Calcitonin expression of high endothelial venules during lymphocyte migration in human pharyngeal tonsil.

The migration routes of lymphocytes through high endothelial venules (HEVs) of control and hypertrophic pharyngeal tonsil (HPT) tissue sections were investigated by immunohistochemistry using the expression of a hormone [calcitonin (CT)] and two calcium-dependent endothelial adhesion molecules (E-selectin and P-selectin), as well as electron microscopy. A marked increase in CT-specific staining was observed in the endothelial cells of HEV in the HPT group compared to the control group. Expressions of E-selectin and P-selectin on HEVs of control group were faint, when compared to the strong expression of these selectins on HEVs of HPT. Electron microscopically, we demonstrated that lymphocytes transmigrated through HEV and observed the close membranous contact between endothelial cells and lymphocytes during this process. We speculate that increasing CT during inflammation may be important for lymphocyte migration through the HEVs via controlling the expression of E-selectin and P-selectin.

Ultrastructure and function of the fractalkine mucin domain in CX(3)C chemokine domain presentation.

Fractalkine (FKN), a CX(3)C chemokine/mucin hybrid molecule on endothelium, functions as an adhesion molecule to capture and induce firm adhesion of a subset of leukocytes in a selectin- and integrin-independent manner. We hypothesized that the FKN mucin domain may be important for its function in adhesion, and tested the ability of secreted alkaline phosphatase (SEAP) fusion proteins containing the entire extracellular region (FKN-SEAP), the chemokine domain (CX3C-SEAP), or the mucin domain (mucin-SEAP) to support firm adhesion under flow. CX3C-SEAP induced suboptimal firm adhesion of resting peripheral blood mononuclear cells, compared with FKN-SEAP, and mucin-SEAP induced no firm adhesion. CX3C-SEAP and FKN-SEAP bound to CX(3)CR1 with similar affinities. By electron microscopy, fractalkine was 29 nm in length with a long stalk (mucin domain), and a globular head (CX(3)C). To test the function of the mucin domain, a chimeric protein replacing the mucin domain with a rod-like segment of E-selectin was constructed. This chimeric protein gave the same adhesion of peripheral blood mononuclear cells as intact FKN, both when immobilized on glass and when expressed on the cell surface. This implies that the function of the mucin domain is to provide a stalk, extending the chemokine domain away from the endothelial cell surface to present it to flowing leukocytes.

The transmembrane domain enhances granular targeting of P-selectin.

P-selectin is an integral membrane glycoprotein that is stored in granules of endothelial cells and platelets. The cytoplasmic domain of P-selectin is known to contain at least part of the signal that directs the protein to storage granules. In order to more fully understand how P-selectin is targeted to the regulated secretory pathway, we have expressed chimeric constructs between P- and E-selectin, a protein which is expressed on the cell surface, in a rat insulinoma cell line. Immunofluorescence studies indicated that replacing the cytoplasmic domain of E-selectin with that of P-selectin resulted in low-level granular expression. In contrast, when both the transmembrane and cytoplasmic domains of E-selectin were replaced with the analogous domains of P-selectin, the granular localization appeared greatly increased. This was confirmed by immunoelectron microscopy which demonstrated a three- to fourfold improvement in granular targeting, i.e. similar to wild-type P-selectin. The transmembrane domain had to be in the context of the P-selectin cytoplasmic domain as this membrane-spanning region could not induce granular targeting on its own. These results describe a novel function for the transmembrane domain of P-selectin in enhancing the efficiency of granular targeting and further implicate protein transmembrane domains in intracellular trafficking.

Precise ultrastructural localization of endothelial leukocyte adhesion molecule-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 in patients with IgA nephropathy.

Using light and electron microscopy, we performed an immunohistochemical study of endothelial leukocyte adhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in 15 patients with IgA nephropathy to clarify the localization of these adhesion molecules. The normal portions of 2 kidneys removed due to localized carcinoma and 3 biopsies from patients without glomerular disease were used as a control. By light microscopy, ELAM-1, VCAM-1, and ICAM-1 all showed positive staining in IgA nephropathy, with the intensity of staining following the sequence ICAM-1 > VCAM-1 > ELAM-1. ELAM-1 and VCAM-1 showed a patchy distribution of moderate staining in the tissues, including the mesangium, crescents, adhesions, and tubules. In contrast, there was marked linear ICAM-1 staining throughout the vascular walls. ELAM-1 and VCAM-1 were positive on the basolateral surfaces of a few proximal tubular epithelial cells in association with inflammatory cell infiltration, while ICAM-1 was found on the brush border. ICAM-1 was positive in the glomerular capillary walls and interstitial vessels of the control kidney tissue, while ELAM-1 and VCAM-1 were virtually absent. By electron microscopy, ELAM-1 positivity on the urinary surface of the parietal/visceral epithelial cells was often associated with adherent mononuclear cells in the urinary space. VCAM-1 positivity was increased in the perinuclear space and/or cytoplasm of mesangial cells as well as at the mesangial cell-endothelial cell interface. These findings suggest that ELAM-1 and VCAM-1 may be more closely related than ICAM-1 to the major histopathological changes occurring in IgA nephropathy, including mesangial expansion, formation of crescents and adhesions, and tubulointerstitial injury.

