Afsaneh Soruri

6-Sulfo LacNAc, a Novel Carbohydrate Modification of PSGL-1, Defines an Inflammatory Type of Human Dendritic Cells

The monoclonal antibody M-DC8 defines a major subset of human blood dendritic cells (DCs). Here we identify the M-DC8 structure as 6-sulfo LacNAc, a novel carbohydrate modification of the P selectin glycoprotein ligand 1 (PSGL-1). In contrast to previously described blood DCs, M-DC8+ DCs lack the cutaneous lymphocyte antigen (CLA) on PSGL-1 and fail to bind P and E selectin. Yet they express anaphylatoxin receptors (C5aR and C3aR) and the Fcgamma receptor III (CD16), which recruit cells to inflammatory sites. While sharing with DC1 the expression of myeloid markers and a potent capacity to prime T cells in vitro, M-DC8+ DCs produce far more TNF-alpha in response to the bacterial endotoxin lipopolysaccharide (LPS). Thus, 6-sulfo LacNAc-expressing DCs appear as a novel proinflammatory DC subset.

Chemoattraction of Macrophages, T Lymphocytes, and Mast Cells Is Evolutionarily Conserved within the Human α-Defensin Family

Human defensins are natural peptide antibiotics. On the basis of the position and bonding of six conserved cysteine residues, they are divided into two families, designated α- and β-defensins. Human α-defensins are expressed predominantly in neutrophils (human neutrophil peptides (HNP) 1–4) or intestinal Paneth cells (human defensins (HD) 5 and 6). Although α-defensins have been implicated in the pathogenesis of inflammatory bowel disease, their immunomodulatory functions are poorly understood. In the present study, HNP-1, HNP-3, and HD5 were found to be potent chemotaxins for macrophages but not dendritic cells using Gαi proteins and MAPK as signal transducers. α-Defensins were also chemoattractive for the human mast cell line HMC-1 but lacked, in contrast to β-defensins, the ability to induce intracellular calcium fluxes. Furthermore, HNP-1, HNP-3, and HD5 comparably mobilized naive as well as memory T lymphocytes. Using the protein kinase C (PKC) inhibitors GF109 and Gö6976, we observed a PKC-independent functional desensitization to occur between human α-defensins, which suggests a common receptor for HNP-1, HNP-3, and HD5 on immune cells. This α-defensin receptor was subject to heterologous desensitization by the PKC activator PMA and to PKC-dependent cross-desensitization by human β-defensins. Conversely, α-defensins desensitized β-defensin-mediated migration of immune cells in a PKC-dependent manner, suggesting unique receptors for both defensin families. Taken together, our observations indicate that chemoattraction of macrophages, T lymphocytes, and mast cells represents an immunomodulatory function which is evolutionarily conserved within the human α-defensin family and tightly regulated by β-defensins.

β‐Defensins chemoattract macrophages and mast cells but not lymphocytes and dendritic cells: CCR6 is not involved

β‐Defensins are natural peptide antibiotics whose immunomodulatory functions are poorly understood. In the present study, macrophages were found to migrate to human β‐defensins (HBD)‐1 to ‐4 using Gαi proteins as well as MAPK ERK, p38 and JNK as signal transducers. In addition, mast cells responded to HBD‐1 to ‐4 with calcium fluxes as well as chemotaxis, which increased upon stimulation with IgE plus antigen or ionomycin. In contrast, human β‐defensins were unable to induce migration of memory lymphocytes and dendritic cells (DC). Similar to HBD, the murine β‐defensin (mBD)‐8 mobilized macrophages and lacked the ability to recruit memory T cells. These findings were unexpected as CCR6 on memory T cells and DC has been previously observed to be a receptor for human β‐defensins. In support of our findings, however, RBL‐2H3 as well as 300.19 cells stably expressing CCR6 proved to be unresponsive to HBD‐2 and ‐3. Intriguingly, our observation of a PKC‐independent homologous desensitization between HBD‐1 to ‐4 suggests a common receptor for HBD. In summary, chemoattraction of macrophages and mast cells is evolutionary conserved within the β‐defensin family despite a considerable sequence variation and distinct antimicrobial activities. However, CCR6 is not a functional receptor for β‐defensins.

Apoptosis of macrophages and T cells in tuberculosis associated caseous necrosis.

