Solar UV irradiation and dermal photoaging.

The skin is increasingly exposed to ambient UV-irradiation thus increasing risks for photooxidative damage with long-term detrimental effects like photoaging, characterized by wrinkles, loss of skin tone and resilience. Photoaged skin displays alterations in the cellular component and extracellular matrix with accumulation of disorganized elastin and its microfibrillar component fibrillin in the deep dermis and a severe loss of interstitial collagens, the major structural proteins of the dermal connective tissue. The unifying pathogenic agents for these changes are UV-generated reactive oxygen species (ROS) which deplete and damage non-enzymatic and enzymatic antioxidant defense systems of the skin. As well as causing permanent genetic changes, ROS activate cytoplasmic signal transduction pathways in resident fibroblasts that are related to growth, differentiation, senescence and connective tissue degradation. This review focuses on the role of UV-induced ROS in the photodamage of the skin resulting in clinical and biochemical characteristics of photoaging. In addition, the relationship of photoaging to intrinsic aging of the skin will be briefly discussed. A decrease in the overall ROS load by efficient sunscreens or other protective agents may represent promising strategies to prevent or at least minimize ROS-induced photoaging.

Evidence for an additional ligand, distinct from B7, for the CTLA-4 receptor.

Activation of T lymphocytes requires the recognition of peptide-major histocompatibility complex complexes and costimulatory signals provided by antigen-presenting cells (APCs). The best-characterized costimulatory molecule to date is the B7 antigen, a member of the immunoglobulin family that binds two receptors, CD28 and CTLA-4, expressed on the T-cell surface. Using the anti-mouse B7 (mB7) monoclonal antibody (mAb) 16-10A1, which we recently developed, we found that mB7 is indeed an important costimulatory ligand for the antigen-specific activation of murine T cells by B lymphocytes. Three lines of evidence suggest, however, the existence of at least one additional ligand for the CTLA-4 receptor. First, a soluble fusion protein of human CTLA-4 and the IgG1 Fc region, termed CTLA4Ig, blocks better than the anti-mB7 mAb the allogeneic stimulation of T cells by unfractionated splenic APCs. Second, saturating amounts of anti-mB7 mAb do not significantly block binding of fluorescein isothiocyanate-conjugated CTLA4Ig to activated splenic APCs. Furthermore, CTLA4Ig but not the anti-mB7 mAb reacts with the M12 and M12.C3 cell lines. The identification of an additional ligand for CTLA-4 may have applications to the treatment of autoimmune disease and transplant-associated disorders.

Expression and function of the costimulatory molecule B7 on murine Langerhans cells: evidence for an alternative CTLA-4 ligand.

We have previously shown, through transfection experiments, that the murine B7 (mB7) molecule, a ligand for the CD28 and CTLA-4 receptors, is a sufficient costimulatory signal for the antigen-specific and major histocompatibility complex (MHC)-restricted activation of murine CD4+ T lymphocytes. In addition to mB7, another ligand with affinity for CTLA-4 has been described on spleen cells. Here we report our studies on the expression and function of these molecules on murine Langerhans cells (LC). Both anti-mB7 monoclonal antibody (mAb) 16-10A1 and human CTLA4Ig (hCTLA4Ig), a chimeric fusion protein consisting of the extracellular domain of human CLTA-4 and the constant domain of human IgG1, detected antigen(s) on cultured but not freshly isolated LC. Preincubation of cultured LC with anti-mB7 mAb did not significantly affect binding of hCTLA4Ig to these cells. This result demonstrate the existence of at least one other ligand for the CLTA-4 receptor on cultured LC. Functional studies revealed that the costimulatory activity of LC was inhibited better by hCTLA4Ig than by the anti-mB7 mAb. This differential effect was seen in the case of both alloreactive and antigen-specific, syngeneic T cell responses. These findings suggest that the non-mB7-ligand for CTLA-4 is functional and participates in the induction of immune responses by LC. Importantly, even synergistic combinations of anti-mB7 mAb and hCTLA4Ig did not inhibit completely the activity of LC. These findings therefore raise the possibility that LC express other costimulatory ligands besides mB7 and related family members.

Costimulation of IL-4 production by murine B7-1 and B7-2 molecules.

We have examined the capacity of murine B7-1 and B7-2 to costimulate the production of IL-4 by murine CD4+ T lymphocytes. Cloned and freshly isolated T cells were incubated with the anti-CD3 mAb 145-2C11 in the presence of Chinese hamster ovary (CHO) cells that stably express murine B7-1 and B7-2 at comparable levels. IL-4 protein levels were measured in culture supernatants by the CT.4S bioassay, and levels of IL-4 mRNA were determined by semiquantitative reverse transcription-PCR. Both B7-1- and B7-2-transfected CHO cells, but not CHO control transfectants, were able to costimulate IL-4 production. Similarly, both B7-1 and B7-2 could up-regulate IFN-gamma mRNA levels. Cell fractionation experiments on freshly isolated CD4+ T lymphocytes revealed that the costimulatory potential of B7-1 and B7-2 for IL-4 production was restricted to CD44high T cells, i.e., the subpopulation that contains recently activated and memory cells. CD44low, naive CD4+ T lymphocytes, could only be induced to produce IL-4 by repeated stimulation with B7 transfectants. In summary, we have not detected qualitative differences in the capacities of murine B7-1 and B7-2 to induce IL-4 production. The results of our experiments, therefore, argue against the recent hypothesis that precursor Th cells are directed toward the Th2 phenotype by B7-2 and toward the Th1 phenotype by B7-1.

