Biomedical subjects
Publications and source records attributed to G Girolomoni.
Allergic contact dermatitis is a prototypic T-cell-mediated disease that has a socio-economic impact in industrialized countries. Here, Andrea Cavani and colleagues highlight recent developments in the T-cell-based effector and regulatory mechanisms of this common skin disorder.
BACKGROUND: Atopic dermatitis (AD) and psoriasis are genetically determined inflammatory skin disorders. Keratinocytes actively participate in cutaneous inflammatory responses by elaborating various chemokines. OBJECTIVE: We investigated the capacity of IL-4, IFN-gamma, and TNF-alpha to modulate the expression of CCL and CXCL chemokines in cultured keratinocytes from patients and healthy individuals, as well as chemokine expression in situ. METHODS: Keratinocyte cultures were established from normal-looking skin of adult patients with AD or psoriasis vulgaris and from healthy subjects. Monocyte chemoattractant protein 1 (MCP-1)/CCL2, RANTES/CCL5, IL-8/CXCL8, and IFN-gamma-induced protein of 10 kd (IP-10)/CXCL10 production was evaluated at the mRNA and protein levels by using RNase protection assay and ELISA, respectively. The expression of the same chemokines was studied in chronic lesional skin by means of immunohistochemistry or in situ hybridization. RESULTS: Only IL-8 mRNA was detected in unstimulated ke-ratinocyte cultures. MCP-1 and IP-10 were potently induced by IFN-gamma, whereas IL-8 and RANTES were preferentially upregulated by TNF-alpha and, to a lesser extent, by IFN-gamma. IL-4 weakly induced IP-10, RANTES, and IL-8 but not MCP-1. Keratinocytes of patients with AD invariably responded with significantly earlier and higher RANTES expression. By contrast, keratinocytes of patients with psoriasis displayed much higher levels of both constitutive and induced IL-8 and a stronger induction of MCP-1 and IP-10. RANTES and MCP-1 mRNA(+) keratinocytes were detected in the basal layer of lesions of patients with AD and psoriasis. IP-10 and IL-8 were consistently upregulated in the epidermis of patients with psoriasis but not in lesions of patients with AD. CONCLUSIONS: Keratinocytes of patients with AD and psoriasis show an intrinsically abnormal and different chemokine production profile and may thus favor the recruitment of distinct leukocyte subsets into the skin.
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Fractalkine (FKN, CX3CL1) is a membrane-bound CX3C chemokine induced by primary proinflammatory signals in vascular endothelial cells (ECs). Here we examined the role of FKN in polarized Th1 or Th2 responses. Proinflammatory signals, including LPS, IL-1, TNF, and CD40 ligand, induced FKN, as did IFN-gamma, which had synergistic activity with TNF. IL-4 and IL-13 did not stimulate the expression of FKN and markedly reduced induction by TNF and IFN-gamma. TNF alone or combined with IFN-gamma also induced release of soluble FKN, which was inhibited by IL-4 and IL-13. In light of this differential regulation of FKN by the master cytokines that control polarized responses, we analyzed the interaction of FKN with natural killer (NK) cells and polarized T-cell populations. NK cells expressed high levels of the FKN receptor CX3CR1 and responded to FKN. CX3CR1 was preferentially expressed in Th1 compared with Th2 cells. Th1 but not Th2 cells responded to FKN. By immunohistochemistry, FKN was expressed on ECs in psoriasis, a Th1-dominated skin disorder, but not in Th2-driven atopic dermatitis. Similarly, ECs in Mycobacterium tuberculosis granulomatous lymphadenitis, but not those in reactive lymph node hyperplasia or in Castelman's disease, showed immunoreactive FKN. These results indicate that regulated expression of FKN in ECs participates in an amplification circuit of polarized type I responses.
