmune diseases and TEN. RESULTS AND CONCLUSION: The current guidelines represent consensual expert opinions and definitions on the use of IVIg reflecting current published evidence and are intended to serve as a decision-making tool for the use of IVIg in dermatological diseases.

[Sarcoidosis : Organ involvement, diagnosis, current treatment]. / Sarkoidose : Organbeteiligung, Diagnostik, aktuelle Therapie.

Sarcoidosis is characterized by the appearance of noncausating, epitheloid cell granulomas, primarily in skin and lung. Hereditary disposition is well known; additional infection-associated triggers play a role for the development of inflammation mediated by T helper (Th)1 cells. Clinically, various disease courses can be observed that are characterized by the formation of skin papules at typical sites of the body which differ in their tendency to be associated with systemic organ involvement. Systemic disease without skin affections is also possible. The diagnosis is based on the typical clinical appearance, biopsy of affected tissue (e.g. skin, lung) and laboratory investigations. Additional systemic involvement needs to be excluded. In most cases, the disease is self-limited, but can also be life threatening due to organ fibrosis. The degree of (extra-)cutaneous involvement and level of discomfort are used to select the type of treatment, which ranges from topical immune suppressive agents to systemic therapy with corticosteroids. In nonresponders, additional modern immunosuppressive/immunomodulating therapeutic options are available.

The registry of the German Network for Systemic Scleroderma: frequency of disease subsets and patterns of organ involvement.

OBJECTIVE: Systemic sclerosis (SSc) is a rare, heterogeneous disease, which affects different organs and therefore requires interdisciplinary diagnostic and therapeutic management. To improve the detection and follow-up of patients presenting with different disease manifestations, an interdisciplinary registry was founded with contributions from different subspecialties involved in the care of patients with SSc. METHODS: A questionnaire was developed to collect a core set of clinical data to determine the current disease status. Patients were grouped into five descriptive disease subsets, i.e. lcSSc, dcSSc, SSc sine scleroderma, overlap-syndrome and UCTD with scleroderma features. RESULTS: Of the 1483 patients, 45.5% of patients had lcSSc and 32.7% dcSSc. Overlap syndrome was diagnosed in 10.9% of patients, while 8.8% had an undifferentiated form. SSc sine scleroderma was present in 1.5% of patients. Organ involvement was markedly different between subsets; pulmonary fibrosis for instance was significantly more frequent in dcSSc (56.1%) than in overlap syndrome (30.6%) or lcSSc (20.8%). Pulmonary hypertension was more common in dcSSc (18.5%) compared with lcSSc (14.9%), overlap syndrome (8.2%) and undifferentiated disease (4.1%). Musculoskeletal involvement was typical for overlap syndromes (67.6%). A family history of rheumatic disease was reported in 17.2% of patients and was associated with early disease onset (P < 0.005). CONCLUSION: In this nationwide register, a descriptive classification of patients with disease manifestations characteristic of SSc in five groups allows to include a broader spectrum of patients with features of SSc.

[Hypocomplementemic urticarial vasculitis syndrome. Successful therapy with intravenous immunoglobulins]. / Hypokomplementämisches Urtikaria-Vaskulitis-Syndrom. Erfolgreiche Therapie mit intravenösen Immunglobulinen.

Autoimmune diseases can initially present as chronic urticaria. We describe the course of a patient with hypocomplementemic urticarial vasculitis syndrome (HUVS) as well as his successful treatment with high-dose intravenous immunoglobulins (IVIG). HUVS was diagnosed clinically and confirmed by histology and laboratory studies. After only one cycle with IVIG (2 g/kg) all HUVS symptoms were significantly decreased.

[Significance of immunologic tolerance in dermatology]. / Bedeutung der immunologischen Toleranz in der Dermatologie.

The immunological tolerance processes enable the organism to distinguish between self and non-self and are, therefore, critical for an efficient immune system. Exogenous or endogenous factors that disturb tolerance mechanisms induce uncontrolled activation of the immune system and the development of autoimmune diseases. In the field of dermatology, the most relevant autoimmune diseases are connective tissue diseases and autoimmune bullous skin disorders. In contrast, increased activity of tolerance shuts down parts of the normal immune response and thus facilitates the development of neoplasia and microbial infections in the skin and other organs. Immunological mechanisms for the induction of tolerance have been studied with the help of experimental models of tolerance to contact allergens. T- and B-cells, as well as antigen presenting cells, in particular dendritic cells, are involved in the immunological mechanisms of tolerance. The modification of autologous immune cells of patients with malignant tumors, allergic and autoimmune diseases might have potential for the development of new therapies.

