In vivo TLR9 inhibition attenuates CpG-induced myocardial dysfunction.

The involvement of toll-like receptor 9 (TLR9), a receptor for bacterial DNA, in septic cardiac depression has not been clarified in vivo. Thus, the aim of the study was to test possible TLR9 inhibitors (H154-thioate, IRS954-thioate, and chloroquine) for their ability to protect the cardiovascular system in a murine model of CpG oligodeoxynucleotide- (ODN-) dependent systemic inflammation. Sepsis was induced by i.p. application of the TLR9 agonist 1668-thioate in C57BL/6 wild type (WT) and TLR9-deficient (TLR9-D) mice. Thirty minutes after stimulation TLR9 antagonists were applied i.v. Survival was monitored up to 18 h after stimulation. Cardiac mRNA expression of inflammatory mediators was analyzed 2 h and 6 h after stimulation with 1668-thioate and hemodynamic parameters were monitored at the later time point. Stimulation with 1668-thioate induced a severe sepsis-like state with significant drop of body temperature and significantly increased mortality in WT animals. Additionally, there was a time-dependent increase of inflammatory mediators in the heart accompanied by development of septic heart failure. These effects were not observed in TLR9-D mice. Inhibition of TLR9 by the suppressive ODN H154-thioate significantly ameliorated cardiac inflammation, preserved cardiac function, and improved survival. This suppressive ODN was the most efficient inhibitor of the tested substances.

Azithromycin induces anti-viral responses in bronchial epithelial cells.

The majority of asthma exacerbations are caused by rhinovirus. Currently the treatment of asthma exacerbations is inadequate. Previous evidence suggests that macrolide antibiotics have anti-inflammatory and antiviral effects; however, the mechanism is unknown. We investigated the anti-rhinoviral potential of macrolides through the induction of antiviral gene mRNA and protein. Primary human bronchial epithelial cells were pre-treated with the macrolides azithromycin, erythromycin and telithromycin, and infected with minor-group rhinovirus 1B and major-group rhinovirus 16. The mRNA expression of the antiviral genes, type I interferon-ß and type III interferon-λ1, interferon-λ2/3, and interferon-stimulated genes (retinoic acid inducible gene I, melanoma differentiation associated gene 5, oligoadenylate synthase, MxA and viperin) and pro-inflammatory cytokines (interleukin (IL)-6 and IL-8), and rhinovirus replication and release were measured. Azithromycin, but not erythromycin or telithromycin, significantly increased rhinovirus 1B- and rhinovirus 16-induced interferons and interferon-stimulated gene mRNA expression and protein production. Furthermore, azithromycin significantly reduced rhinovirus replication and release. Rhinovirus induced IL-6 and IL-8 protein and mRNA expression were not significantly reduced by azithromycin pre-treatment. In conclusion, the results demonstrate that azithromycin has anti-rhinoviral activity in bronchial epithelial cells and, during rhinovirus infection, increases the production of interferon-stimulated genes.

AIDS and Langerhans cells: CD4 antigenic site density modification evidenced by single-cell immunogold labeling.

Langerhans cells (LC) are CD4-positive antigen-presenting cells within the human epidermis and thus potentially may be infected by the causative agent of AIDS (acquired immunodeficiency syndrome), called HIV (human immunodeficiency virus). Because CD4 antigens have been demonstrated to be decreased in HIV-infected lymphocytes, we wondered whether some immunologic markers of LC might be modified in seropositive patients. For this purpose, LC, obtained from clinically unaffected skin of ARC (AIDS-related complex) and AIDS patients, were subjected to anti-CD4 (OKT4) and anti-HLA class II monoclonal antibodies (anti-DR: BL1 and anti-DQ). The density of the antigenic sites/LC recognized by the antibodies was evaluated by employing the electron microscopic immunogold labeling procedure. The density of CD4 molecules/cell measured in LC of ARC and AIDS patients by direct count of gold particles bound to the cell membrane was found to be dramatically decreased among AIDS LC, whereas a small subset of ARC LC strongly expressed this antigen. In contrast, the density of HLA class II (DR and DQ) antigenic determinants was found unchanged in comparison with that of healthy donors. In addition to the quantitative modifications of the CD4 molecule expression by ARC and AIDS LC, the observation in these cell populations of several surface protrusions suggesting viral buds affords evidence that LC are a target for HIV.

HLA class I antigen (heavy and light chain) expression by Langerhans cells and keratinocytes of the normal human epidermis: ultrastructural quantitation using immunogold labelling procedure.

