Dieter Armerding

Cutaneous lymphocyte antigen is a specialized form of PSGL-1 expressed on skin-homing T cells

T cells play a pathogenic role in many inflammatory and certain malignant skin diseases, including psoriasis, atopic and allergic contact dermatitis, and cutaneous T-cell lymphoma. Memory T cells that infiltrate the skin express a unique skin-homing receptor called cutaneous lymphocyte-associated antigen (CLA), a carbohydrate epitope that facilitates the targeting of T cells to inflamed skin. CLA is defined by both its reactivity with a unique monoclonal antibody, HECA-452, and its activity as a ligand for E-selectin, but the structure of the protein component of CLA has not previously been defined. Here we report that CLA is an inducible carbohydrate modification of P-selectin glycoprotein ligand-1 (PSGL-1), a known surface glycoprotein that is expressed constitutively on all human peripheral-blood T cells. Cultured peripheral-blood T cells can be differentiated into CLA-bearing cells, which bind both E-selectin and P-selectin, or CLA-negative cells, which bind P-selectin but do not bind E-selectin, suggesting that there is independent regulation of selectin-binding phenotypes. We propose that differential post-translational modification of a single cell-surface receptor, PSGL-1, mediated by fucosyltransferase VII, serves as a mechanism for regulating tissue-specific homing of memory T cells.

Functional Cutaneous Lymphocyte Antigen Can Be Induced in Essentially All Peripheral Blood T Lymphocytes

The cutaneous lymphocyte–associated antigen (CLA) is a skin–homing receptor expressed on a minority of memory–type peripheral blood T (PBT) lymphocytes. Induction of high–level CLA expression in PBT has previously been difficult to accomplish in vitro. Here we report that constitutive CLA expression could be readily induced in virtually all PBT by various polyclonal activators. There was no requirement for accessory cells or addition of other mediators except for IL–2 for maintaining cell survival. Absence of serum in the culture medium was important for optimal induction of CLA. The number of T cells adhering to E–selectin as well as tethering and shear stress resistance under hydrodynamic flow increased in correlation with the level of cell surface CLA expressed. Clonal analysis of CLA induction revealed that in serum–containing medium, which permits the majority of T cells to expand, only a minority of clones did not express CLA. Such T cells could be induced to highly express CLA within 8 days by switching from serum–containing to serum–free medium. This cell–surface phenotype change was closely associated with acquisition of E–selectin ligand activity. Fucosyltransferase VII, which is believed to be important for the generation of the CLA epitope on the P–selectin glycoprotein ligand–1 (PSGL–1) backbone, was shown to be significantly increased in CLA–positive versus CLA–negative T cell populations by PCR analysis. Our findings are consistent with the idea that restriction of CLA expression after activation, rather than positive selection of predetermined T cell subpopulations exposed to restrictive stimulatory conditions in unique microenvironments, may be important in vivo.

Induction of IgE and IgG1 in human B cell cultures with staphylococcal superantigens: Role of helper T cell interaction, resistance to interferon-gamma

Non-antigen-specific activation of human B lymphocytes for IgE production in vitro requires the presence of interleukin 4 and non-cognate physical interaction with T cells. The latter can be replaced by antibodies directed against the B cells CD40 structure. Antigen-specific induction of immunoglobulin responses, including IgE, is difficult in human lymphocyte cultures. Thus, we developed a model system which might resemble physiological B lymphocyte stimulation by antigen. Co-cultures of purified tonsillar B cells from normal donors with non-HLA matched T helper clones obtained from the skin of atopic dermatitis patients produced significant levels of IgE and IgG1 after stimulation with appropriate types of staphylococcal exotoxins, provided that IL-4 was also induced in the T cells. Such responses were further enhanced by addition of low doses of anti-CD40 antibodies. Concentrations of anti-CD40, optimal for stimulation of B cells in the absence of T helper lymphocytes, were less effective in this regard and even inhibitory in some experiments. Most powerful immunoglobulin induction was observed when the cultures were spiked with low amounts of IL-4 and anti-CD40 which did not elicit substantial immunoglobulin production in the absence of the staphylococcal exotoxins. Induction of IL-2 in T/B cell cultures by superantigens without production of appreciable quantities of IL-4 provoked considerable IgG1 titer but no IgE. High amounts of interferon-gamma generated by the T cells in vitro in the presence of superantigens did not appear to interfere with immunoglobulin induction. Addition of recombinant interferon at the beginning of the culture period at doses which effectively suppressed IL-4 plus anti-CD40 induced immunoglobulin responses did not inhibit T helper and superantigen dependent B cell activation. Superantigen mediated B cell stimulation for immunoglobulin production was dependent on cell-cell contact. The experimental results presented suggest that this cellular interaction did not necessarily involve T-B cell bridging by superantigens.