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The recruitment of T cells into the skin is regulated by chemokines released by resident cells. In this study, we found that normal human keratinocytes activated with Th1-derived supernatant (sup) expressed early (6-12 h) IP-10/CXCL10, MCP-1/CCL2, IL-8/CXCL8, and I-309/CCL1 mRNAs and with slower kinetics (24-96 h), RANTES/CCL5 and MDC/CCL22 mRNAs. Upon stimulation with the Th1 sup, keratinocytes secreted high levels of RANTES, IP-10, MCP-1, and IL-8 and moderate levels of I-309 and MDC. Although much less efficiently, Th2 sup could also induce keratinocyte expression of IL-8, IP-10, RANTES, and MCP-1 but not of I-309 and MDC. TARC/CCL17 was not significantly induced by any stimuli. Sup from keratinocytes activated with Th1-derived cytokines elicited a strong migratory response of Th1 cells and a limited migration of Th2 cells, whereas sup from Th2-activated keratinocytes promoted a moderate migration of Th1 and Th2 lymphocytes. Thus, keratinocytes appear considerably more sensitive to Th1- than to Th2-derived lymphokines in terms of chemokine release and can support the preferential accumulation of Th1 lymphocytes in the skin.
Allergic contact dermatitis (ACD) is the result of an exaggerated immune reaction to haptens mediated by skin-homing T cells, but the effector mechanisms responsible for the tissue damage are poorly understood. Here we studied the capacity of distinct subsets of hapten-specific T cells to induce apoptosis in autologous keratinocytes. Skin- and blood-derived nickel-specific CD8+ T cytotoxic 1 (Tc1) and Tc2 clones as well as CD4+ Th1 and Th2 expressed the cutaneous lymphocyte-associated Ag and exhibited strong MHC-restricted cytotoxicity against nickel-coupled B lymphoblasts, as detected by the [3H]TdR release assay. Both Tc1 and Tc2 clones, but not CD4+ T cells, displayed a significant cytotoxic activity against resting nickel-modified keratinocytes. Following IFN-gamma treatment, keratinocytes expressed MHC class II and ICAM-1 and became susceptible to Th1-mediated, but not Th2-mediated, cytotoxicity. The molecules of the two major cytotoxic pathways, Fas ligand (FasL) and perforin, were expressed by Tc1, Tc2, and Th1 cells, whereas Th2 cells expressed only FasL. Experiments performed in the presence of specific inhibitors of the perforin (concanamycin A) and FasL (brefeldin A) pathway indicated that perforin-mediated killing dominated in Tc1 and Tc2, and FasL-mediated cytotoxicity prevailed in Th2 clones, with a more heterogeneous behavior in the case of Th1 cells. Finally, perforin mRNA was expressed in ACD lesional skin, as assessed by RT-PCR analysis. In aggregate, our results indicate that keratinocytes can be target of multiple hapten-specific CTL responses, that may have distinct roles in the epidermal injury during ACD.
IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC) belong to the non-glutamate-leucine-arginine motif CXC chemokine family and act solely through the CXCR3 receptor for potent attraction of T lymphocytes. In this study, we evaluated the capacity of the T cell-derived cytokines IL-4, IL-10, and IL-17 to modulate IP-10, Mig, and I-TAC in cultured human keratinocytes and CXCR3 expression in T cells from allergic contact dermatitis (ACD). IL-4, but not IL-10 or IL-17, significantly up-regulated IFN-gamma- or TNF-alpha-induced IP-10, Mig, and I-TAC mRNA accumulation in keratinocytes and increased the levels of IP-10 and Mig in keratinocyte supernatants. Immunohistochemistry of skin affected by ACD revealed that >70% of infiltrating cells were reactive for CXCR3 and that CXCR3 staining colocalized in CD4+ and CD8+ T cells. Nickel-specific CD4+ and CD8+ T cell lines established from ACD skin produced IFN-gamma and IL-4 and expressed moderate to high levels of CXCR3. Finally, CXCR3 agonistic chemokines released by stimulated keratinocytes triggered calcium mobilization in skin-derived nickel-specific CD4+ T cells and promoted their migration, with supernatant from keratinocyte cultures stimulated with IFN-gamma and IL-4 attracting more efficaciously than supernatant from keratinocytes activated with IFN-gamma alone. In conclusion, IL-4 exerts a proinflammatory function on keratinocytes by potentiating IFN-gamma and TNF-alpha induction of IP-10, Mig, and I-TAC, which in turn may determine a prominent recruitment of CXCR3+ T lymphocytes at inflammatory reaction sites.
