Matrix metalloproteinases-7, -8, -9 and TIMP-1 in the follow-up of diisocyanate-induced asthma.

BACKGROUND: Diisocyanate-induced asthma (DIA) is known to be associated with poor prognosis. We wished to clarify if matrix metalloproteinases (MMP)-7, -8 or -9 or tissue inhibitor of matrix metalloproteinases (TIMP-1) are associated with the functional or inflammatory outcome in DIA patients. METHODS: This is a longitudinal study where 17 patients with DIA diagnosed by a specific challenge test to diisocyanates were monitored. Exposure to diisocyanates was terminated seven (mean) months before the challenge test. The studies included spirometry, histamine challenge test and bronchoscopy. MMP-7, MMP-8, TIMP-1 [Enzyme-linked immunosorbent assay (ELISA)- and immunofluorometric assay-methods], MMP-9 (ELISA and zymography), interferon-gamma, tumour necrosis factor-alpha, interleukin-6, -8, -15, -17, CXCL-5/ENA-78, monocyte chemoattractant protein-1 and macrophage inhibitory factor (MIF) (ELISA) were assayed from bronchoalveolar lavage (BAL) fluid. Inhaled steroid therapy was initiated after the examinations, which were repeated at 6 months and at 3 years during the treatment. The results were compared with those of 15 healthy controls. RESULTS: Inhaled steroid medication increased BAL levels of MMP-9 and MMP-9/TIMP-1 and decreased MMP-7 and MMP-7/TIMP-1. The increase in MMP-9 levels was associated with a decline in the TH-2 type inflammation. CONCLUSIONS: Our data suggest that reduced TH-2 type inflammation in DIA after inhaled steroid medication is reflected as elevated MMP-9 and MMP-9/TIMP-1 levels in BAL. MIF may be the inducer of MMP-9. This might point to some protective role for MMP-9 in DIA.

Inflammation and functional outcome in diisocyanate-induced asthma after cessation of exposure.

BACKGROUND: The clinical outcome of diisocyanate-induced asthma has been found to be poor despite cessation of exposure. Our aim was to study the outcome of diisocyanate-induced asthma after initiation of inhaled steroid treatment at a mean period of 7 months (range 2-60 months) after cessation of exposure by following up lung function and bronchial inflammation. METHODS: Bronchoscopy was performed on 17 patients 2 days after a positive inhalation challenge test, after which budesonide 1600 mug a day was started. Bronchoscopy, spirometry, and histamine challenge tests were repeated at 6 months and on average 3 years. The results were also compared with those obtained from 15 healthy control subjects. RESULTS: Nonspecific bronchial hyperreactivity diminished significantly (P = 0.006); however, forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) values decreased, with a median yearly reduction of FEV1 of 79 ml. The count of mast cells in bronchial mucosa decreased (P = 0.012) and that of macrophages increased (P = 0.001). Interleukin-4 level in mucosa was during the first year significantly higher than in controls but its level decreased in the follow-up. Interleukin-6, interleukin-15, and tumour necrosis factor alpha messenger-RNA levels were significantly higher in hyperreactive patients than in nonhyperreactive patients at the end of the follow-up. CONCLUSION: Our results indicate that inflammation may persist in diisocyanate-induced asthma despite inhaled steroid medication. However, TH2-type inflammation diminished. Persistent nonspecific bronchial hyperreactivity was associated with proinflammatory acting cytokines produced mainly by macrophages. Considering the poor prognosis of the disease the findings could be utilized to develop the follow-up and treatment of diisocyanate-induced asthma.

Adenovirus mediated intra-articular expression of collagenase-3 (MMP-13) induces inflammatory arthritis in mice.

OBJECTIVES: To better understand the role of collagenase-3 (MMP-13) in joint inflammation by investigating the consequences of transient overexpression of human collagenase-3 (matrix metalloproteinase-13 (MMP-13)), introduced by adenoviral gene delivery, in the mouse knee joint. METHODS: A single dose (5x10(7) pfu) of recombinant adenovirus coding either for beta-galactosidase (RAdLacZ) or human MMP-13 (RAdMMP-13) was injected intra-articularly into the knee joint of adult mice. The joints were analysed at frequent intervals up to 4 weeks by histology, immunohistochemistry, and RNA analysis. RESULTS: When RAdLacZ reporter virus was used, adenoviruses efficiently infected synovial cells, chondrocytes of articular cartilage, and hypertrophic chondrocytes of the growth plate. The infection was transient as no reporter gene activity was detected 3 weeks after the injection. After RAdMMP-13 injection into the knee joints, expression of human MMP-13 in joint tissues resulted in an arthritis characterised by recruitment of inflammatory cells and increased production of cytokines and chemokines, synovial hyperplasia, and pannus formation. After the loss of MMP-13 transgene expression at 3 weeks, these inflammatory changes began to diminish. CONCLUSIONS: MMP-13 has a role in the onset of inflammatory reaction in synovium. However, damage to articular cartilage was only rarely detected after the short term overexpression of MMP-13.

