Induction of skewed Th1/Th2 T-cell differentiation via subcutaneous immunization with Freund's adjuvant.

CD4+ T cells differentiate into at least two distinct subsets, Th1 and Th2, that are characterized by their cytokine-producing profiles. In this study, we attempted to delineate whether and how CD4+ T-cell responses could be skewed in one direction or another. BALB/c mice were immunized with chicken ovalbumin (OVA) emulsified with either incomplete or complete Freund's adjuvant (IFA or CFA). When lymph node cells were assessed on day 7, antigen specific proliferation was similarly observed both in the mice immunized with IFA and CFA. In contrast, on day 28 there was a less significant response in the mice primed with IFA than in those primed with CFA. ELISA analyses revealed more Th1 predominant cytokine production by T cells immunized with OVA+CFA rather than in IFA, which resulted in balanced IFN-gamma and IL-4 production. Flow cytometric analyses of intracellular cytokines confirmed that T cells from mice primed with CFA produced Th1 cytokines more predominantly. When lymph node dendritic cells (DC) were compared for their co-stimulatory molecule expression, priming with CFA and IFA similarly upregulated CD80 and CD86 expression by lymph node DC, and no significant differences were observed in CD40, 54, 80 and 86 expression between the DC harvested from IFA and CFA immunized mice. In addition, both priming with IFA and CFA similarly induced IL-12 production by DC. Thus, although the reason(s) for the preferential induction of a Th1/Th2 response remains unknown, these results indicate that a relatively Th1/Th2 skewed response is differentially induced by different types of adjuvants, and induction of a Th1 skewed response may be responsible for long lasting cellular immunity.

Activation through CD40 ligation induces functional Fas ligand expression by Langerhans cells.

Langerhans cells (LC) are professional antigen-presenting cells of dendritic cell (DC) lineage and are critical for the induction of primary immune responses in skin. Following antigenic stimulation, LC migrate to regional lymph nodes and induce antigen-specific activation of T cells. After primary expansion, the majority of T cells undergo Fas/Fas ligand (FasL)-mediated apoptotic cell death, thereby suppressing their excessive expansion. Although recent investigations have indicated an immunoregulatory function for DC, whether LC could be involved in Fas/FasL-mediated suppression of activated T cells is still unclear. In this study, we found that LC express FasL after activation triggered through CD40 molecules on their surface, but not by stimulation with LPS or IFN-gamma. The functional significance of FasL expression by LC was demonstrated using two different assays for apoptosis induced in Jurkat cells. The apoptosis in Jurkat cells was completely blocked by anti-FasL blocking antibody, suggesting a Fas/FasL-mediated mechanism. These results indicate a new feedback mechanism to down-regulate T cell activation by LC through the interaction of the TNF receptor/ligand superfamily, CD40/CD40L and Fas/FasL.

Contact hypersensitivity.

Contact hypersensitivity is a simple in vivo assay of cell-mediated immune function in which exposure of epidermal cells to exogenous haptens results in a delayed-type hypersensitive reaction that can be measured and quantified. The Langerhans cell is the critical antigen-presenting cell in this reaction which initiates sensitization to haptens by presenting antigens to CD4-bearing T lymphocytes which, in turn, secrete lymphokines and recruit other cells to the site of the reaction. In the protocol described here, mice are shaved and the skin of their abdomens is exposed to a hapten. After 6 days (the afferent phase), the baseline ear thickness is measured prior to initiation of the efferent phase. Finally, the ear is treated epicutaneously with the hapten solution and ear thickness is measured in approximately 24 hr. The change in ear thickness after allergen treatment can be used to calculate the percent suppression of contact hypersensitivity.

Dermatological research in the 21st century: our fantastic future.

During the second half of the twentieth century, dermatology came of age. Just as clinical dermatology, through new diagnostic and therapeutic modalities, matured to a point where the dermatologist could affect peoples' lives profoundly, so too did dermatological research enhance our understanding of skin diseases enormously. Dermatology should not be viewed as a scientific discipline-advances come from fundamental scientific areas such as cell and molecular biology, biochemistry, immunology, microbiology, technology, the clinical sciences and others. Thus, dermatology and skin biology live within a universe of science, only a small part of which is dermatology and skin biology, and our patients and our science are dependent on integration and interdigitation with the universe of science for future success. In this lecture I will elaborate on where I think the next 10-20 years will take us in this universe.

Generation of mature dendritic cells from a CD14+ cell line (XS52) by IL-4, TNF-alpha, IL-1 beta, and agonistic anti-CD40 monoclonal antibody.