We report a 60-year-old man with familial scleroatrophic syndrome of Huriez who developed squamous cell carcinomas on the affected skin of the right palm. Immunohistochemical analysis showed a marked reduction in the number of CD1a+, Lag+ and S100+ epidermal Langerhans cells, but not of CD1b+ and factor XIIIa+ dermal dendritic cells, limited to palmoplantar skin. The Langerhans cell depletion was not associated with an abnormal skin content of mRNA for factors involved in Langerhans cell development or recruitment in the epidermis, including granulocyte/macrophage colony-stimulating factor, transforming growth factor-beta1 and macrophage inflammatory protein-3alpha. The results indicate that other as yet unknown mechanisms may account for the reduced number of Langerhans cells in the affected skin of such patients.
Interleukin-17 is a T-cell-derived cytokine, detected in skin affected by allergic contact dermatitis and psoriasis, which regulates keratinocyte expression of adhesion molecules and chemokines. In this study, we have analyzed whether interleukin-17 production segregates with a particular T helper (Th) cell subset, and have examined the capacity of interleukin-17 to modulate the activation of keratinocytes induced by Th1 and Th2 cytokines. A panel of 80 nickel-specific CD4+ T cell clones (36 Th0, 30 Th1, and 14 Th2) was isolated from peripheral blood or lesional skin of allergic contact dermatitis patients. Significant amounts (> 50 pg per ml) of interleukin-17 were released by about 50% of activated Th0, Th1, and Th2 cells. Interleukin-17 alone and in cooperation with interleukin-4, or to a lesser extent with interferon-gamma, decreased the interleukin-1 receptor antagonist to interleukin-1alpha ratio in the supernatants as well as in cell lysates from keratinocytes. In addition, interleukin-17 stimulated the release of growth-regulated oncogene-alpha, granulocyte-macrophage colony stimulating factor, and interleukin-6, with synergistic or additive effects when used together with interferon-gamma or interleukin-4. Interleukin-17 and interleukin-4 also increased stem cell factor release, a function that was inhibited by interferon-gamma. Moreover, interleukin-17 and interleukin-4 enhanced interferon-gamma-induced expression of intercellular adhesion molecule 1, but not CD40, on keratinocytes. The constitutive expression of interleukin-17 and interferon-gamma receptors on keratinocytes was not modulated by interleukin-17, interferon-gamma, or interleukin-4, whereas the interleukin-4 receptor was significantly downregulated by interferon-gamma. As a whole, the results indicate that interleukin-17 can participate relevantly in T-cell-mediated skin immune responses by amplifying both interferon-gamma- and interleukin-4-induced activation of keratinocytes.
Keratinocytes of patients with atopic dermatitis produce high amounts of granulocyte/macrophage colony-stimulating factor, a factor essential for dendritic cell function and thus for the development of skin immune responses. In contrast to keratinocytes cultured from nonatopic, healthy individuals, granulocyte/macrophage colony-stimulating factor mRNA could be detected in unstimulated cultures of atopic dermatitis keratinocytes, and phorbol myristate acetate induced much greater granulocyte/macrophage colony-stimulating factor mRNA levels in these cells, although the decay kinetics were not altered. Using reporter gene (chloramphenicol acetyl transferase) analysis, a minimal granulocyte/macrophage colony-stimulating factor promoter was shown to confer constitutive and phorbol-myristate-acetate-induced regulation of transcriptional activity in keratinocytes, and significantly higher levels of chloramphenicol acetyl transferase activity were measured in lysates of unstimulated and phorbol-myristate-acetate-treated atopic dermatitis keratinocytes than in control keratinocyte cultures. Electrophoretic mobility shift assays showed that low levels of NF-kappa B binding activity could be induced by phorbol myristate acetate in both normal and atopic dermatitis keratinocytes. By contrast, activator protein 1 complexes were efficiently induced, and they were invariably present at higher levels in nuclear lysates of atopic dermatitis keratinocytes. Atopic dermatitis keratinocyte nuclear lysates had higher constitutive levels of c-Jun, and phorbol myristate acetate promoted an earlier and stronger expression of c-Jun, JunB, and of the phosphorylated forms of c-Fos. A dysregulated activation of activator protein 1 may be implicated in the molecular mechanisms leading to increased granulocyte/macrophage colony-stimulating factor expression in atopic dermatitis keratinocytes. J Invest Dermatol 115:1134-1143 2000
