Heparan sulfate heterogeneity in skeletal muscle basal lamina: demonstration by phage display-derived antibodies.

The basal lamina (BL) enveloping skeletal muscle fibers contains different glycoproteins, including proteoglycans. To obtain more information on the glycosaminoglycan moiety of proteoglycans, we have selected a panel of anti-heparan sulfate (HS) antibodies from a semisynthetic antibody phage display library by panning against glycosaminoglycan preparations derived from skeletal muscle. Epitope recognition by the antibodies is strongly dependent on O- and N-sulfation of the heparan sulfate. Immunostaining with these antibodies showed a distinct distribution of heparan sulfate epitopes in muscle basal lamina of various species. Clear differences in staining intensity were observed between neural, synaptic, and extrasynaptic basal laminae. Moreover, temporal and regional changes in abundancy of heparan sulfate epitopes were observed during muscle development both in vitro and in vivo. Taken together, these data suggest a role for specific heparan sulfate domains/species in myogenesis and synaptogenesis. Detailed analysis of the functions of heparan sulfate epitopes in muscle morphogenesis has now become feasible with the isolation of antibodies specific for distinct heparan sulfate epitopes.

A profile of differentially expressed genes in primary colorectal cancer using suppression subtractive hybridization.

As a step towards understanding the complex differences between normal cells and cancer cells, we have used suppression subtractive hybridization (SSH) to generate a profile of genes overexpressed in primary colorectal cancer (CRC). From a 35¿ omitted¿000 clone SSH-cDNA repertoire, we have screened 400 random clones by reverse Northern blotting, of which 45 clones were scored as overexpressed in tumor compared to matched normal mucosa. Sequencing showed 37 different genes and of these, 16 genes corresponded to known genes in the public databases. Twelve genes, including Smad5 and Fls353, have previously been shown to be overexpressed in CRC. A series of known genes which have not previously been reported to be overexpressed in cancer were also recovered: Hsc70, PBEF, ribophorin II and Ese-3B. The remaining 21 genes have as yet no functional annotation. These results show that SSH in conjunction with high throughput screening provides a very efficient means to produce a broad profile of genes differentially expressed in cancer. Some of the genes identified may provide novel points of therapeutic intervention.

Activation of murine epidermal TCR-gamma delta+ T cells by keratinocytes treated with contact sensitizers.

The vast majority of TCR gamma delta+, CD4-, CD8- T cells resident in the adult mouse epidermis expresses tissue-specific V-region genes (V gamma 3/V delta 1) in the absence of junctional diversity. The role this unique T cell population plays in the immune surveillance of the skin is not clear. It has been shown that dendritic epidermal T cells (DETC) were activated by stressed keratinocytes and that stimulated DETC produced, for example, a keratinocyte-specific growth factor. To investigate whether DETC are involved in the induction of a contact allergy, we examined the influence of contact sensitizers and nonsensitizing contact irritants on the DETC response toward epidermal symbionts. We show that 9 of 15 cloned DETC are specifically activated, apparently in a non-MHC-restricted way, to proliferate in the presence of keratinocytes or unseparated epidermal cells, which were treated with a sensitizing agent either in vivo or in vitro. All seven tested contact sensitizing substances activated all of the reactive DETC, while keratinocytes/epidermal cells treated with nonsensitizing irritants were as nonstimulatory as vehicle controls. We demonstrate that direct cell to cell contact of DETC and stimulatory keratinocytes/epidermal cells was required and that the TCR was involved in the induction of DETC proliferation. This specific reactivity of DETC toward keratinocytes or epidermal cells pretreated with a contact sensitizer may be indicative of participation of epidermal T cells in the induction of a contact sensitivity and points to a possible role of DETC in the skin immune system.

Activation of phenotypically heterogeneous murine T cell receptor gamma delta + dendritic epidermal T cells by self-antigen(s).