Activation of CD4+ T lymphocytes form interleukin 2-deficient mice by costimulatory B7 molecules.

Interleukin 2 (IL-2)-deficient (IL-2-/-) mice develop hemolytic anemia and chronic inflammatory bowel disease. Importantly, the induction of disease in IL-2-deficient mice is critically dependent on CD4+ T cells. We have studied the requirements of T cells from IL-2-deficient mice for costimulation with B7 antigens. Stable B7-1 or B7-2 chinese hamster ovary (CHO) cell transfectants could synergize with anti-CD3 monoclonal antibody (mAb) to induce the proliferation of CD4+ T cells from IL-2-/- mutant mice. Further mechanistic studies established that B7-induced activation resulted in surface expression of the alpha chain of the IL-2 receptor. B7-induced proliferation occurred independently of IL-4 and was largely independent of the common gamma chain of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors. Finally, anti-B7-2 but not anti-B7-1 mAb was able to inhibit the activation of IL-2-/- T cells induced by anti-CD3 mAb in the presence of syngeneic antigen-presenting cells. The results of our experiments indicate that IL-2-/- CD4+ T cells remain responsive to B7 stimulation and raise the possibility that B7 antagonists have a role in the prevention/treatment of inflammatory bowel disease.

Expression and function of the murine B7 antigen, the major costimulatory molecule expressed by peritoneal exudate cells.

The murine B7 (mB7) protein is a potent costimulatory molecule for the T-cell receptor (TCR)-mediated activation of murine CD4+ T cells. We have previously shown that stable mB7-transfected Chinese hamster ovary (CHO) cells but not vector-transfected controls synergize with either anti-CD3 monoclonal antibody-induced or concanavalin A-induced T-cell activation, resulting ultimately in lymphokine production and proliferation. We now have generated a hamster anti-mB7 monoclonal antibody. This reagent recognizes a protein with an apparent molecular mass of 50-60 kDa. The mB7 antigen is expressed on activated B cells and on peritoneal exudate cells (PECs). Antibody blocking experiments demonstrate that mB7 is the major costimulatory molecule expressed by PECs for the activation of murine CD4+ T cells. This suggests an important role for mB7 during immune-cell interactions. We have also surveyed a panel of murine cell lines capable of providing costimulatory activity. Our results indicate that mB7 is the major costimulatory molecule on some but not all cell lines and that there may be additional molecules besides mB7 that can costimulate the activation of murine CD4+ T cells.

Effects of cyclosporin A, FK 506, and mycalamide A on the activation of murine CD4+ T cells by the murine B7 antigen.

The murine B7 (mB7) protein is a potent co-stimulatory molecule for the activation of murine CD4+ T cells. We have previously shown that stable mB7-transfected Chinese hamster ovary (CHO) cells synergize with either anti-CD3 monoclonal antibodies (mAb) or concanavalin A to stimulate T cell activation. In addition, mB7 can synergize with phorbol 12-myristate 13-acetate (PMA) to induce T cell activation in an alternative pathway. In the present report we describe the effects of three immunosuppressive drugs, cyclosporin A (CsA), FK 506, and mycalamide A on mB7-mediated T cell activation. The immunophilin ligands CsA and FK 506 block activation of murine CD4+ T cells by the combination of anti-CD3 mAb and CHO-mB7 cells but do not affect activation by CHO-mB7 cells and PMA. These results support the relevance of pharmacological studies of immunosuppressive compounds in the murine model prior to their application in man. In contrast to the effects of CsA and FK 506, mycalamide A blocks activation of murine CD4+ T cells by anti-CD3 mAb and mB7 as well as activation by mB7 and PMA. On a molar basis, mycalamide A appears to be at least 10-fold more potent than FK 506 which is 100-fold more potent than CsA. Because of its effects on multiple activation pathways and because of its potency, mycalamide A could have considerable therapeutic potential.

Murine B7 antigen provides a sufficient costimulatory signal for antigen-specific and MHC-restricted T cell activation.