We established a model system to generate mature dendritic cells (DC) from a GM-CSF-dependent cell line, XS52, which had been isolated from the epidermis of newborn BALB/c mice. Screening of various soluble factors revealed that IL-4 induces phenotypic maturation of XS52 (as evaluated by enhanced expression of class II, CD40, CD80, CD86, CD11c, and loss of expression of CD14) in a time-dependent manner. The addition of TNF-alpha, IL-1 beta, and agonistic anti-CD40 mAb further enhanced expression of these maturation markers. Consistent with their phenotypic maturation, these cells (termed XS-DC) exhibited potent Ag-presenting capacity to both naive and primed T cells. In addition, injection of hapten-conjugated XS-DC induced contact hypersensitivity in vivo, suggesting their potential as tools for vaccination. Expression of CD14 by the starting cell population, the requirement for GM-CSF and IL-4, and the relatively long culture period are the common characteristics shared between our cells and human monocyte-derived DC, whose analogues in mice have not been identified. Because large numbers of skin-associated mature DC devoid of other cell lineages are easily obtained, this model system may facilitate the study of molecular events associated with maturation of DC and the use of DC for immunization.

Human herpesvirus 6 infects dendritic cells and suppresses human immunodeficiency virus type 1 replication in coinfected cultures.

Human herpesvirus 6 (HHV-6) has been implicated as a cofactor in the progressive loss of CD4(+) T cells observed in AIDS patients. Because dendritic cells (DC) play an important role in the immunopathogenesis of human immunodeficiency virus (HIV) disease, we studied the infection of DC by HHV-6 and coinfection of DC by HHV-6 and HIV. Purified immature DC (derived from adherent peripheral blood mononuclear cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4) could be infected with HHV-6, as determined by PCR analyses, intracellular monoclonal antibody staining, and presence of virus in culture supernatants. However, HHV-6-infected DC demonstrated neither cytopathic changes nor functional defects. Interestingly, HHV-6 markedly suppressed HIV replication and syncytium formation in coinfected DC cultures. This HHV-6-mediated anti-HIV effect was DC specific, occurred when HHV-6 was added either before or after HIV, and was not due to decreased surface expression or function of CD4, CXCR4, or CCR5. Conversely, HIV had no demonstrable effect on HHV-6 replication. These findings suggest that HHV-6 may protect DC from HIV-induced cytopathicity in AIDS patients. We also demonstrate that interactions between HIV and herpesviruses are complex and that the observable outcome of dual infection is dependent on the target cell type.

Immunization through dermal delivery of protein-encoding DNA: a role for migratory dendritic cells.

The early mechanisms by which DNA-dependent immunization occurs remain poorly understood. We determined whether intradermal injection of a cytomegalovirus (CMV) promoter-driven plasmid encoding hen egg lysozyme (pCMV:HEL) induced sensitization against the encoded protein, and whether cutaneous dendritic cells (DC) were involved in this sensitization. Both humoral and cellular responses to HEL were observed. DC that migrated from skin explant culture 3 days after injection of pCMV:HEL DNA contained mRNA encoding HEL. They induced a 3.5-7-fold increase in [3H]thymidine incorporation by HEL protein-primed CD4+ T cells compared to that induced by DC from mice injected with control plasmid. DC emigrating from skin explants recovered from pCMV:HEL injected mice also sensitized naive mice after adoptive transfer and induced the generation of CTL. Thus following DNA delivery within the dermis, DC can induce primary and secondary immune responses.

Contact hypersensitivity in MHC class II-deficient mice depends on CD8 T lymphocytes primed by immunostimulating Langerhans cells.

Studies to determine if CD4+ and/or CD8+ T cells are critical for the initiation and propagation of contact hypersensitivity (CHS) reactions have yielded conflicting results regarding their roles. We studied the induction and expression of CHS to trinitrochlorobenzene (TNCB) using major histocompatibility complex class II-deficient mice that display normal numbers of CD8+ T cells but lack CD4+ T cells. CHS to TNCB, detected as an increase in ear thickness 24 h after epicutaneous challenge, was significantly enhanced in major histocompatibility complex class II-deficient mice compared with wild-type controls. Ear swelling responses in major histocompatibility complex class II-deficient mice were decreased by treatment with anti-CD8 antibody or by injection of wild-type CD4+ T cells. To further characterize mechanisms involved in the initiation of CHS responses, phenotypical and functional characteristics of both freshly isolated and cultured Langerhans cells were studied. Like Langerhans cells from wild-type controls, Langerhans cells from major histocompatibility complex class II-deficient mice upregulated B7-1 and B7-2 costimulatory molecules and enhanced major histocompatibility complex class I expression upon short-term culture. Cultured Langerhans cells induced a 3.5-fold increase in the stimulation of autologous hapten-specific CD8+ T cell proliferation compared with fresh Langerhans cells. Finally, TNP-coupled Langerhans cells from major histocompatibility complex class II-deficient mice primed naive mice to TNCB after transfer. These results demonstrate that hapten-specific CD8+ T cells are sufficient for the expression of CHS and that CD8 priming does not require the presence of CD4+ T cells or major histocompatibility complex class II antigen. Key words: CD8 lymphocytes/MHC class II deficiency.