Humoral and cellular responses to gliadin in wheat-dependent, exercise-induced anaphylaxis.

BACKGROUND: Wheat-dependent, exercise-induced anaphylaxis (WDEIA) is a severe allergy where wheat ingestion together with physical exercise induces anaphylaxis. We have previously shown that patients with WDEIA have IgE antibodies against gliadin proteins and identified omega-5 gliadin (Tri a 19) as a major allergen. OBJECTIVE: The aim of this study was to examine gliadin-specific IgG subclass, IgA and IgE antibodies, basophil histamine release and cell-mediated responses in WDEIA. METHODS: Sera and peripheral blood mononuclear cells (PBMC) were obtained from patients with WDEIA and from controls without wheat allergy. Serum antibodies to crude gliadin extract (CGE) and purified omega-5 gliadin were measured by ELISA and basophil reactivity by histamine-release test. Gliadin-induced cell-mediated responses were assessed by lymphocyte proliferation assay, and cytokine mRNA expression with real-time quantitative PCR. RESULTS: All patients with WDEIA, but none of the controls, had IgE antibodies to CGE and omega-5 gliadin. Both allergens released high levels of histamine from the basophils of patients with WDEIA. Levels of IgA antibodies to CGE and omega-5 gliadin were significantly elevated in the patients, but the distribution of IgG subclass antibodies showed no statistically significant differences between the two groups. Proliferative responses of PBMC to CGE were increased in patients with WDEIA, and stimulation of PBMC with CGE caused, both in patients and in controls, a clear induction of IL-10 mRNA. Compared with the controls, induction of IL-10 mRNA expression in patients with WDEIA was significantly (P < 0.01) suppressed. CONCLUSION: These results suggest that, in addition to IgE antibodies against omega-5 gliadin, specific IgA antibodies may be involved in the pathogenesis of WDEIA. Decreased expression of IL-10 mRNA in PBMC during gliadin stimulation may facilitate the development of gliadin-specific T cell responses.

High endothelial cells synthesize and degrade sLex. Putative implications for L-selectin-dependent recognition.

L-selectin guides lymphocytes into peripheral lymphoid tissues by recognizing glycoprotein ligands decorated with 6-sulfated sialyl Lewis x (sulfo sLex). Here we have used a rat peripheral lymph node high endothelial cell line (Ax) to study in detail the synthesis, expression and degradation of sLex epitope. We show here that Ax cells possess active alpha(1,3)fucosyltransferase Fuc-TVII, the enzyme responsible for the final fucosylation of sialyl-N-acetyllactosamine during sLex synthesis, and express sLex on the cell surface. Furthermore, these cells degrade sLex, primarily by desialylating it to neutral Lex epitopes by alpha(2,3)sialidase(s).

Biosynthesis of sialylated and fucosylated selectin ligands of HL-60 cells in vitro. Midchain alpha3-fucose units inhibit terminal alpha6-sialylation but not alpha3-sialylation of polylactosamines.

Polylactosamines Neu5Ac alpha2-3'Lex beta1-3'Lex beta1-3'Lex and Neu5Ac alpha2-3'LNbeta1-3'Lex beta1-3'Lex [Lex, Gal beta1-4(Fuc alpha1-3)GlcNAc; LN, Gal beta1-4GlcNAc] decorate selectin counterreceptors in human HL-60 cells. Here, we show that HL-60 cell lysates catalyze distal alpha3-sialylation of LNbeta1-3'LNbeta1-3'LN and LNbeta1-3'Lex beta1-3'Lex efficiently, outlining two potential sets of biosynthetic pathways leading to the selectin ligands. In one set, alpha3-sialylation precedes internal fucosylation of the polylactosamine backbone, whereas in the other one, internal fucosylation is initiated before alpha3-sialylation. In contrast to alpha3-sialylation, LNbeta1-3'Lex beta1-3'Lex was alpha6-sialylated much less efficiently than LNbeta1-3'LNbeta1-3'LN by HL-60 cell lysates. Hence, internal fucosylation may regulate the extent of alpha6-sialylation of polylactosamines in these cells.