Alopecia areata: immunohistochemistry and ultrastructure of infiltrate and identification of adhesion molecule receptors.

BACKGROUND: Alopecia areata (AA) is a noncicatricial alopecia with still unknown pathogenesis, but increasing evidence suggests that an immunologic process might be responsible for the disease. MATERIALS AND METHODS: Nineteen patients with AA were studied with ten of them in the progressive phase of the disease and nine in the stabilized phase. Biopsies of both affected and unaffected skin were taken. For immunohistochemistry, monoclonal antibodies directed against CD3, CD4, CD8, CD10a, CD36, and HLA-DR antigens, were used, as well as antibodies directed against adhesion molecules ICAM-1, ELAM-1 and LFA-1. For electron microscopy (EM), specimens were fixed in glutaraldehyde-sodium cacodylate buffer, post-fixed in osmium tetroxide, and stained with uranyl acetate. For statistical analysis, sections from involved and uninvolved skin of each patient for each antibody, the sign test, Fisher's F-test, and the Tukey-Kramer test were used. RESULTS: There was a rich infiltrate of CD4+ cells and CD1a+ cells, particularly in the perivascular zone of both unaffected and affected skin (here in the perivascular and in the peribulbar zone) in the progressive phase of AA. In the stabilized phase the infiltrate was scant, both in unaffected and affected skin and limited to the peribulbar area. Receptors of adhesion molecules (ICAM-2, ELAM-1, LFA-1) were strongly expressed, mainly at the microvascular level in both unaffected and affected skin in the progressive phase, but were only weakly or not at all expressed in the stabilized phase, again in unaffected and affected skin. Ultrastructural data confirmed the immunohistochemical findings and showed close contacts between infiltrating lymphocytes and Langerhans'-lineage cells mainly in the progressive phase. CONCLUSIONS: Our results suggest that: 1) an immunologic process, apparently carried out by CD4+ lymphocytes and by dendritic CD1a+ and CD36+ cells, may play a key role at least in the early phase of the disease involving primarily microvessels and later on the bulbar area; 2) the expression of adhesion molecule receptors is involved at the beginning of the disease by mediating the adherence of leukocytes to endothelial cells and subsequent trafficking into the dermis.

Expression of adhesion molecules on the microvasculature of the pharyngeal tonsil (adenoid).

We have examined the vasculature of 12 human adenoids and the expression and distribution of four endothelial adhesion molecules, ICAM-1, VCAM-1, P-selectin and E-selectin, in tissue sections using histology, immunocytochemistry and immunoelectron microscopy (IEM). The connective tissue septa and septula contained arterioles, veins and efferent lymphatics. Branches of arterioles supplied lymphoid follicles and divided into sub- and intraepithelial capillary plexuses which drained into interfollicular venules, mostly high endothelial venules (HEV), before joining larger veins. No afferent lymphatics were observed entering the adenoid. Although ICAM-1 was widely distributed in the tissue, it was preferentially expressed on luminal aspects of HEV. E-selectin was found only in a few areas on HEV and subepithelial capillaries, whereas P-selectin was strongly expressed on segments of HEV, adjacent small venules and a few follicular capillaries. IEM showed the localisation of VCAM-1 on the components of the perivascular sheath, but not on the endothelium, of some HEV and capillaries. Its strongest expression was on follicular dendritic cells (FDC). These findings showed that in addition to HEV, lymphocyte-binding molecules are expressed on other segments of adenoid vasculature and their distribution and intensity of expression varies. In the non-inflamed adenoid, the VCAM-1 does not seem to participate in the adhesive mechanism of recirculating lymphocytes to the endothelium which, in this study, lacked the expression of VCAM-1 in all vessels.

Expression of E- and P-selectins by vascular endothelial cells in human tonsils.

Selectins play an important role in the adhesion between vascular endothelial cells and leukocytes in immuno-inflammatory events. We investigated the immunohistochemical localization of E- and P-selectins in the human palatine tonsils at light and electron microscopic levels. E-selectin was expressed by high endothelial venules (HEV) in a linear pattern in heavily inflamed tonsils with focal infection and extensive neutrophilic infiltration, and much less in hypertrophic tonsils without focal infection. P-selectin expression was found on HEV to the same extent in both disease states in a dot pattern in the cytoplasm. Immunoelectron microscopically, E- and P-selectins were localized along the luminal plasma membrane and in the perinuclear space in HEV endothelial cells. The present study suggests that increased expression of E- and P-selectin in vascular endothelial cells is a significant feature of inflamed tonsils induced by focal infection. E- and P-selectins may participate in different roles in the inflammatory process of the tonsils.