Heterogeneous MHC II restriction pattern of autoreactive desmoglein 3 specific T cell responses in pemphigus vulgaris patients and normals.

Pemphigus vulgaris is a life threatening bullous autoimmune disease of the skin mediated by autoantibodies against desmoglein 3 (Dsg3) on epidermal keratinocytes. Pemphigus vulgaris patients exhibit T cell responses against Dsg3 that may serve as a target to modulate the production of pathogenic autoantibodies. Healthy carriers of major histocompatibility complex class II alleles identical or similar to those that are highly prevalent in pemphigus vulgaris, namely DRbeta1*0402 and DRbeta1*1401, also mount T cell responses against Dsg3. We thus wanted to determine whether these prevalent major histocompatibility complex class II alleles restricted Dsg3 specific T cell responses. A CD4+ T cell line from the DRbeta1*0402+ patient PV9 was stimulated by Dsg3 with DRbeta1*0402+ L cells as antigen-presenting cells. A CD4+ T cell line and six CD4+ T cell clones from the DR11/14+ patient PV8, and six CD4+ T cell clones from the DR11+ healthy donor C6, required DR11/ DQbeta1*0301+ peripheral blood mononuclear cells but not DR11+ L cells as antigen-presenting cells and were strongly inhibited by anti-DQ antibodies, indicating that they were restricted by HLA-DQbeta1*0301. A CD4+ T cell line and three T cell clones from the DR11+ healthy donor C11 were differentially stimulated by Dsg3 with L cells expressing one of several DR11 alleles. T cell recognition of Dsg3 was thus not only restricted by the pemphigus vulgaris associated DRbeta1*0402 allele, but also by several DR11 alleles, some of which are highly homologous to DRbeta1*0402, and by HLA-DQbeta1*0301.

Recognition of desmoglein 3 by autoreactive T cells in pemphigus vulgaris patients and normals.

Pemphigus vulgaris (PV) is an autoimmune blistering disease of the skin and is caused by autoantibodies against desmoglein 3 (Dsg3) on epidermal keratinocytes. Because the production of autoantibodies is presumably T cell dependent, Dsg3-specific T cell reactivity was investigated in 14 PV patients and 12 healthy donors. Peripheral blood mononuclear cells from seven PV patients with active disease showed a primary in vitro response to a recombinant protein containing the extracellular portion (EC1-5) of Dsg3, whereas two of seven PV patients in remission or under immunosuppressive treatment exhibited only secondary (2 degrees) or tertiary (3 degrees) T cell responses to Dsg3. T cell responses to Dsg3 were also observed in four of 12 healthy individuals upon 2 degrees or 3 degrees stimulation with Dsg3. Both PV patients and healthy responders were either positive for DRbeta1*0402 -- which is highly prevalent in PV -- or positive for DR11 alleles homologous to DRbeta1*0402. Two CD4+ Dsg3-specific T cell lines and 12 T cell clones from two PV patients and two CD4+ T cell lines and eight T cell clones from two normals were also stimulated by a Dsg3 protein devoid of the EC2-3 (deltaN1), suggesting that epitopes were located in the EC1, EC4, and/or EC5. Using Dsg3 peptides, one immunodominant peptide (residues 161-177) was also identified in the EC2. These observations demonstrate that T cell responses to Dsg3 can be detected in PV patients and in healthy donors carrying major histocompatibility class II alleles identical or similar to those highly prevalent in PV.

Productive infection of dendritic cells by HIV-1 and their ability to capture virus are mediated through separate pathways.