Inhibition of human allergic T-cell responses by IL-10-treated dendritic cells: differences from hydrocortisone-treated dendritic cells.

BACKGROUND: Dendritic cells (DCs) are able to induce human allergic T(H)1 responses as well as T(H)2 responses. OBJECTIVE: In this study, we examined the effect of antiinflammatory agents such as IL-10 and hydrocortisone (HC) on the accessory function of DCs and the resulting T-cell response, especially that of T(H)2 cells. METHODS: Naive and memory CD4(+) T cells from atopic donors were stimulated with autologous allergen-pulsed DCs generated from CD14(+) monocytes by culture with GM-CSF/IL-4 and fully matured with IL-1 beta, TNF-alpha, and PGE(2) in the presence or absence of IL-10 or HC. RESULTS: IL-10-treated DCs and, to a lesser extent, HC-treated DCs showed a decreased expression of MHC II molecules, the costimulatory molecule CD86, and the DC-specific marker CD83, as well as a strongly reduced IL-12 secretion. Consequently, T-cell proliferation was reduced after stimulation with IL-10- or HC-treated DCs alike. However, pretreatment of DCs with IL-10 inhibited the production of T(H)1 and T(H)2 cytokines by T cells, whereas HC-treated DCs inhibited production of IFN-gamma but induced an increased release of IL-4 and no change in IL-5. Both effects were long-lasting; cytokine production remained low (which was due not to enhanced apoptosis but to functional hyporesponsiveness) or even increased after restimulation with fully matured DCs. CONCLUSION: These data indicate that IL-10- or HC-treated DCs differ in their ability to influence human allergic T-cell responses. This has major implications for therapeutic strategies aiming at the downregulation of proallergic T(H)2 responses.

Dendritic cells as a tool to induce anergic and regulatory T cells.

The induction of antigen-specific T-cell tolerance in the thymus and its maintenance in the periphery is crucial for the prevention of autoimmunity. As well as their stimulatory functions, there is growing evidence that dendritic cells, acting as professional antigen-presenting cells, also maintain and regulate T-cell tolerance in the periphery. This control function is exerted by certain maturation stages and subsets of different ontogeny, and can be influenced by immunomodulatory agents. What is the current state of knowledge of the "immunoregulatory" properties of dendritic cells and how might tolerance-inducing dendritic cells be relevant to therapeutic applications in humans?

Induction of dendritic cell maturation and modulation of dendritic cell-induced immune responses by prostaglandins.

Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system. In this study we investigated the effects of various prostaglandins (PG) on the stimulatory capacity of DC. DC were generated from peripheral progenitor cells in the presence of IL-4 and GM-CSF and stimulated with IL-1, IL-6 and TNF-alpha on day 7. Simultaneously, PG (PGD(2), PGE(1), PGE(2), PGF(2 alpha), PGI(2)) were added at various concentrations (10(-5) to 10(-9) M) on day 7. In all experiments, PGE(2) had the most potent influence on the maturation of the DC, followed by other PG in the order PGE(1) > PGD(2) > PGF(2 alpha) > PGI(2). In addition, the expression of the surface molecules CD40, CD54, CD58, CD80, CD83, CD86 and the MHC class II molecules was upregulated after stimulation with PG. Analysis of DC supernatants after treatment with PG demonstrated significantly higher amounts of the proinflammatory cytokines IL-1 beta, IL-6, TNF-alpha, and IL-12. Addition of PG to DC induced a markedly enhanced proliferation of both naive and activated CD4(+) and CD8(+) T cells in alloantigen-induced MLR assays. Assessment of coculture supernatants after restimulation revealed significantly higher amounts of the Th1-cytokines IL-2 and IFN-gamma and only minimal amounts of IL-4 compared to control cells. No production of IL-10 was observed. The effects of PG on the maturation of DC and enhanced T-cell proliferation could be mimicked by db-cAMP and forskolin, indicating that they were due to elevated cAMP levels. Collectively, our data show that members of the PG family promote the differentiation of DC and enhance their capacity to induce a Th1 immune response.

Genetic immunization of mice with human tyrosinase-related protein 2: implications for the immunotherapy of melanoma.