Complementary acceptor and site specificities of Fuc-TIV and Fuc-TVII allow effective biosynthesis of sialyl-TriLex and related polylactosamines present on glycoprotein counterreceptors of selectins.

The P-selectin counterreceptor PSGL-1 is covalently modified by mono alpha2,3-sialylated, multiply alpha1,3-fucosylated polylactosamines. These glycans are required for the adhesive interactions that allow this adhesion receptor-counterreceptor pair to facilitate leukocyte extravasation. To begin to understand the biosynthesis of these glycans, we have characterized the acceptor and site specificities of the two granulocyte alpha1,3-fucosyltransferases, Fuc-TIV and Fuc-TVII, using recombinant forms of these two enzymes and a panel of synthetic polylactosamine-based acceptors. We find that Fuc-TIV can transfer fucose effectively to all N-acetyllactosamine (LN) units in neutral polylactosamines, and to the "inner" LN units of alpha2,3-sialylated acceptors but is ineffective in transfer to the distal alpha2,3-sialylated LN unit in alpha2,3-sialylated acceptors. Fuc-TVII, by contrast, effectively fucosylates only the distal alpha2,3-sialylated LN unit in alpha2,3-sialylated acceptors and thus exhibits an acceptor site-specificity that is complementary to Fuc-TIV. Furthermore, the consecutive action of Fuc-TIV and Fuc-TVII, in vitro, can convert the long chain sialoglycan SAalpha2-3'LNbeta1-3'LNbeta1-3'LN (where SA is sialic acid) into the trifucosylated molecule SAalpha2-3'Lexbeta1-3'Lexbeta1-3'Lex (where Lex is the trisaccharide Galbeta1-4(Fucalpha1-3)GlcNAc) known to decorate PSGL-1. The complementary in vitro acceptor site-specificities of Fuc-TIV and Fuc-TVII imply that these enzymes cooperate in vivo in the biosynthesis of monosialylated, multifucosylated polylactosamine components of selectin counterreceptors on human leukocytes.

In vitro experimental studies of sialyl Lewis x and sialyl Lewis a on endothelial and carcinoma cells: crucial glycans on selectin ligands.

Extravasation from the blood of malignant tumour cells that form metastasis and leukocytes that go into tissues require contact between selectins and their sialyl Lewis x and sialyl Lewis a (sLe(x) and sLe(a) respectively) decorated ligands. Endothelial cells have been shown to express sLe(x) epitopes in lymph nodes and at sites of inflammation, and this is crucial for the selectin-dependent leukocyte traffic. Besides the ability to synthesize sLe(x) on sialylated N-acetyllactosamine via the action of alpha(1,3)fucosyltransferase(s), endothelial cells can also degrade sLe(x) to Lewis x through the action of alpha(2,3)sialidase(s). In addition, several epithelial tumors possess the machinery to synthesize sLe(x), which facilitates their adhesion to endothelial E- and P-selectin.

Targeting of active rat alpha 2,3-sialyltransferase to the yeast cell wall by the aid of the hsp 150 delta-carrier: toward synthesis of sLe(x)-decorated L-selectin ligands.

Interactions between selectins and their oligosaccharide-decorated ligands play a crucial role in the initiation of leukocyte extravasation. We have shown that synthetic multivalent sialyl Lewis x glycans inhibit strongly the adhesion of lymphocytes to endothelium at sites of inflammation. However, enzyme-assisted synthesis of these oligosaccharides si hampered by the lack of sufficient amounts of specific glycosyltransferases. We report here the construction of Saccharomyces cerevisiae strains expressing the soluble catalytic ectodomain of rat Gal(beta)1-3/4GlcNac alpha 2,3-sialyltransferase (ST3Ne) fused to the C-terminus of the hsp150 delta-carrier polypeptide. The hsp150 delta-carrier, which is an N-terminal fragmented of a natural secretory protein of yeast, is able to confer secretion-competence to several heterologous proteins, which otherwise remain in the yeast endoplasmic reticulum. The ST3Ne portion of the hsp 150 delta-ST3Ne fusion protein adopted an enzymatically active conformation and was N-glycosylated and disulfide-bonded. Hsp150 delta-ST3Ne was secreted with a half-time of about 7.5 min and remained intercalated in the cell wall, which covers the yeast plasma membrane. About 110 mU of sialyltransferase per litre was produced in 16 h. Whole live yeast cells were able to transfer sialic acid from CMP-NeuNAc to N-acetyllactosamine yielding alpha 2,3-sialyl-N-acetyllactosamine, as evidenced by paper chromatography, cleavage by linkage-specific sialidase, and NMR analysis. Our data suggest that yeast cells externalizing mammalian glycosyltransferases with the aid of the hsp150 delta-carrier could provide a source of enzymes for synthesis of valuable oligosaccharides.

