Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to lymphoid organs and inflamed skin.

Chemerin is a chemotactic agent that was recently identified as the ligand of ChemR23, a serpentine receptor expressed by activated macrophages and monocyte-derived dendritic cells (DCs). This paper shows that blood plasmacytoid and myeloid DCs express functional ChemR23. Recombinant chemerin induced the transmigration of plasmacytoid and myeloid DCs across an endothelial cell monolayer. In secondary lymphoid organs (lymph nodes and tonsils), ChemR23 is expressed by CD123(+) plasmacytoid DCs and by CD1a(+) DC-SIGN(+) DCs in the interfollicular T cell area. ChemR23(+) DCs were also observed in dermis from normal skin, whereas Langerhans cells were negative. Chemerin expression was selectively detected on the luminal side of high endothelial venules in secondary lymphoid organs and in dermal endothelial vessels of lupus erythematosus skin lesions. Chemerin(+) endothelial cells were surrounded by ChemR23(+) plasmacytoid DCs. Thus, ChemR23 is expressed and functional in plasmacytoid DCs, a property shared only by CXCR4 among chemotactic receptors. This finding, together with the selective expression of the cognate ligand on the luminal side of high endothelial venules and inflamed endothelium, suggests a key role of the ChemR23/chemerin axis in directing plasmacytoid DC trafficking.

Replication-deficient mutant Herpes Simplex Virus-1 targets professional antigen presenting cells and induces efficient CD4+ T helper responses.

Both neutralizing antibodies and cytotoxic T-cells are necessary to control a viral infection. However, vigorous T helper responses are essential for their elicitation and maintenance. Here we show that a recombinant replication-deficient Herpes Simplex Virus (HSV)-1 vector encoding the Human Immunodeficiency Virus (HIV)-1 matrix protein p17 (T0-p17) was capable of infecting professional antigen presenting cells (APCs) in vitro and in vivo. The injection of T0-p17 in the mouse dermis generated a strong p17-specific CD4+ T helper response preceding both p17-specific humoral and effector T cell responses. Moreover, we show that T0-p17 infection did not interfere with the endogenous processing of the transgene encoded antigen, since infected APCs were able to evoke a strong recall response in vitro. Our results demonstrate that replication-deficient HSV vectors can be appealing candidates for the development of vaccines able to trigger T helper responses.

Increased DC trafficking to lymph nodes and contact hypersensitivity in junctional adhesion molecule-A-deficient mice.

Junctional adhesion molecule-A (JAM-A) is a transmembrane adhesive protein expressed at endothelial junctions and in leukocytes. In the present work, we found that DCs also express JAM-A. To evaluate the biological relevance of this observation, Jam-A(-/-) mice were generated and the functional behavior of DCs in vitro and in vivo was studied. In vitro, Jam-A(-/-) DCs showed a selective increase in random motility and in the capacity to transmigrate across lymphatic endothelial cells. In vivo, Jam-A(-/-) mice showed enhanced DC migration to lymph nodes, which was not observed in mice with endothelium-restricted deficiency of the protein. Furthermore, increased DC migration to lymph nodes was associated with enhanced contact hypersensitivity (CHS). Adoptive transfer experiments showed that JAM-A-deficient DCs elicited increased CHS in Jam-A(+/+) mice, further supporting the concept of a DC-specific effect. Thus, we identified here a novel, non-redundant role of JAM-A in controlling DC motility, trafficking to lymph nodes, and activation of specific immunity.

Claudin 4 identifies a wide spectrum of epithelial neoplasms and represents a very useful marker for carcinoma versus mesothelioma diagnosis in pleural and peritoneal biopsies and effusions.

We evaluated the usefulness of the tight-junction associated protein Claudin 4 (CL-4) in the diagnosis of mesothelioma and mimickers, analyzing biopsies from 454 tumors, including 82 mesotheliomas, 336 carcinomas of different origin (278 primary, 58 metastatic to serosae), 36 nonepithelial spindle cell neoplasms, as well as 97 cytological samples from reactive effusions (12), mesothelioma (23) and metastatic carcinomas (62). CL-4 was consistently negative in normal and reactive mesothelium, as well as in all 82 mesotheliomas. In contrast, strong reactivity was found in 57/58 serosal metastasis, and in 245/278 primary carcinomas, with uppermost expression (150/153) in those most frequently involved in the differential with mesothelioma (lung, breast, gastrointestinal tract, pancreas, ovary, primary serous papillary carcinoma of peritoneum). On effusions, reactive and neoplastic mesothelial cells were regularly negative, while metastatic tumor cells stained positively in 60/62 (96.8%) cases. Among spindle cell neoplasms, only 2/9 biphasic synovial sarcomas and 4/4 follicular dendritic cell sarcomas stained positively. Results indicate that CL-4 reacts with the majority of epithelial neoplasms that often metastasize to serous membranes, representing a pancarcinoma marker with extremely high sensitivity and specificity. CL-4 may be considered a primary immunohistochemical reagent to rule out the diagnosis of mesothelioma.