The melanosomal protein TRP2 expressed by melanocytes and most melanoma cells is an attractive, clinically relevant model antigen for the experimental development of melanoma immunotherapy in mice. A peptide shared by murine and human TRP2 can be recognized by melanoma-reactive CTL in C57BL/6 mice, as well as in human melanoma patients. Previous experiments demonstrated that gene gun immunization of mice with plasmid DNA encoding autologous murine TRP2 was unable to induce protective immunity against B16 melanoma cells naturally expressing TRP2. In the present study, we investigated whether the use of cDNA encoding xenogeneic human TRP2, which is highly homologous to murine TRP2, would be more effective. Genetic immunization of mice with human TRP2 resulted in coat depigmentation as a sign of autoimmune-mediated destruction of melanocytes and provided significant protection against metastatic growth of B16 melanoma Induction of protective immunity was associated with TRP2-reactive antibodies and CD8+ T cells. Furthermore, immunization with recombinant adenovirus was more effective than immunization with plasmid DNA using the gene gun. Our results provide new insights for the development of antigen-specific immunotherapy of melanoma.

Efficient transduction of mature CD83+ dendritic cells using recombinant adenovirus suppressed T cell stimulatory capacity.

We have developed a culture method for the foreign serum-free generation of highly immunostimulatory, CD83+ human dendritic cells (DC). In this study, we evaluated the feasibility and consequences of endogenously expressing antigens in mature DC using adenoviral vectors. Transduction of DC with Ad-EGFP demonstrated endogenous fluorescence in 50-85% of CD83+ DC. Ad-transduced DC stimulated the proliferation of allogeneic CD8+ and CD4+ T cells at low DC: T cell ratios. However, at high DC: T cell ratios the stimulatory capacity of Ad-transduced DC was suppressed. This immunosuppressive effect was confirmed by demonstrating that the stimulatory function of untreated DC could be suppressed in a dose-dependent manner by addition of Ad-transduced DC. Furthermore, transwell experiments suggested that direct cell contact was required. Taken together, our results demonstrate the feasibility of efficiently expressing antigens in CD83+ DC using adenoviruses. However, immunosuppressive effects must be considered and carefully studied before Ad-transduced DC are employed for clinical trials. Gene Therapy (2000) 7, 249-254.

Fetal calf serum-free generation of functionally active murine dendritic cells suitable for in vivo therapeutic approaches.

UNLABELLED: Standard protocols to generate mouse dendritic cells (DC) generally use culture medium supplemented with fetal calf serum; however, reinjection in vivo of DC cultured in fetal calf serum results in priming to xenogeneic proteins that clearly limits the use of such DC. We therefore established a fetal calf serum-free culture system for the generation of murine DC from bone marrow precursors. DC can be generated fetal calf serum-free using RPMI supplemented with 1.5% syngeneic mouse serum. Although the yield of DC grown under fetal calf serum-free conditions was somewhat lower than that of the standard culture, large numbers of DC could be generated without the exposure to xenogeneic proteins. The yield of fetal calf serum-free cultured DC was further enhanced by addition of the proinflammatory cytokines TNF-alpha and IL-1beta with the combination resulting in up to 10% more DC. Phenotypically, CD11c + DC cultured fetal calf serum-free homogenously coexpressed the DC-specific molecule DEC-205 as well as the costimulatory molecules CD40, CD80, and CD86. In contrast, only a subpopulation of the CD11c + DC cultured in fetal calf serum-containing medium coexpressed these molecules. Functionally, fetal calf serum-free DC showed strong stimulatory capacity for naïve allogeneic CD4 + and CD8 + T cells. Importantly, fetal calf serum-free DC showed spontaneous in vivo migratory activity. Moreover, 5 x 105 subcutaneously injected TNBS-conjugated fetal calf serum-free DC were able to mediate contact sensitivity. Furthermore, the intravenous or subcutaneous injection of a single dose of 5 x 105 OVA-pulsed fetal calf serum-free DC resulted in the induction of an OVA-specific immune response in naïve TCR transgenic animals. Thus DC cultured under fetal calf serum-free conditions are suitable instruments for in vivo therapeutic approaches, especially in autoimmune models. KEYWORDS: DC vaccines/dendritic cell development/fetal calf serum-free culture conditions for DC/in vivo therapeutic DC approaches.

Ineffective elimination of Leishmania major by inflammatory (MRP14-positive) subtype of monocytic cells.