We have previously shown that the murine B7 (mB7) molecule, when expressed in Chinese hamster ovary cells in stable fashion, can costimulate with anti-CD3 mAb or Con A to induce T cell activation. We have now derived, by gene transfection, Chinese hamster ovary cell lines that express the I-Ad molecule, either alone or in context with mB7. We have analyzed these transfectants for their capacity to present Ag to murine CD4+ T lymphocytes. I-Ad/mB7-double transfectants were able to stimulate mixed lymphocyte reactions and to present peptide Ag to specific T cells. Chinese hamster ovary cells that expressed only the I-Ad molecule were not able to stimulate T cell proliferation in these systems. Thus, the mB7 protein is a sufficient costimulatory molecule for the physiologic, Ag-dependent/MHC-restricted activation of murine CD4+ T cells. Stimulation of T cell bulk cultures resulted predominantly in the production of IL-2 and not of IL-4. The costimulatory activity of mB7 is not, however, restricted to the IL-2-secreting subset. We have identified one IL-4-secreting T cell clone, CDC35, which is responsive to mB7 triggering. Finally, we present experiments that suggest that mB7 and peptide/MHC complexes need to be expressed on the same cell for optimal induction of T cell activation.

Murine B7 antigen provides an efficient costimulatory signal for activation of murine T lymphocytes via the T-cell receptor/CD3 complex.

We demonstrate that the murine B7 (mB7) protein is a potent costimulatory molecule for the activation of resting murine CD4+ T cells through the T-cell receptor (TCR)/CD3 complex. Stable mB7-transfected Chinese hamster ovary cells, but not vector-transfected controls, synergize with anti-CD3 monoclonal antibody and Con A-induced T-cell activation, resulting ultimately in proliferation. mB7 exerted its effect by inducing production of interleukin 2 and expression of the interleukin 2 receptor. Thus, mB7 costimulates T-cell activation through the TCR/CD3 complex by positively modulating the normal pathway of T-cell expansion. In contrast to the pronounced effect of mB7 on the activation of T cells through the TCR/CD3 complex, the mB7-transfected CHO cell line costimulated T-cell activation via the glycosylphosphatidylinositol-anchored proteins Thy-1 and Ly-6A.2 only inefficiently. Finally, the combination of a calcium ionophore and mB7 is not sufficient to cause T-cell proliferation, while the combination of a calcium ionophore and phorbol 12-myristate 13-acetate (PMA) stimulates T cells efficiently. The signals that mB7 and PMA provide for murine T lymphocyte activation are therefore not interchangeable, although both costimulate activation through the TCR/CD3 complex.

Vascular endothelial growth factor causally contributes to the angiogenic response upon ultraviolet B irradiation in vivo.

BACKGROUND: Ultraviolet (UV)-B irradiation has been shown to be an inducer of vascular endothelial growth factor (VEGF) in primary keratinocytes and epidermal cell lines in vitro. OBJECTIVES: To determine the expression pattern and the causal role of VEGF in the UVB-mediated angiogenic response in vivo in human skin and in a mouse model. METHODS: Skin biopsies or epidermal lysates thereof were studied for VEGF expression following UVB irradiation at a dose of 50 or 60 mJ cm-2, respectively, using immunostaining and a VEGF-specific highly sensitive sandwich enzyme-linked immunosorbent assay. The VEGF-dependent increase in vessels upon repetitive UVB irradiation was studied in skh-1 hairless mice using immunostaining for factor VIII-related antigen (FVIII RAG) in the presence and absence of intraperitoneally injected neutralizing VEGF antibodies. RESULTS: VEGF was found to be induced in the epidermis following UVB irradiation of human and mouse skin. Repetitive UVB irradiation of skh-1 hairless mice resulted in an increase in FVIII RAG positive vessels in the skin. UVB-induced angiogenic response could be partly abrogated by neutralizing antibodies against VEGF, while isotype-matched IgG control antibodies did not reveal any suppressive effect. CONCLUSIONS: Our results support previous in vitro data and show the in vivo relevance of VEGF as a paracrine inducer of cutaneous vessels after UVB irradiation.

Chronological ageing and photoageing of the fibroblasts and the dermal connective tissue.

In recent years, the exposure of human skin to environmental and artificial UV irradiation has increased dramatically. This is due not only to increased solar UV irradiation as a consequence of stratospheric ozone depletion, but also to inappropriate social behaviour with the use of tanning salons still being very popular in the public view. Besides this, leisure activities and a lifestyle that often includes travel to equatorial regions add to the individual annual UV load. In addition to the common long-term detrimental effects such as immunosuppression and skin cancer, the photo-oxidative damage due to energy absorption of UV photons in an oxygenized environment leads to quantitative and qualitative alterations of cells and structural macromolecules of the dermal connective tissue responsible for tensile strength, resilience and stability of the skin. The clinical manifestations of UV/reactive oxygen species (ROS)-induced disturbances result in photoaged skin with wrinkle formation, laxity, leathery appearance as well as fragility, impaired wound healing capacities and higher vulnerability. Strategies to prevent or at least minimize ROS-induced photo-ageing and intrinsic ageing of the skin necessarily include protection against UV irradiation and antioxidant homeostasis.