Allergen-specific challenge was first shown to induce ICAM-1 expression on epithelial cells (ECs) of conjunctiva in allergic patients. The data have also been confirmed in the nose. We then checked for the presence of ICAM-1 consequent to natural exposure to allergens. For both conjunctivitis and rhinitis due to pollen we confirmed, during the pollen season, the presence of ICAM-1 on ECs. We demonstrated the endogenous source of ICAM-1 by in situ hybridization both in vitro and in vivo. ICAM-1 could be an activation marker on ECs, or could, enhanced EC susceptibility to bind offending cells such as eosinophils (LFA-1+). ICAM-1 is also a receptor for the vast majority of rhinoviruses, which are known to provoke, mainly in children, asthma attacks. Since we found that mite allergens can induce ICAM-1 on ECs, even during clinical latency, allergy may be considered as a primary event leading to asthma (through rhinovirus infection) and non-specific hyperreactivity.

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SummaryHighly purified CD1 -3 -4'8 -human thymocytes were obtained by panning techniques combined with cell depletion with antibody-coated magnetic beads. Most of these cells expressed cytoplasmic CD3 antigen, as assessed by mAbs known to react with the CD3E chain. After culture with low doses of PMA (0.5 ng/ml) and subsequent addition (at 24 h) of recombinant interleukin 2 (rIIr2; 100 U/ml) cells underwent extensive proliferation (40-60-fold of the initial cell input after 2 wk). The majority of the proliferating cells were CD3 -TCR -. The remaining cells (5-40%) were represented by CD3+TCR y /6+ (BB3 -A13+) cells. Further removal of CD3 +TCR-y/8+ cells resulted in highly purified CD3 -populations that further proliferated in culture with no substantial phenotypic changes. When CD3+ thymocytes were cultured under the same experimental conditions, only CD3+ TCR a/ß+ cells could be detected, thus indicating that PMA did not agect the surface expression of the CD3/TCR complex, but rather induced preferential growth of CD3 -thymocytes . Surface marker analysis of cultured CD3 -thymocytes showed that they were homogeneously CD7+, whereas low proportions of cells expressed CD2 and CD8 antigens. Among the natural killer (NK) cell markers, CD56 was highly expressed by all cells, whereas CD16, CD57, CD11b, NKH2, and GL183 were absent . Importantly, these cells were different from peripheral NK cells, as 80-95% of them expressed cytoplasmic CD3 antigen. Functional analysis revealed a strong cytolytic activity against both NK-sensitive (K562) and NK-resistant (M14, Daudi) human target cells. In a redirected killing assay against the FcyR+ P815 cells, mAbs specific for triggering molecules including CD3, CD2, and CD16 failed to augment target cell lysis, while a strong cytolytic effect was induced by PHA. In addition, PHA alone or in combination with PMA induced tumor necrosis factor a (TNF-a) and interferon y (IFN-y) (but not 11,2) production by CD3 -thymocytes. Cloning of fresh CD1 -3 -4 -8 -thymocytes in the presence of PMA and rIIr2 resulted in CD3 -CD56+ clones that displayed a pattern of cytolytic activity and lymphokine production similar to that of the polyclonal populations. Northern blot analysis of transcripts coding for CD3/TCR molecules revealed the presence of CD3r, e, and y transcripts, while CD3E was undetectable. Mature transcripts for both y and 6 TCR chains could be detected, whereas no TCR-a mRNA and only a truncated (1.0 kb) form of TCR ß mRNA were revealed. These data suggest that CD3 -cultured thymocytes may be, at least in part, representative of a T cell maturation stage that precedes the surface expression of the CD3/TCR molecular complex. Southern blot analysis of the TCR-6 gene rearrangements revealed that the cultured CD3 -thymocytes retained a germline configuration.

The expression of intercellular adhesion molecule-1 (CD54 or ICAM-1) on epithelial cells during acute or chronic inflammation may favor the interaction between epithelial cells and leukocytes expressing the natural ligands of ICAM-1, LFA-1 (CD11a/CD18), and Mac-1 (CD11b/CD18). We have evaluated in vitro the expression of ICAM-1 by a conjunctival (WK) and an intestinal (I407) human continuous epithelial cell line. Cells were cultured for 24 h in the presence or absence of IFN-gamma, TNF-alpha, IL-1 beta, IL-4, IL-6, IL-8, IL-10, and TGF-beta 1. Both epithelial cell lines showed a constitutive expression of ICAM-1. IFN-gamma at 500 U/ml and TNF-alpha at 200 ng/ml upregulated ICAM-1 expression; IL-1 beta at 100 pg/ml upregulated ICAM-1 on WK cells only. Cells cultured in the presence of both IFN-gamma and TNF-alpha exhibited a mean fluorescence intensity far greater than those cultured with IFN-gamma or TNF-alpha alone. I407 and WK cells were able to release soluble ICAM-1. IFN-gamma and TNF-alpha enhanced the release of sICAM-1. IL-4, IL-6, IL-8, IL-10, and TGF-beta 1 did not affect either ICAM-1 expression or sICAM-1 release. In conclusion, continuously cultured human epithelial cells may express ICAM-1 on their surface and release it in culture medium. These phenomena are upregulated by proinflammatory cytokines.