Myeloid-related protein (MRP) 14, an intracellular protein involved in calcium-dependent activation of myeloid cells, presents a differentiation marker for a subtype of macrophages. In experimental leishmaniasis, BALB/c mice succumb to visceral dissemination after infection with L. major, due to a Th2 cell response, while C57Bl/6 mice develop protective immunity associated with a Th1 cell response. We have previously shown that resistance in (C57Bl/6 mice was also associated with a significantly lower percentage of MRP14-positive cells in the infiltrate than in susceptible BALB/c mice. In C57Bl/6 mice, weekly injections of bone marrow (BM) cells enriched with MRP14-positive cells (d1 of culture) did not reverse, but prolonged the course of infection, associated with increased local parasite spread. In BALB/c mice a single dose of an antiphlogistic agent (dexamethasone or lipoxygenase inhibitor) was associated with reduction of infiltrating MRP14-positive cells and also with a decrease of parasite loads in footpads, lymph nodes as well as spleens, and with delayed progression of disease, Double labeling experiments in vitro revealed that at least 43.1% of MRP14-positive mononuclear cells in BM cultures (8h) had phagocytosed parasites after 4 h of co-incubation. Activation by IFN-gamma (20 U/ml) for 24h and 48h did not significantly reduce parasite load in these cells. In contrast, 77.0% of F4/80-positive macrophages (6d of culture) were infected with L. major parasites and these cells responded to activation with IFN-gamma (20 U/ml) with significant reduction of parasite load (25.3%). The protein MRP14 did not have an effect on parasite survival in vitro. Thus, the impaired capability of MRP14-positive cells to kill L. major upon stimulation may be one reason for the adverse course of infection observed with their increased appearance.

Interleukin-10-treated human dendritic cells induce a melanoma-antigen-specific anergy in CD8(+) T cells resulting in a failure to lyse tumor cells.

Dendritic cells (DC) are critically involved in the initiation of primary immune processes, including tumor rejection. In our study, we investigated the effect of interleukin-10 (IL-10)-treated human DC on the properties of CD8(+) T cells that are known to be essential for the destruction of tumor cells. We show that IL-10-pretreatment of DC not only reduces their allostimulatory capacity, but also induces a state of alloantigen-specific anergy in both primed and naive (CD45RA+) CD8(+) T cells. To investigate the influence of IL-10-treated DC on melanoma-associated antigen-specific T cells, we generated a tyrosinase-specific CD8(+) T-cell line by several rounds of stimulation with the specific antigen. After coculture with IL-10-treated DC, restimulation of the T-cell line with untreated, antigen-pulsed DC demonstrated peptide-specific anergy in the tyrosinase-specific T cells. Addition of IL-2 to the anergic T cells reversed the state of both alloantigen- or peptide-specific anergy. In contrast to optimally stimulated CD8(+) T cells, anergic tyrosinase-specific CD8(+) T cells, after coculture with peptide-pulsed IL-10-treated DC, failed to lyse an HLA-A2-positive and tyrosinase-expressing melanoma cell line. Thus, our data demonstrate that IL-10-treated DC induce an antigen-specific anergy in cytotoxic CD8(+) T cells, a process that might be a mechanism of tumors to inhibit immune surveillance by converting DC into tolerogenic antigen-presenting cells.

Dendritic cell-based genetic immunization in mice with a recombinant adenovirus encoding murine TRP2 induces effective anti-melanoma immunity.

BACKGROUND: The induction of cellular immune responses to melanocyte-specific enzymes such as the tyrosinase family of proteins is the goal of various clinical studies for the immunotherapy of melanoma. Tyrosinase-related protein-2 (TRP2) is an attractive model antigen for preclinical studies in C57BL/6 mice because it is naturally expressed by the murine B16 melanoma and can be recognized by self-reactive cytolytic T lymphocytes (CTL). Here we describe efforts to develop genetic immunization with dendritic cells (DC) for the immunotherapy of melanoma in this clinically relevant system. METHODS: Recombinant adenoviruses encoding green fluorescent protein (Ad-EGFP) and murine TRP2 (Ad-mTRP2) were constructed using Cre-loxP-mediated recombination. DC were generated in vitro from precursors in bone marrow and transduced with Ad-EGFP or Ad-mTRP2. Mice were immunized by direct injection of adenovirus or by injection of Ad-transduced DC. Induction of tumor immunity was assessed by intravenous challenge with B16 melanoma cells and enumeration of experimentally induced lung metastases. RESULTS: Flowcytometric analysis of DC transduced with Ad-EGFP demonstrated endogenous fluorescence due to cytoplasmatic expression of EGFP in 30-60% of cells. Ad-EGFP-transduced DC simultaneously displayed the DC-specific marker NLDC145 and high levels of MHC and costimulatory molecules on their cell surface. Transduction of DC with Ad-mTRP2 resulted in strong intracellular expression of TRP2 which could be readily detected by immunostaining. Importantly, immunization of mice with cultured Ad-mTRP2-transduced DC completely prevented the development of lung metastases following an intravenous challenge with B16 melanoma cells. This striking protective effect was observed with both the intravenous and the subcutaneous route of DC immunization. In vivo depletion of T-cell subsets suggested that the protective effect of an immunization with Ad-mTRP2-transduced DC involved both CD8+ and CD4+ T-cells. CONCLUSIONS: Our results demonstrate that DC-based genetic immunization of mice with TRP2, a clinically relevant melanocyte-specific self-antigen, induces effective cellular immunity and prevents metastatic growth of B16 melanoma cells in vivo.