Immunity against mycobacteria is almost exclusively confined to epithelioid cell granulomas, where a long‐lasting but labile balance exists between host and bacilli. The relationship between immunity and mycobacteria results in regression, growth, or caseation of granulomas. To prove whether caseation is associated with apoptosis, biopsy specimens of patients with tuberculosis were analysed by electron microscopy and by in situ end‐labelling combined with immunofluorescence. Apoptotic cells were not detected in regressive granulomas. Whereas productive granulomas without histologically recognizable caseous necrosis revealed only single apoptotic cells, large numbers of apoptotic CD68+ macrophages and apoptotic CD3+, CD45RO+ T cells were observed within caseous foci. As prime candidates undergoing and/or eliciting apoptosis, vital cells surrounding caseous foci were characterized. Immunohistochemistry showed that the majority of vital CD68+ macrophages surrounding caseous foci are negative for the anti‐apoptotic protein bcl2, but positive for the pro‐apoptotic protein bax. In situ hybridization combined with immunofluorescence demonstrated that the majority of the adjacent lymphocytes are activated CD3+, CD45RO+ cells expressing the pro‐inflammatory cytokine interferon γ (IFNγ) and the death ligand FasL. These results suggest that caseation is strongly associated with apoptosis of macrophages and T lymphocytes; that the onset of apoptosis in macrophages may be promoted by the lack of bcl2 and the abundance of bax; and that activation‐induced cell death (AICD) may be responsible for the apoptosis of T cells. Copyright

Characterization of C5aR expression on murine myeloid and lymphoid cells by the use of a novel monoclonal antibody

The anaphylatoxin C5a is a potent proinflammatory stimulus with immunomodulatory activities. Expression of its receptor C5aR (CD88) has been detected on cells of myeloid origin such as granulocytes and monocytes/macrophages. However, controversial results exist on the expression of C5aR on T and B lymphocytes as well as on mature dendritic cells (DC). The aim of the present study was to characterize expression of C5aR protein on myeloid and lymphoid cells in the mouse. For this purpose, rat monoclonal antibodies with specificity against the murine C5aR were generated. Using these reagents a distinct amount of C5aR antigen was observed on neutrophils and macrophages. In contrast, C5aR protein was not detectable on resting or stimulated murine T or B lymphocytes. Furthermore, no C5aR protein could be observed on splenic CD11c positive DC which have been classified in the literature as relatively mature. Taken together, our results suggest that in the mouse expression of C5aR protein may be restricted to leukocytes of myeloid origin whereas previous evidence for C5aR expression on lymphoid cells may be reevaluated.

Anaphylatoxin C5a Induces Monocyte Recruitment and Differentiation into Dendritic Cells by TNF-α and Prostaglandin E2-Dependent Mechanisms

Although monocytes can be directed to develop into dendritic cells (DC) in vitro, the molecular mechanisms that induce their transformation in vivo are largely unknown. In the present study we employed an in vivo SCID mouse model to investigate the impact of two proinflammatory chemotaxins, the anaphylatoxin C5a and the chemokine macrophage inflammatory protein-1α (CCL3), on the differentiation of human monocytes and immature DC generated from monocytes in the presence of GM-CSF and IL-4. Both C5a and macrophage inflammatory protein-1α recruited human monocytes and immature DC into the peritoneal cavity of SCID mice, but only C5a induced their differentiation into phenotypically mature DC by 48 h after injection. Macrophages derived from monocytes by in vitro culture were resistant to C5a-mediated transformation in vivo. The effect of C5a was indirect, since C5a-stimulated TNF-α and PGE2 were found to be obligatory as well as sufficient to induce differentiation of monocytes. In contrast to monocytes, in vitro generated immature DC required TNF-α, but not PGE2, for their C5a-mediated maturation in vivo. C5a-transformed monocytes represented an inflammatory type of DC, as they constitutively secreted high amounts of TNF-α, but also retained the capacity to release the Th1 cytokine IL-12 p70 upon stimulation with CD40 ligand. In summary, we identified for the first time a cascade of inflammatory signals that can induce the transformation of monocytes into DC in vivo. This novel function emphasizes the important immunoregulatory role of C5a at the interface of innate and adaptive immunity.

T lymphocytes and altered keratinocytes express interferon-γ and interleukin 6 in lichen planus

Abstract Lichen planus is asumed to represent a delayed hypersensitivity reaction, in the course of which cytokines control the proliferation and differentiation of cytotoxic T lymphocytes which attack the epidermis and cause apoptosis of undifferentiated keratinocytes. Since interferon-γ and interleukin 6 are known to be markedly generated in lichen planus, we investigated the cellular localization of these cytokines in affected skin/oral mucosa biopsy specimens using in situ hybridization for interferon-γ and in situ reverse transcription-polymerase chain reaction for interleukin 6 mRNA. In the upper subepithelial connective tissue interferon-γ mRNA was noted within proliferating CD3 + T lymphocytes. In this tissue compartment interleukin 6 mRNA was detected in infiltrating CD4 + and CD8 + T lymphocytes. In the epithelium, expression of interferon-γ mRNA and interleukin 6 mRNA was observed in the basal and suprabasal keratinocytes of altered skin/oral mucosa. In contrast, normal skin did not reveal any interferon-γ or interleukin 6 expression, although a few CD4 + and CD8 + T lymphocytes were noted in the dermis as well as the epidermis. These findings indicate that in lichen planus the proinflammatory cytokines interferon-γ and interleukin 6 are produced not only by activated T lymphocytes but also by altered keratinocytes, and suggest that stimulated keratinocytes may amplify the course of lichen planus.