The contribution of T helper (Th) and T cytotoxic (Tc) type 1 lymphocytes in the expression of allergic contact dermatitis to haptens has been amply documented. Conversely, the existence of T cell-based regulatory mechanisms has been poorly investigated. Here, we examined the properties of a subset of nickel-specific CD4+ T cells displaying the cytokine profile (IL-10 , IL-5 , IFN-gamma+/-, IL-4+/-) of T regulatory cells 1 (Tr1) and with the potential to down-modulate immune responses to nickel. Tr1 clones were isolated from skin challenged with NiSO4 and peripheral blood of nickel-allergic patients, and from the blood of healthy individuals. Tr1 clones expressed CD25, CD28, CD30, CD26, and the IL-12 receptor beta2 chain upon activation, whereas the lymphocyte activation antigen-3 was present on 50% of the clones. Monocytes precultured with Tr1 cells in the presence of nickel, or treated with Tr1-derived supernatant, exhibited a markedly diminished capacity to stimulate nickel-specific Th1 responses. Tr1 supernatants also blocked the differentiation of dendritic cells (DC) from monocytes, as well as DC maturation and IL-12 production induced by lipopolysaccharide. As a consequence, the ability of DC to stimulate nickel-specific Th1 and Tc1 responses was greatly impaired. These inhibitory effects were completely prevented by IL-10, but not IL-5, neutralization. In aggregate, the results indicate that Tr1 cells can potently regulate the expression of Th1-mediated allergic diseases via release of IL-10.
We investigated the expression of purinoceptors in human dendritic cells, providing functional, pharmacological, and biochemical evidence that immature and mature cells express P2Y and P2X subtypes, coupled to increase in the intracellular Ca(2+), membrane depolarization, and secretion of inflammatory cytokines. The ATP-activated Ca(2+) change was biphasic, with a fast release from intracellular stores and a delayed influx across the plasma membrane. A prolonged exposure to ATP was toxic to dendritic cells that swelled, lost typical dendrites, became phase lucent, detached from the substrate, and eventually died. These changes were highly suggestive of expression of the cytotoxic receptor P2X(7), as confirmed by ability of dendritic cells to become permeant to membrane impermeant dyes such as Lucifer yellow or ethidium bromide. The P2X(7) receptor ligand 2',3'-(4-benzoylbenzoyl)-ATP was a better agonist then ATP for Ca(2+) increase and plasma membrane depolarization. Oxidized ATP, a covalent blocker of P2X receptors, and the selective P2X(7) antagonist KN-62 inhibited both permeabilization and Ca(2+) changes induced by ATP. The following purinoceptors were expressed by immature and mature dendritic cells: P2Y(1), P2Y(2), P2Y(5), P2Y(11) and P2X(1), P2X(4), P2X(7). Finally, stimulation of LPS-matured cells with ATP triggered release of IL-1 beta and TNF-alpha. Purinoceptors may provide a new avenue to modulation of dendritic cells function.
Bacteria are being actively investigated as vaccine carriers for inducing or boosting protective immune responses. In this study, human monocyte-derived dendritic cells (DCs) and normal B cells were compared for their capacity to present the C fragment of tetanus toxin (TTFC), expressed on the surface of recombinant Streptococcus gordonii, to specific CD4(+) T lymphocytes. DCs were more efficient than B cells at presenting soluble TTFC and remarkably more capable of presenting bacterium-associated TTFC both in terms of the amount of antigen required to obtain a given T-cell response and on a per-cell basis. This difference was associated with a much lower capacity of B cells to endocytose soluble TTFC and phagocytose recombinant S. gordonii. In addition, S. gordonii induced the phenotypic maturation of DCs but not of B cells. The results thus indicate that DCs but not B cells play a crucial role in the amplification of class II-restricted immune responses induced by immunization with recombinant gram-positive bacteria.