Using an immunogold labelling procedure, we quantified the density of major histocompatibility (MHC) class I antigens on the surface of Langerhans cells (LCs) and keratinocytes of the normal human epidermis. According to ultrastructural features, keratinocytes were divided into three subpopulations: stratum basalis (SBK), stratum spinosum (SSK), and stratum granulosum keratinocytes (SGK), and analyzed separately. For this purpose, three monoclonal antibodies (MCAs) were employed: an anti-HLA A,B,C, and anti-B2-microglobulin (B2-m), and a polymorphic anti-HLA A2 Aw69 MCA. Under electron microscopy, quantitative analysis demonstrated: (a) the presence of a high amount of HLA monomorphic determinants on SBK and SSK and moderate but significant labelling of SGK; (b) the very weak density of MHC class I antigens on the surface of epidermal LCs; (c) the expression, at an identical level, of the HLA heavy chain common determinant (HLA A,B,C), B2-m, and the alloantigen HLA A2 by all epidermal cells (ECs) apart from SGKs and LCs that presented far fewer HLA A2 sites than monomorphic determinants (B2-m and HLA A,B,C); (d) the absence of HLA class I on corneocytes and a moderate labelling of melanocytes. A knowledge of the precise quantitative distribution of HLA class I antigens among various cell subpopulations of the normal human epidermis would be very useful for the study and follow-up of cutaneous malignancies that are known to lose these molecules as well as for the understanding of immune responses, especially allospecific, that involve the skin.

Effects of trypsin on the in situ identification of epidermal cell membrane antigens.

In this work the role of trypsin in revealing epidermal cell surface antigens, with the use of immunological markers, was investigated. Two monoclonal antibodies (MCA) were used, the first: D47, belongs to the first cluster of differentiation and recognizes a membrane antigen of human thymocytes; the second HLA-ABC-m3, is an anti-HLA-B27 MCA. Preliminary treatment with various concentrations of trypsin was performed on frozen skin sections and followed by indirect immunofluorescence. D47 reacted with epidermal dendritic cells only after trypsin pretreatment of skin sections. In addition a mild preliminary trypsinization was shown to increase in situ immunoreactivity of MCA HLA-ABC-m3 with epidermal cells. Best results were obtained when trypsin concentrations ranging from 2.5 to 5 micrograms/ml were applied for 10 min at 37 degrees C. Preliminary trypsinization may be of interest for a better exposure of some surface antigens to immunohistochemical markers.

Reactivity of anti-HLA class I polymorphic monoclonal antibodies with normal human skin.

In this work the reactivity of 16 monoclonal antibodies raised against different HLA class I specificities was tested with human skin of healthy donors of known HLA typing. By indirect immunofluorescence, six antibodies reacted strongly with keratinocytes carrying the corresponding alloantigens. The reactivity of 3 other antibodies which was weak or absent using indirect immunofluorescence, was enhanced by various amplification systems such as avidin-biotin-peroxidase method, biotin-streptavidin-fluorescein complex and especially preliminary trypsin treatment that revealed alloantigens masked in the epidermis. The immunostaining of 4 antibodies was negative regardless of the method used. Some of the antibodies we tested cross-reacted with cytoplasmic antigens of keratinocytes. This study has allowed to select a battery of monoclonal antibodies which can specifically detect alloantigens on keratinocytes and will be useful for the recognition the cell origin in allografting experiments.

Expression of class I-MHC antigens by cultured human epidermis and epidermal allografts.

In vitro grown class II-MHC antigen free epidermal sheets were used as epidermal allografts (EAG) across a major histocompatibility barrier in 20 non-immunosuppressed recipients suffering from leg ulcers. Class I antigens were expressed on cell membranes of basal cell layer only on the epidermal sheets. After grafting, patchy areas of membrane fluorescence were observed among cells from the suprabasal layers on the epidermis from skin biopsies taken between days 5 and 28. All cells of the basal and the suprabasal layers expressed class I antigens on biopsies taken after day 28, as on normal human epidermis. This work demonstrates that class I antigens are expressed by epidermal cells in cultures used for grafting. The absence of rejection cannot be explained by the absence of class I-MHC antigens in EAG.

Progressive replacement of human cultured epithelial allografts by recipient cells as evidenced by HLA class I antigens expression.

Human keratinocytes may be grown in vitro into living epithelia devoid of Langerhans cells and MHC class II antigens. These epithelia have been shown to be usable as epidermal allografts in patients with dermal wounds, without any apparent sign of rejection in the 12-month follow-up study. To evidence a progressive replacement by recipient cells of the grafted keratinocytes, we employed anti-MHC class I antigen monoclonal antibodies directed against tissue specificities expressed by either donors or recipients. At 2 and 4 weeks after grafting, some small epithelial cell islets from the recipient phenotype were clearly identified among cells from a donor origin by indirect immunofluorescence. At 6 months, all keratinocytes present at the grafted areas were labelled by antibodies directed toward recipient specificities only. This replacement may be related to the fact that, when placed on such superficial dermal wounds, the allografts are likely colonized by epithelial cells proliferating from residual recipient dermal appendages.