There is substantial evidence that dendritic cells (DC) residing within epithelial surfaces (e.g., Langerhans cells) are the initial cells infected with HIV after mucosal exposure to virus. To study DC-HIV interactions in detail, we propagated Langerhans cell-like DC from cord blood CD34(+) cells and from adult blood plastic-adherent PBMC in the presence of cytokines (GM-CSF, IL-4, and/or TNF-alpha). DC pulsed overnight with HIVBaL or HIVIIIB were infected productively with both viral subtypes (as assessed by PCR, supernatant p24 protein levels, electron microscopy, and antibody staining). Productive infection could be blocked by anti-CD4 mAbs, RANTES (regulated upon activation, normal T cell expressed and secreted) (for HIVBaL), stromal cell-derived factor-1 (for HIVIIIB), or azidothymidine added during the HIV pulse, as well as by blocking DC proliferation. However, pulsing DC with HIV under these blocking conditions had no effect on the ability of DC to capture virus and transmit infection to cocultured antigen-stimulated CD4(+) T cells. Thus, we show by several criteria that (a) productive infection of DC and (b) the ability of DC to capture virus are mediated through separate pathways. We suggest that strategies designed to block mucosal transmission of HIV should consider interfering with both virus infection and virus capture by DC.

Cytokine gene expression during the elicitation phase of contact sensitivity: regulation by endogenous IL-4.

Recent studies have focused on characterizing the cytokine profile produced in the epidermis during the sensitization phase of contact sensitivity (CS). Some prior studies have also identified altered individual cytokine mRNA profiles in skin or draining lymph nodes (or several cytokine mRNA profiles in the epidermis) during the elicitation phase of CS. In this study we determined the dynamics of appearance of a battery of cytokine mRNA levels in both the epidermis and dermis during the elicitation phase of CS. We isolated mRNA from dispase-separated epidermis and dermis of TNCB-sensitized and naive BALB/c mice at various times after TNCB challenge. Changes in IFN-gamma and IL-4 mRNA levels (by semiquantitative RT-PCR) were more reproducible and dramatic than those of other cytokines studied (IL-1beta, IL-2, IL-10, and IL-12 p40). Compared to naive mice, sensitized mice had significantly elevated IL-4 mRNA signals 9 and 24 h (dermis), and 24 h (epidermis), after TNCB challenge. The increased IL-4 mRNA levels were mast-cell-independent, because sensitized mast-cell-deficient mice showed similar increases in IL-4 mRNA. To examine the role of endogenous IL-4 in CS elicitation, sensitized mice were treated with anti-IL-4 mAb 1 h before challenge. In accord with prior studies, anti-IL-4 mAb-pretreated mice showed increased ear swelling 24 h after challenge compared to mice pretreated with isotype control mAb. Anti-IL-4 mAb pretreatment also enhanced IFN-gamma, IL-2, IL-12 p40, and IL-1beta (but not IL-10) mRNA signals in the dermis of sensitized and challenged mice. These data indicate that IL-4 is produced in murine skin during the elicitation phase of CS and is an important down-modulator of inflammation. IL-4 may blunt CS by regulating local production of proinflammatory cytokines.

UVB induces IL-12 transcription in human keratinocytes in vivo and in vitro.

Human epidermal cells produce a wide range of cytokines, including those characteristic of Th2-like responses such as interleukin (IL)-4 and IL-10. As well, keratinocytes have recently been shown to produce Th1-like cytokines such as IL-12. Exposure to UVB has profound effects on the skin and systemic immune system, which is in part mediated by secretion of tumor necrosis factor (TNF)-alpha by epidermal cells. Because IL-12 induces production of TNF-alpha by certain cells of the immune system, we sought to determine whether UVB is an inducer of IL-12 gene expression in epidermal cells. Human epidermal cells were exposed to UVB radiation in vivo, isolated by suction blister technique and trypsinization and transcription of the IL-12 p35 and p40 chains was examined by RT-PCR. We found the p35 chain of IL-12 to be constitutively expressed and the p40 chain inducible by UVB irradiation. Because epidermis consists of a heterogenous cell population with distinct cytokine repertoires, we sought to determine the cellular source of the IL-12 message after UVB exposure. After depleting UVB-exposed epidermal cells for DR+ cells, no reduction in the IL-12 activity was detected, suggesting that keratinocytes are a source of IL-12 transcripts in UVB-exposed human epidermis. This was supported by the up-regulation of IL-12 p40 transcripts in UV-irradiated cultured keratinocytes that were devoid of DR+ cells. Up-regulation of IL-12 p40 gene expression by UVB as demonstrated here, taken together with the finding that keratinocytes also up-regulate IL-10 transcription, suggests that there is a complex interplay between Th1- and Th2-like epidermis-derived cytokines following exposure to UVB.