Tonsillar B Cells Do Not Express PSGL-1, but a Significant Fraction Displays the Cutaneous Lymphocyte Antigen and Exhibits Effective E- and P-Selectin Ligand Activity

Skin-homing T cells are defined by the expression of the cutaneous lymphocyte-associated antigen (CLA) which enables the cells to selectively bind to vascular endothelial E-selectin close to sites of cutaneous inflammation, an initial step in the effective extravasation from blood into the inflamed tissue. Essentially all CLA on T cells decorates the backbone of the P-selectin glycoprotein ligand-1 (PSGL-1). In this study we show that human peripheral blood B cells (PBBC) and tonsillar B cells (TBC) do not display PSGL-1 in fluorescence-activated cell sorter analysis using different murine monoclonal antibodies and polyclonal rabbit anti-PSGL-1 antiserum. A significant population of TBC, however, expresses a HECA-452-reactive epitope. These cells represent nonactivated IgM+/IgG– mature B lymphocytes. Up to 50% of the TBC in a given preparation strongly bind to E- and up to 79% to P-selectin. The shear stress resistance in a parallel-plate flow chamber system was high. Neuraminidase treatment of TBC totally and O-sialoglycoprotein endopeptidase partially diminished HECA-452 reactivity and reduced E- but not P-selectin ligand activities. Mocarhagin had no effect in the assays. The data suggest a different ligand for P-selectin and a distinct glycoprotein carrier for the E-selectin ligand as compared to T cells or other leukocytes. Adhesion to P-selectin, however, still required sulfation of the ligand for function. Western blots of TBC cell lysates detected a >240-kD HECA-452-reactive material that was resistant to reducing conditions. Anti-PSGL-1 did not reveal immunoreactive material in these cell lysates. B cell activation did neither significantly change HECA positivity nor induce PSGL-1 expression. Cultured, activated TBC, however, maintained expression of the integrin α4β7. Human peripheral blood B cells had similar cell surface characteristics to TBC. Our observations suggest that several adhesion molecules may be involved in B cell homing which include CLA, the P-selectin ligand, and structures such as α4β7.

Phorbol Ester, Prostaglandin E2, Forskolin and Okadaic Acid Differentially Modulate lnterleukin-4- versus lnterleukin-2-Dependent Immunoglobulin Induction in Human Cellular Models, in Contrast to Other Selected Modifiers of Cellular Activation

Interleukin 2 (IL2) and 4 (IL4) are the most important mediators for immunoglobulin (Ig) synthesis of human B lymphocytes. There is no obvious difference with regard to Ig isotypes induced by either lymphokine except for IgE: only IL4 induces this allergic antibody type. Monoclonal anti-CD40 antibodies enhance both IL2- and IL4-dependent Ig induction. Searching for drugs which may inhibit induction of IgE but not of rather non-pathogenic immunoglobulins, we selected commercial compounds which are commonly used as probes for transmembrane signalling pathways in other cellular systems. They included modulators of protein kinase C and intracellular calcium, inducers of cAMP, and inhibitors of protein tyrosine kinase, protein serine/threonine phosphatases and phosphodiesterases. The data presented suggest that IL2- and IL4-mediated B cell activation can be differentially modulated. Phorbol ester at non-cell-toxic doses inhibited IL4- but not IL2-dependent Ig induction. Prostaglandin E2 potently enhanced IgE production stimulated with IL4 alone but was inhibitory in the presence of anti-CD40 as a co-stimulatory signal. IgG1 responses elicited with IL2 plus anti-CD40, in contrast, were not affected. All other compounds did not discriminate between IL2- versus IL4-mediated Ig induction.

Induction of cognate and non-cognate T-cell help for B-cell IgE production in relation to CD40 ligand expression.

Nonactivated, fixed peripheral blood T cells (PBT) from healthy donors or patients with X-linked-hyper-IgM (HIGM) syndrome, or cloned T cells provided effective help for tonsillar B lymphocytes for induction of IgE or other immunoglobulin (Ig) isotypes. Helper activity was mediated by staphylococcal superantigens adsorbed to the T cells prior to fixation and required presence of IL-4 in the cultures. We demonstrated that the T cells neither expressed detectable CD40 ligand at the beginning of the superantigen treatment nor 24 h later. Phorbol ester (PMA) plus Ca-ionophore treatment efficiently induced CD40L. Such T cells did not, however, provide any help for B-cell activation in some experiments or stimulated only low responses in others. Antibodies against CD2, CD3 and ICAM-1 adsorbed to fixed T cells prior to coculturing inhibited helper activity. A soluble CTLA4 construct was also inhibitory. Our results suggest a pathway of B-cell activation independent of CD40L expressed on T cells.