Tissue-specific sensitivity to AID expression in transgenic mouse models.

Activation-induced cytidine deaminase (AID), an enzyme with homology to members of the APOBEC family, is involved in somatic hypermutation (SHM) of immunoglobulin (Ig) genes, either by direct deamination of DNA or by an indirect action through its putative RNA editing activity. AID is able to mutate both Ig-like reporter constructs and selected non-Ig genes in normal B cells and in other cells when ectopically overexpressed in mammalian cells and transgenic mice. However, in spite of the fact that in these transgenic animals AID activity was driven by an ubiquitous promoter, only T lymphomas and lung adenomas occurred. In the present work, we constructed three sets of transgenic mice in which AID was under the control of lck, HTLV-I and MMTV promoters, respectively. The lck/AID mice developed thymic lymphomas with variable but high efficiency, while no tumor was detected in HTLV-I/AID mice after two years of monitoring. Four MMTV/AID founder mice died with an atypical clinical picture, although no mammary tumor was found. These findings suggest that additional factors, present in thymocytes but not in other tissues or in lymphoid cells at different stages of differentiation, are needed for AID to fully manifest its tumorigenic potential in mouse. Alternatively, the display of full AID mutagenic and transforming activity could be related to the existence of physiologic DSBs which occur in both thymocytes and switching B cells.

Cytotoxic molecule expression and epithelial cell apoptosis in oral and cutaneous lichen planus.

We evaluated the expression of T cell-restricted intracellular antigen (Tia-1), granzyme B, and perforin by lymphocytes and the degree of epithelial apoptosis in oral and cutaneous lichen planus (LP) in 51 untreated cases, including 27 oral LP (OLP) and 24 cutaneous LP (CLP) cases. The number of total dermal-positive lymphocytes in OLP and CLP was similar, indicating similar activity of the inflammatory process. Intraepithelial Tia-1-positive, perforin-positive, and granzyme B-positive lymphoid cells were more numerous in OLP than in CLP (P < .05). The epithelial cell apoptotic index (AI) was increased significantly in OLP (P < .05), particularly in erosive-atrophic variants. A linear correlation between AI and the mean +/- SEM number of intraepithelial and dermal perforin+ cells (6.85 +/- 2.44 and 27.48 +/- 10.19, respectively), per 10 high-power fields for OLP and for CLP (1.17 +/- 0.88 and 10.42 +/- 5.74, respectively), was found (intraepithelial, r = 0.50; dermal, r = 0.51; P < .01). These data suggest a pivotal role for perforin in triggering epithelial cell apoptosis. The differences of infiltrating cytotoxic cells and related AI observed in OLP and CLP are in keeping with the clinical behaviors that distinguish these LP variants.

Mismatch repair proteins expression and microsatellite instability in skin lesions with sebaceous differentiation: a study in different clinical subgroups with and without extracutaneous cancer.

Muir-Torre syndrome (MTS) is defined as the association of a sebaceous tumor or keratoacanthoma and an extracutaneous neoplasm, mainly from the gastrointestinal or genitourinary tracts. MTS is related to hereditary non-polyposis colorectal cancer (HNPCC), a syndrome with germline mutations in the mismatch repair (MMR) gene(s), leading to microsatellite instability (MSI). In this study, using immunohistochemistry and a microsatellite instability assay, we analyzed the incidence of MMR gene abnormalities in 79 sebaceous lesions from 70 patients, 26 of whom also had an extracutaneous visceral neoplasm. We were unable to investigate the family histories of our patients regarding other tumors in order to assess which of our cases met the Amsterdam criteria. Defective MMR protein expression (MMR-) was found in 18/70 (25.7%) patients, with an identical distribution between those having an isolated skin tumor (11/44, 25.0%) and those with an extracutaneous cancer (7/26, 25.4%). In the sporadic group, MMR negative lesions were significantly more frequent in extrafacial areas (P = 0.03). High concordance was found between MMR expression in sebaceous lesions and the extracutaneous neoplasm in the same patient (20/23, 86.9%), as well as between MMR expression and microsatellite status (18/20, 90%). In conclusion, this study confirms the value of immunohistochemistry to identify MMR defective tumors. However, since only a minority of sebaceous neoplasms in patients who also have an extracutaneous cancer display MMR defects, these techniques are of limited value for the identification of "clinically defined" MTS.