We investigated the phenotype and function of thyroid tumor-, metastatic lymph node-, and peripheral blood-derived T lymphocytes of four patients with papillary thyroid cancer. Both phenotypic analysis of freshly isolated cells and clonal analysis, using a high efficiency cloning technique, were performed. For comparison, intrathyroid and peripheral blood T lymphocytes derived from patients with autoimmune thyroid diseases (Graves' disease and Hashimoto's thyroiditis) have been studied. In papillary cancer, the phenotype of thyroid and lymph node-derived T lymphocytes did not differ from that of peripheral blood lymphocytes of the same patients or lymphocytes from normal peripheral blood. At variance with respect to autoimmune thyroid disease, activation markers were poorly represented. The functional analysis of T cell clones showed similar proportions of interleukin-2-producing (helper-inducer) clones in thyroid, lymph node, and peripheral blood, slightly lower than in Hashimoto's thyroiditis and slightly higher than in Graves' disease. With regard to effector function, we found lower proportion of clones with cytolytic activity in a lectin-dependent assay compared to that in Hashimoto's thyroiditis. Interestingly, however, the proportions of cytolytic clones displaying cytolytic activity against the neoplastic cell line K562 (natural killer-like activity) or fresh unrelated tumor cells (lymphokine-activated killer activity) were relatively high in thyroid cancer infiltrates.

Thyroid follicular cells (TFC) abundantly express a variety of immunologically relevant surface molecules in Hashimoto's thyroiditis (HT), for example, MHC antigens and adhesion molecules such as ICAM-1. Cytokines produced by infiltrating type 1 helper and cytotoxic T cells are importantly involved in de novo expression or up-regulation of such molecules. We recently demonstrated that TFC from HT patients almost invariably bear on their surface two additive functional molecules: Fas/Apo1/CD95, an important participant in apoptosis, and B7.1, a member of a family of "co-stimulatory" molecules that are crucial for efficient antigen presentation. To date, 12 out of 14 surgical HT thyroid specimens that we studied by immunohistochemistry showed B7.1-positive TFC, and all showed Fas-positive TFC, different from Graves' disease (GD) or nonautoimmune specimens. We have investigated the role of a number of cytokines (IL-1 beta, TNF-alpha, IL-4, IL-6, IL-10, IL-12, TGF-beta 1, IFN-gamma) in regulating B7.1 and Fas expression. The experiments were performed by immunofluorescence flow cytometry on TFC purified from nontoxic goiter specimens which were Fas- and B7.1-negative at baseline, and one B7.1/Fas-positive HT specimen. IFN-gamma (500 U/mL) and TNF-alpha (200 ng/mL) were unable to induce de novo expression of B7.1 or Fas on cultured TFC. At higher doses (2000 U/mL and 800 ng/mL, respectively), they were unable to induce B7.1, but potentiated the spontaneous expression of Fas. Type 2 cytokines (IL-4 and IL-10) were unable to induce Fas or B7.1 on TFC at all, or to down-regulate Fas or B7.1 when expressed. On the other hand, IL-1 beta was the only cytokine able to induce Fas expression on Fas-negative TFC at doses ranging from 100 to 1000 pg/mL. Moreover, at a dose of 400 pg/mL, it was also able to induce B7.1. We demonstrated by immunohistochemistry that IL-1 beta is abundantly present on HT thyroids, including follicular structures. It is conceivable that IFN-gamma, or other cytokines secreted by infiltrating T-lymphocytes, are able to promote IL-1 beta secretion by TFC. In conclusion, a crucial role of IL-1 beta in "destructive" organ-specific autoimmunity may be suggested both for the perpetuation of the autoimmune reaction (induction of efficient autoantigen presentation by TFC, via co-stimulatory molecules) and in induction of tissue damage via "suicide" Fas/FasL-mediated TFC interaction.

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