Contact tolerance.

Contact tolerance describes an immunological state which is caused by ordinary contact allergens painted in doses too low to sensitize, either once or repeatedly, onto healthy intact skin. The tolerance state accomplished by this means in BALB/c and C57BI/6 mice was found to be mediated by hapten-specific T cells that adoptively transferred tolerance to naive recipients. Furthermore, these cells were shown to be sensitive to cyclophosphamide, to express the Lyt2+ (CD8) phenotype, and to produce, upon restimulation in vitro, predominantly anti-inflammatory cytokines such as IL-4, IL-5 and IL-10. These data indicate that contact tolerance of the low zone type is actively mediated by Th2-like CD8 T cells rather than arising as a consequence of clonal anergy. The induction of contact tolerance appeared to be strictly dose-dependent. As opposed to sensitizing doses of allergen, subsensitizing doses did not involve epidermal Langerhans cells discernibly. This was suggested by their normal ultrastructure, their unaffected adenosine triphosphatase system, the inefficacy of functional blocking and excision experiments. Both radiolabeled and fluorescent contact sensitizers were observed to readily enter the bloodstream, thereby being dispersed throughout the body. Presumably, contact tolerance is induced systemically rather than locally. The presence of hapten-specific tolerance can only be uncovered through a subsequent attempt to sensitize. If the attained sensitization turns out to be significantly lower than that of immunologically naive controls, and if sensitization to chemically unrelated sensitizers is not impaired, hapten-specific tolerance does exist. Thus, contact tolerance is a result obtained from experimental sensitization in animals. Nonetheless, it is assumed to occur also in humans, although it is not demonstrable unless different proofs of existence become available.

Induction of tolerance by IL-10-treated dendritic cells.

Dendritic cells (DC) form a specialized system for presenting Ag to naive or quiescent T cells and consequently play a central role in the induction of T and B cell immunity. In this study we used DC generated from peripheral progenitors to analyze the effect of IL-10 on the accessory function of human DC. We demonstrate that immature DC, harvested on days 9 to 11 and exposed to IL-10 for the last 2 days of culture, show a strongly reduced capacity to stimulate a CD4+ T cell response in an allogeneic MLR in a dose-dependent manner. In contrast, fully mature DC are completely resistant to the effects of IL-10. These results were obtained in both an alloantigen-induced MLR and an anti-CD3 mAb-induced response of primed and naive (CD45RA+) CD4+ T cells. FACS analysis revealed inhibition of the up-regulation of the costimulatory molecules CD58 and CD86 and the specific DC marker CD83 in DC pretreated with IL-10. These data suggest that IL-10 inhibited the development of fully mature DC. Furthermore, DC precultured with IL-10, but not controls, induced a state of alloantigen-specific anergy in CD4+ T cells and of peptide-specific anergy in the influenza hemagglutinin-specific T cell clone HA1.7. Analysis of the supernatants of these anergic T cells revealed a reduced production of IL-2 and IFN-gamma compared with that in control cells. Collectively, these data suggest that IL-10 converts immature DC into tolerogenic APC, which might be a useful tool in the therapy of patients with autoimmune or allergic diseases.

Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions.