Imbalance between activin A and follistatin drives postburn hypertrophic scar formation in human skin.

Hypertrophic scarring is a skin disorder characterized by persistent inflammation and fibrosis that may occur after wounding or thermal injury. Altered production of cytokines and growth factors, such as TGF-beta, play an important role in this process. Activin A, a member of the TGF-beta family, shares the same intra-cellular Smad signalling pathway with TGF-beta, but binds to its own specific transmembrane receptors and to follistatin, a secreted protein that inhibits activin by sequestration. Recent studies provide evidences of a novel role of activin A in inflammatory and repair processes. The aim of this study was to evaluate the importance of activin A and follistatin expression in the different phases of scar evolution. Immunostaining of sections obtained from active phase hypertrophic scars (AHS) revealed the presence of a high number of alpha-SMA(+) myofibroblasts and DC-SIGN(+) dendritic cells coexpressing activin A. Ex-vivo AHS fibroblasts produced more activin and less follistatin than normal skin or remission phase hypertrophic scar (HS) fibroblasts, both in basal conditions and upon TGF-betas stimulation. We demonstrate that fibroblasts do express activin receptors, and that this expression is not affected by TGF-betas. Treatment of HS fibroblasts with activin A induced Akt phosphorylation, promoted cell proliferation, and enhanced alpha-SMA and type I collagen expression. Follistatin reduced proliferation and suppressed activin-induced collagen expression. These results indicate that the activin/follistatin interplay has a role in HS formation and evolution. The impact of these observations on the understanding of wound healing and on the identification of new therapeutic targets is discussed.

G-CSF treatment of severe congenital neutropenia reverses neutropenia but does not correct the underlying functional deficiency of the neutrophil in defending against microorganisms.

The treatment of children affected by severe congenital neutropenia (SCN) with G-CSF strongly reduces the risk of sepsis by reversing neutropenia. However, SCN patients who respond to the treatment with the growth factor still have an elevated risk of succumbing to sepsis. Because the disease is usually caused by heterozygous mutations of ELA2, a gene encoding for neutrophil elastase (NE), we have investigated in G-CSF-responder and nonresponder patients affected by SCN the expression of polypeptides that constitute the antimicrobial machinery of these cells. In peripheral blood-derived neutrophils of patients with heterozygous mutations of ELA2 who were treated with G-CSF, NE was nearly absent as detected by immunofluorescence and immunoblotting, suggesting that production of the mutant protein interferes with normal gene expression. This defect was associated with abnormal expression of other granule-associated proteins such as myeloperoxidase, lactoferrin, cathepsin G, and human-neutrophil-peptide. Moreover, in one patient with partial response to G-CSF, we observed an impairment of neutrophil antimicrobial activity against Candida albicans, and, to a lower extent against Escherichia coli. Thereby, we propose that the treatment with G-CSF is not sufficient to correct all of the functional deficiency of neutrophils, and this might account for the consistent risk of infections observed in SCN patients.

Receptor-type protein tyrosine phosphatase gamma (PTPgamma), a new identifier for myeloid dendritic cells and specialized macrophages.

Protein tyrosine phosphatase (PTPgamma) is a receptor-like molecule with a known role in murine hematopoiesis. We analyzed the regulation of PTPgamma expression in the human hematopoietic system, where it was detected in human peripheral blood monocytes and dendritic cells (DCs) of myeloid and plasmacytoid phenotypes. Its expression was maintained during in vitro monocyte differentiation to dendritic cells (moDC) and was further increased after maturation with bacterial lipopolysaccharide (LPS), CD40L, and TNFalpha. But PTPgamma was absent when monocytes from the same donor were induced to differentiate in macrophages. B and T lymphocytes did not express PTPgamma. Rather, PTPgamma mRNA was expressed in primary and secondary lymphoid tissues, and the highest expression was in the spleen. PTPgamma was detected by immunohistochemistry in subsets of myeloid-derived DCs and specialized macrophages (tingible bodies, sinus and alveolar macrophages). Classic macrophages in infective or reactive granulomatous reactions did not express PTPgamma. Increased PTPgamma expression was associated with a decreased ability to induce proliferation and interferon-gamma secretion in T cells by moDCs from patients with advanced pancreatic cancer. Taken together, these results indicate that PTPgamma is a finely regulated protein in DC and macrophage subsets in vitro and in vivo.