Keratosis lichenoides chronica (KLC) is a rare chronic dermatosis characterized by lichenoid hyperkeratotic papules arranged in a linear and reticular pattern, and seborrheic-dermatitis-like lesions on the face. Less frequently, palmoplantar keratoderma, nail dystrophies, mucosal as well as eye lesions are present. KLC affects adults and very few cases have been reported in childhood. Although infrequently, KLC has been associated with systemic diseases, including chronic infectious diseases, kidney disorders and lymphoma. Here we report the case of an adult KLC patient with skin, nail and mucosal involvement, and onset in the first year of life who developed a leg panniculitis and a mantle cell lymphoma. Following chemotherapy for the lymphoma, panniculitis resolved completely, and skin and mucosal KLC lesions ameliorated.
Nerve growth factor (NGF) receptors are expressed in different cell types outside the nervous system, and increasing evidence indicates that NGF can act as a regulatory molecule during inflammatory and immune responses. In this study, we show that triggering of the high-affinity NGF receptor TrkA with agonists protects monocytes from apoptosis induced by gliotoxin or UVB radiation. TrkA stimulation up-regulates the expression of the anti-apoptotic Bcl-2 family members, Bcl-2, Bcl-XL, and Bfl-1. On the other hand, TrkA stimulation does not change the expression of MHC, CD80, CD86, CD40, and CD54 molecules, nor the antigen-presenting function of monocytes. In addition, during in vitro monocyte to dendritic cell differentiation TrkA expression is progressively lost, suggesting that NGF selectively affects monocyte but not dendritic cell survival.
Atopic dermatitis (AD) is a chronic inflammatory disease which results from complex interactions between genetic and environmental mechanisms. An altered lipid composition of the stratum corneum is responsible for the xerotic aspect of the skin and determines a higher permeability to allergens and irritants. Keratinocytes of AD patients exhibit a propensity to an exaggerated production of cytokines and chemokines, a phenomenon that can have a major role in promoting and maintaining inflammation. Specific immune responses against a variety of environmental allergens are also implicated in AD pathogenesis, with a bias towards Th2 immune responses. In particular, dendritic cells expressing membrane IgE receptors play a critical role in the amplification of allergen-specific T cell responses. Cross-linkage of specific IgE receptors on dermal mast cells provokes the release and synthesis of a vast series of mediators. Following their recruitment and activation into the skin, eosinophils are also thought to contribute relevantly to tissue damage. Thus, a complex network of cytokines and chemokines contributes to establishing a local milieu that favors the permanence of inflammation in AD skin.
Recombinant Streptococcus gordonii expressing on the surface the C-fragment of tetanus toxin was tested as an Ag delivery system for human monocyte-derived dendritic cells (DCs). DCs incubated with recombinant S. gordonii were much more efficient than DCs pulsed with soluble C-fragment of tetanus toxin at stimulating specific CD4+ T cells as determined by cell proliferation and IFN-gamma release. Compared with DCs treated with soluble Ag, DCs fed with recombinant bacteria required 102- to 103-fold less Ag and were at least 102 times more effective on a per-cell basis for activating specific T cells. S. gordonii was internalized in DCs by conventional phagocytosis, and cytochalasin D inhibited presentation of bacteria-associated Ag, but not of soluble Ag, suggesting that phagocytosis was required for proper delivery of recombinant Ag. Bacteria were also very potent inducers of DC maturation, although they enhanced the capacity of DCs to activate specific CD4+ T cells at concentrations that did not stimulate DC maturation. In particular, S. gordonii dose-dependently up-regulated expression of membrane molecules (MHC I and II, CD80, CD86, CD54, CD40, CD83) and reduced both phagocytic and endocytic activities. Furthermore, bacteria promoted in a dose-dependent manner DC release of cytokines (IL-6, TNF-alpha, IL-1beta, IL-12, TGF-beta, and IL-10) and of the chemokines IL-8, RANTES, IFN-gamma-inducible protein-10, and monokine induced by IFN-gamma. Thus, recombinant Gram-positive bacteria appear a powerful tool for vaccine design due to their extremely high capacity to deliver Ags into DCs, as well as induce DC maturation and secretion of T cell chemoattractans.