Culture conditions for human dendritic cells (DC) have been developed by several laboratories. Most of these culture methods, however, have used conditions involving fetal calf serum (FCS) to generate DC in the presence of granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. Recently, alternative culture conditions have been described using an additional stimulation with monocyte-conditioned medium (MCM) and FCS-free media to generate DC. As MCM is a rather undefined cocktail, the yield and quality of DC generated by these cultures varies substantially. We report that a defined cocktail of tumor necrosis factor (TNF)-alpha, IL-1beta and IL-6 equals MCM in its potency to generate DC. Addition of prostaglandin (PG)E2 to the cytokine cocktail further enhanced the yield, maturation, migratory and immunostimulatory capacity of the DC generated. More importantly, culture conditions also influenced the outcome of the T cell response induced. DC cultured with TNF-alpha/IL-1/IL-6 or MCM alone induced CD4+ T cells that release intermediate levels of interferon (IFN)-gamma and no IL-4 or IL-10. Production of IFN-gamma was significantly induced by addition of PGE2, while no effect on production of IL-4 or IL-10 was observed. Even more striking differences were observed for CD8+ T cells. While MCM conditions only induced IFN-gamma(low), IL-4(neg) cells, TNF-alpha/IL-1/IL-6 promoted growth of IFN-gamma(intermediate), IL-4(neg) CD8+ T cells. Addition of PGE2 again only further polarized this pattern enhancing IFN-gamma production by alloreactive CD8+ T cells in both cultures without inducing type 2 cytokines. Taken together, the data indicate that the defined cocktail TNF-alpha/IL-1/IL-6 can substitute for MCM and that addition of PGE2 further enhances the yield and quality of DC generated. TNF-alpha/IL-1, IL-6 + PGE2-cultured DC seem to be optimal for generation of IFN-gamma-producing CD4/CD8+ T cells.

E-selectin expression in experimental models of inflammation in mice.

E-selectin (CD62E, formerly termed ELAM-1) is a cytokine-inducible adhesion molecule which mediates the binding of neutrophils, monocytes, and skin homing T-cells. The murine homologue of E-selectin has been cloned. A monoclonal antibody (21KC10) was used here to study immunohistochemically the expression and regulation of murine E-selectin in vitro and in vivo. As described for the human system, there was no staining of normal endothelium in skin and other tissues. LPS and tumour necrosis factor-alpha (TNF-alpha), but not interleukin-4 (IL-4) or interferon-gamma (IFN-gamma), induced a transient expression of E-selectin, both when injected in vivo and when added to endothelial cell lines in vitro. To analyse temporal expression of E-selectin under pathophysiological conditions in vivo, we chose two murine models of inflammation: allergic (ACD) and irritant contact dermatitis (ICD). Expression of E-selectin was found to be induced on vascular endothelium of post-capillary venules in both ACD and ICD. In ICD, maximal staining of endothelial cells occurred earlier than in ACD. Expression of E-selectin during ICD and ACD was then compared between strains of mice which differ with regard to the intensity of their inflammatory reaction. BALB/c mice, which in contrast to C57BI/6 mice show a denser infiltrate and prolonged influx of granulocyte and monocytes, revealed a more pronounced and more prolonged expression of E-selectin than C57BI/6 mice. This held true for both ACD and ICD, and in each case, peak expression of E-selectin was associated with the highest density of the leukocytic infiltrate. This study thus reveals regulatory mechanisms involved in the expression of murine E-selectin in vivo and in vitro. It also demonstrates a correlation between endothelial expression of E-selectin and the genetically determined intensity of the inflammatory response.

Induction of low zone tolerance to contact allergens in mice does not require functional Langerhans cells.

Epidermal Langerhans cells are known to be the major controlling element in the development of contact hypersensitivity. Haptenic molecules permeating the skin are taken up locally by Langerhans cells and then presented to T lymphocytes in the regional lymph nodes. Despite the presence of functional Langerhans cells, however, subsensitizing doses of hapten applied epicutaneously induce tolerance. We examined epidermal Langerhans cells at the site of contact with picryl chloride or oxazolone in BALB/c and C57B1/6 mice with regard to their responding to either subsensitizing or sensitizing doses of allergen. Subsensitizing doses did not interfere with the membranous adenosine triphosphatase system on Langerhans cells, known to relate to functional readiness of the cell. Accordingly, on electron microscopy the ultrastructure of Langerhans cells was found to be like that in untreated skin. In contrast, sensitizing doses caused a significant depletion of adenosine triphosphatase-positive Langerhans cells, and electron microscopy revealed marked cellular activation of Langerhans cells, with enlarged nuclei and increased numbers of mitochondria and Birbeck granules. Furthermore, subsensitizing doses induced tolerance regardless of whether Langerhans cells were functionally intact or had their function blocked arbitrarily. Blocking was achieved either by preceding ultraviolet B irradiation at the site of application or by painting of a sensitizer before painting another sensitizer on the same site. Moreover, not even surgical removal of the site within minutes after painting could prevent the induction of tolerance. The data suggest that subsensitizing doses of contact allergens painted on normal murine skin bypass involvement of epidermal Langerhans cells.