IL-4 Down-Regulates Anaphylatoxin Receptors in Monocytes and Dendritic Cells and Impairs Anaphylatoxin-Induced Migration In Vivo

Anaphylatoxins mobilize leukocytes to the sites of inflammation. In the present study we investigated the impact of GM-CSF, IL-4, and IFN-γ on anaphylatoxin receptor expression in monocytes and dendritic cells (DC). IL-4 was identified as the strongest down-regulator of the receptors for C5a and C3a in monocytes and monocyte-derived DC (MoDC). To study the impact of IL-4 on anaphylatoxin-induced chemotaxis, an in vivo migration model was established. For this purpose, human monocytes and MoDC were injected i.v. into SCID mice that at the same time received anaphylatoxins into the peritoneal cavity. A peritoneal influx of human monocytes could be demonstrated by 4 h after injections of C5a and C3a. In line with receptor down-regulation, IL-4 treatment inhibited in vivo mobilization of human monocytes and MoDC in response to C5a and C3a. In addition to its effects on human cells, IL-4 reduced C5a receptors in murine bone marrow-derived DC and impaired recruitment of labeled bone marrow-derived DC in syngeneic BALB/c mice to i.p. injected C5a. Overall, these data suggest that inhibition of a rapid anaphylatoxin-induced mobilization of monocytes and DC to inflamed tissues represents an important anti-inflammatory activity of the Th2 cytokine IL-4.

CD137 ligand reverse signaling has multiple functions in human dendritic cells during an adaptive immune response.

T cell activation via dendritic cells (DC) is an important step in the adaptive immune response, which requires DC maturation, migration to lymph nodes and presentation of antigen to T cells. CD137 receptor expressed on activated T cells is a potent costimulatory molecule. Here, we investigated the functions of CD137 ligand (CD137L) in human monocyte‐derived DC during an immune response. Cross‐linking of CD137L on DC leads to cell maturation in an autocrine fashion, mostly via release of TNF‐α. Reverse signaling of CD137L also mediates migration of DC via up‐regulation of the CCR7 chemokine receptor, demonstrated by an in vivo MIP‐3β‐dependent SCID mouse migration model. Finally, CD137L‐activated DC induce differentiation of human T cells into potent Th1 effectors. Cocultivation of autologous T cells and CD137L‐activated DC in an antigen‐specific reaction leads to T cell proliferation and the release of IL‐12p70 and IFN‐γ. These findings deliver new insights into the multiple effects of reverse signaling of CD137L in human DC during the initiation of an adaptive immune response, including the key features of DC maturation, migration and, ultimately, antigen‐specific T cell differentiation.

C5a Receptor and Interleukin-6 Are Expressed in Tissue Macrophages and Stimulated Keratinocytes but not in Pulmonary and Intestinal Epithelial Cells

The anaphylatoxin derived from the fifth component of the human complement system (C5a) mediates its effects by binding to a single high-affinity receptor (C5aR/CD88), the expression of which has been traditionally thought to be restricted to granulocytes, monocytes, macrophages (Mphi), and cell lines of myeloid origin. Recent immunohistochemical data suggested that human bronchial and alveolar cells express C5aR as well. To reexamine the tissue distribution of human C5aR expression, transcription of the C5aR gene was investigated in normal and pathologically affected human lung (bronchopneumonia, tuberculosis), large intestine (acute appendicitis, Crohns disease), and skin (pyogenic granuloma, lichen planus) using in situ hybridization. In contrast to previous evidence, C5aR mRNA could not be detected in pulmonary or intestinal epithelial cells, whereas keratinocytes in inflamed but not in normal skin revealed detectable levels of C5aR transcripts. Additionally, it could be documented that only migrating Mphi express C5aR mRNA, whereas sessile Mphi in normal tissues and epithelioid/multinucleated Mphi found in granulomatous lesions do not. Because C5a has been demonstrated to upregulate the expression of interleukin (IL)-6 in human monocytes, we also studied IL-6 gene transcription in parallel to the C5aR. IL-6 mRNA was detectable in many tissue Mphi. Surprisingly, a tight co-expression of C5aR and IL-6 mRNA was observed in keratinocytes from lesions of pyogenic granuloma and lichen planus. These results point to an as yet unknown role for C5a in the pathogenesis of skin disorders beyond its well-defined function as a chemoattractant and activator of leukocytes.