Adult murine epidermis contains a population of Thy-1+, CD45+, CD3+, CD4- and CD8- in situ primarily T cell receptor (TCR) V gamma 3+/V delta 1+ dendritic epidermal T cells (DETC). In the present study, cell surface phenotypes as well as functional properties of DETC were characterized by using in vitro mitogen-stimulated short-term- (10 days) and cloned long-term-cultured (> 1 year) DETC lines. Phenotypic characterization revealed that > 80% of the short-term-cultured cells were routinely TCR gamma delta +, CD4-, CD8-. The majority expressed the V gamma 3 TCR. Seventy-five percent of the lines contained detectable numbers of V gamma 2+ (5-7%) and V gamma 2-3- cells (2-3%). Four different types of stable TCR gamma delta +, CD4-, CD8- long-term-cultured clones were found: TCR V gamma 3+, V gamma 2+, V gamma 2-3- and V gamma 2-3-MHC-class-II+ clones. Nine of the 15 DETC clones tested were activated to proliferate in the presence of dendritic cells or macrophages, while keratinocytes were not stimulatory. This reactivity of DETC was mediated by the TCR and was apparently not MHC-restricted. Only the V gamma 3+, V gamma 2-3- or V gamma 2-3-MHC-class-II+DETC clones were self-responsive. Short-term-cultured DETC clones were self-responsive. Short-term-cultured DETC lines also exhibited the same reactivity. There was no specific cytokine profile of self-reactive DETC. Upon mitogen stimulation, short-term-cultured DETC lines secreted interleukin (IL)-2, IL-3, IL-4 and interferon (IFN)-gamma. In contrast, none of the long-term-cultured DETC clones produced IL-4. Four different profiles were discernible for the other lymphokines tested, irrespective of the clones' phenotype and reactivity towards accessory cells. The manifested profiles were IL-2, IL-3 and IFN-gamma, IL-2 and IL-3, IFN-gamma only and none of the tested lymphokines. The results suggest that DETC may comprise functionally different subsets and support the notion that DETC may exhibit immunorelevant activities in conjunction with natural neighboring cells.

IL-1 serves as a secondary signal for IL-9 expression.

IL-9 is produced in vitro by activated CD4+ T cell lines of the Th2 subtype and by naive CD4+ T cells. Here we show that T cell lines stimulated with Con A in the presence of accessory cells (AC) such as irradiated spleen cells or bone marrow-derived macrophages produced substantially more IL-9 than T cells stimulated with Con A alone. These data suggest that AC influence the production of IL-9 through accessory signals that result in an at least 10-fold increase of IL-9 secretion by the respective T cells. Addition of IL-1 to T cells activated by Con A, PHA, or anti-CD3 antibodies revealed that this monokine was responsible for the potentiation of IL-9 production. This finding was confirmed by applying anti-IL-1 antibodies. The production of other lymphokines, namely, IL-3, IL-4, and IL-6, by activated T cells was not or only marginally enhanced in the presence of AC or IL-1, thus indicating that the synthesis of IL-9 is regulated differently from that of other Th2-derived lymphokines. Furthermore, it was demonstrated by Northern blot analysis that IL-1 increases IL-9 expression at the pretranslational level. Because IL-1 alone failed to induce the production of IL-9 by T cells, this monokine acts as a costimulator in combination with a T cell receptor-mediated signal.

Establishment of different T cell sublines using either interleukin 2 or interleukin 4 as growth factors.

Purified protein derivative reactive T cell lines were established under identical conditions with the exception that different lymphokines, namely interleukin (IL) 2 and IL 4 were employed as growth factors. IL 2 favored the development of T cell lines (LNC.2) which upon activation by concanavalin A (Con A) secreted predominantly lymphokines characteristic of TH1 cells. By contrast, T cell lines established with the aid of IL 4 as growth factor (LNC.4) produced mainly lymphokines representative of TH2 cells. Apart from their pattern of lymphokine secretion LNC.2 and LNC.4 T cells were found to differ in their proliferative response to lymphokines and Con A. LNC.2 T cells proliferated only marginally in the presence of IL 4, Con A or a combination of Con A and IL 1. Furthermore, the IL 2-dependent proliferation of LNC.2 T cells was slightly but significantly diminished by IL 4. In contrast, LNC.4 T cells showed a substantial IL 4-induced proliferative response which was on the one hand synergistically enhanced by minimal amounts of IL 2 and, on the other hand, strongly inhibited by interferon-gamma. In addition, LNC.4 T cells displayed a strong proliferation when stimulated by low concentrations of Con A in the presence of IL 1 as co-stimulator.

TCGF III/P40 is produced by naive murine CD4+ T cells but is not a general T cell growth factor.

Several antigen-specific T cell lines were found to secrete a lymphokine upon activation by antigen or lectin that was provisionally termed T cell growth factor III (TCGF III) because it induced the proliferation of a CD4+ T cell clone independently from IL2 and IL4. Amino acid sequence analysis (and the functional properties of TCGF III) revealed that TCGF III was identical with a recently identified lymphokine termed P40. TCGF III/P40 was not only produced by long-term cultured T cell lines but also upon stimulation of freshly isolated Mlsa-reactive T cells. In addition, naive CD4+ T cells secreted TCGF III/P40 upon activation by lectin or allo-major histocompatibility complex structures. However, in spite of its growth-promoting activity for a CD4+ T cell clone this lymphokine does not appear to function as a general growth factor for T cells.