Interleukin-15 mRNA is expressed by human keratinocytes Langerhans cells, and blood-derived dendritic cells and is downregulated by ultraviolet B radiation.

Interleukin (IL)-15 is a recently described cytokine that shares many functional activities with IL-2; however, unlike IL-2, IL-15 is produced by monocytes/macrophages, and not by lymphocytes. In this report, we assessed IL-15 mRNA expression by freshly isolated human epidermal cells, as well as by negatively selected keratinocytes and positively selected Langerhans cells, utilizing reverse transcription and polymerase chain reaction. In addition, cultured keratinocytes, immortalized keratinocytes (HaCaT cells), and dendritic cells expanded from adult peripheral blood in the presence of granulocyte/macrophage-colony stimulating factor and IL-4 were examined for IL-15 transcripts. Using cultured keratinocytes, we also studied the regulation of IL-15 mRNA expression by ultraviolet B radiation in vitro. Freshly isolated keratinocytes, HaCaT cells, and cultured keratinocytes all constitutively expressed IL-15 mRNA, and IL-15 expression was downregulated by ultraviolet B radiation in cultured keratinocytes in a time- and dose-dependent manner. In addition, IL-15 transcripts were constitutively expressed by freshly isolated Langerhans cells. IL-15 produced by keratinocytes, Langerhans cells, and other tissue-specific dendritic cells may be important in attracting and activating antigen-specific Th1 T cells. Furthermore, ultraviolet B-induced downregulation of keratinocyte IL-15 production may contribute to the relative state of immunosuppression induced by sun exposure.

Modulation of T cell responses to recall antigens presented by Langerhans cells in HIV-discordant identical twins by anti-interleukin (IL)-10 antibodies and IL-12.

Decreased antigen (Ag)-specific T cell (TC) proliferation and IL-2 production are detected in all stages of HIV disease. To determine whether dendritic cell dysfunction and/or abnormal cytokine production contribute to HIV-induced immune dysregulation, we studies TC responses to recall Ags (influenza virus and tetanus toxoid) presented by Langerhans cells (LC) in six pairs of HIV-discordant identical twins, and the modulation of these responses by anti-IL-10 (alphaIL-10) mAbs and IL-12. LC from HIV+ twins induced IL-2 comparable to normal LC in cultures containing TC from uninfected twins. In contrast, IL-2 production was markedly decreased in cultures containing TC from HIV+ twins. IL-12 enhanced Ag-specific IL-2 production by TC from two patients with CD4+ counts > 600. In contrast, alphaIL-10 mAbs enhanced IL-2 production in influenza virus-stimulated cultures containing TC from two patients with CD4+ counts < 20. Thus, these findings suggest that immunologic dysfunction of dendritic cells does not contribute to impaired secondary immune responses in HIV+ individuals. Although few patients were studied, partial immune reconstitution in vitro, as demonstrated here, may help to predict those individuals who might benefit from cytokines or antibodies against cytokines as immunotherapy for HIV disease.

Murine epidermal Langerhans cells do not express the low-affinity receptor for immunoglobulin E, FcEpsilonRII (CD23).

The low-affinity receptor for IgE, FcepsilonRII (CD 23), plays an important role in IgE-mediated disorders such as allergy, atopy, and parasitic infections. In humans, the FcepsilonRII B isoform on epidermal Langerhans cells is thought to be an important accessory molecule in the allergy-specific T cell activation in atopic dermatitis. Since considerable knowledge about the accessory function of Langerhans cells for T-cell activation stems from mouse models, and since an IgE-bearing Langerhans cell mouse model would be useful in studying the pathophysiology of atopic dermatitis, we determined whether FcepsilonRII was also present on murine Langerhans cells. FcepsilonRIIa, which is the major FcepsilonRII isoform in mice, was found to be constitutively expressed on spleen cells from normal mice but was not present on epidermal Langerhans cells. When interleukin-4, a known inducer of FcepsilonRII, was administered in vivo, FcepsilonRII-specific mRNA and protein was significantly unregulated in spleen cells but not in Langerhans cells. De novo synthesis of FcepsilonRII was also induced in vitro by interleukin-4 on spleen cells, but not on epidermal cells. The presence of a recently cloned murine counterpart of the human Fc-epsilon- R. II isoform on murine Langerhans cells could also be excluded on the protein and mRNA level because of the high degree of homology to mouse Fc-epsilon-R IIA. Taken together the data indicate that murine epidermal Langerhans cells do not express the low-affinity receptor for IgE.