Role of dendritic cell-derived CXCL13 in the pathogenesis of Bartonella henselae B-rich granuloma.

Dendritic cells (DCs) initiate adaptive immunity and regulate the inflammatory response by producing inflammatory chemokines. This study was aimed to elucidate their role in the pathogenesis of the suppurative granuloma induced by Bartonella henselae infection, which characterizes cat scratch disease (CSD). In vitro DC infection by B. henselae results in internalization of bacteria, phenotypic maturation with increased expression of HLA-DR and CD86, and induction of CD83, CD208, and CCR7. In comparison to LPS-activated DCs, B henselae-infected DCs produce higher amounts of IL-10, whereas the production of IL-12p70 is reduced. Infected DCs also produce high levels of CXCL8 and CXCL13, 2 chemokines active respectively on neutrophils and B lymphocytes. These results provide the molecular basis for the morphogenesis of CSD granuloma, which typically contains high numbers of neutrophils and B cells. Remarkably, CSD granulomas in vivo contain CXCL13-producing DCs. We further demonstrate that the B cells in CSD granulomas are represented by monocytoid B cells and, worth noting, they express T-bet, a transcription factor able to induce a T-independent immunoglobulin (Ig) class switch in B lymphocytes. These findings suggest that the humoral immune response to B henselae initiates in the extrafollicular areas of infected lymph nodes and is regulated by DCs.

Innate immunity defects in Hermansky-Pudlak type 2 syndrome.

Adaptor protein-3 (AP-3) is an ubiquitous cytoplasmic complex that shuttles cargo proteins from the trans-Golgi and a tubular-endosomal compartment to endosome-lysosome-related organelles. Lack of the beta3A subunit of this complex causes Hermansky-Pudlak syndrome type 2, an autosomal recessive disease characterized by partial albinism, prolonged bleeding tendency, and immunodeficiency. To investigate the pathogenesis of immunodeficiency, we studied natural killer (NK) cells and neutrophil functions in 2 previously unreported siblings affected by Hermansky-Pudlak type 2 syndrome. In both patients we observed a dramatic reduction of cytolytic activity of freshly isolated and of IL-2-activated NK cells. Levels of perforin were reduced in unstimulated NK cells, thereby accounting for the impairment of NK cytolitic activity. In addition, analysis of neutrophils in these patients demonstrated that intracellular elastase content was largely reduced while CD63 expression on plasma membrane was substantially increased. Taken together, these observations suggest that type 2 Hermansky-Pudlak syndrome is characterized by defects of innate immunity.

Recruitment of dendritic cells in oral lichen planus.

Using immunohistochemistry the presence of different dendritic cell (DC) subsets was analysed in 16 biopsies from patients with oral lichen planus (OLP). A significant increase of CD1a+/Langerin+ Langerhans cells, DC-SIGN+ DC and CD123+/BDCA2+ plasmacytoid DCs (PDCs) was found in the epithelium and in the stroma of OLP biopsies compared to normal oral mucosa. A proportion of DCs were mature DC-LAMP+ and expressed S100 or CD11c, typically found in the interdigitating DCs of nodal T-cell areas. Double staining revealed that mature DCs co-expressed CCR7, thus indicating the development of a nodal migratory phenotype upon maturation. Significant recruitment of PDCs producing IFN-alpha was demonstrated by the expression of MxA within the lichenoid inflammatory infiltrate and close cell-to-cell contacts between PDCs and mature DCs were observed, with a significant correlation between the numbers of these two populations. Moreover, PDCs were also found to contain Granzyme-B, an associated-cytotoxic granule protein, inducing target cell apoptosis. Taken together, these results suggest that PDCs may promote maturation of DCs and amplify the cytotoxicity of lymphoid cells. Finally, the recruitment of different subtypes of DC, such as Langerhans cells, stromal DC-SIGN+ DCs and PDCs, associated with a significant proportion of mature DCs, acquiring a CCR7+ 'migratory' phenotype, indicate that they may play a pivotal role in the development of the lichenoid inflammatory infiltrate that occurs typically in OLP.