Expression and function of alpha 2,3-sialyl- and alpha 1,3/1,4-fucosyltransferases in colon adenocarcinoma cell lines: role in synthesis of E-selectin counter-receptors.

We show here that colon-carcinoma cell lines adhere to E-selectin via sialyl Lewis x and sialyl Lewis a (s(Lex) and s(Lea)) oligosaccharides and that this adhesion can be enhanced by TNF stimulation. To study in greater detail this endothelial binding, we analysed the mRNA expression and function of the enzymes participating in the generation of s(Lex) and s(Lea on cancer cells. These oligosaccharides are synthesized by sequential action of alpha 2,3 sialyl (alpha 2,3-ST) and alpha 1,3/1/4 fucosyltransferases (alpha 1,3/1,4-FT) on existing (poly)N-acetyllactosamine chains. We report here that mRNAs of 2 recently cloned alpha 2,3-STs and 4 alpha 1,3/1,4-FTs are expressed in adenocarcinoma cells. In functional assays alpha 2,3-ST and alpha 1,3- or 1,4-FT activities were observed in adenocarcinoma cell lysates to exogenous N-acetyllactosamine and lacto-N-biose acceptors and to their sialylated derivatives, leading to the synthesis of the sialyl-N-acetyllactosamine and s(Lex) or the sialyllacto-N-biose and s(Lea), respectively. Furthermore, the inflammatory cytokine TNF could enhance some alpha 2,3-ST and alpha 1,3/1,4-FT activities capable of generating E-selectin counter-receptors. Taken together, these data show that COLO 205 and HT-29 adenocarcinoma cell lines adhere to E-selectin in a TNF-inducible manner via their cell-surface s(Lex) and s(Lea). These cells also express mRNA as well as inducible enzyme activities of several alpha 2,3-STs and alpha 1,3/1,4-FTs responsible for the final steps in the synthesis of s(Lex) and s(Lea).(ABSTRACT TRUNCATED AT 250 WORDS)

De novo expression of endothelial sialyl Lewis(a) and sialyl Lewis(x) during cardiac transplant rejection: superior capacity of a tetravalent sialyl Lewis(x) oligosaccharide in inhibiting L-selectin-dependent lymphocyte adhesion.

Acute organ transplant rejection is characterized by a heavy lymphocyte infiltration. We have previously shown that alterations in the graft endothelium lead to increased lymphocyte traffic into the graft. Here, we demonstrate that lymphocytes adhere to the endothelium of rejecting cardiac transplants, but not to the endothelium of syngeneic grafts or normal hearts analyzed with the in vitro Stamper-Woodruff binding assay. Concomitant with the enhanced lymphocyte adhesion, the cardiac endothelium begins to de novo express sialyl Lewis(a) and sialyl Lewis(x) (sLea and sLex) epitopes, which have been shown to be sequences of L-selectin counterreceptors. The endothelium of allografts, but not that of syngeneic grafts or normal controls, also reacted with the L-selectin-immunoglobulin G fusion protein, giving further proof of inducible L-selectin counterreceptors. The lymphocyte adhesion to endothelium could be significantly decreased either by treating the lymphocytes with anti-L-selectin antibody HRL-1, or by treating the tissue sections with sialidase or anti-sLea or anti-sLex monoclonal antibodies. Finally, we synthetized enzymatically several members of the sLex family oligosaccharides and analyzed their ability to block lymphocyte adhesion to cardiac endothelium. The monovalent sLex (a tetramer), divalent sLex (a decamer), and tetravalent sLex (a 22-mer) could all significantly reduce lymphocyte binding, but the inhibition by the tetravalent sLex-construct was clearly superior to other members of the sLex family. The crucial control oligosaccharides, sialyl lactosamines lacking fucose but being otherwise similar to the members of sLex family, had no effect on lymphocyte binding.

Alpha 2,3-sialyl and alpha 1,3-fucosyltransferase-dependent synthesis of sialyl Lewis x, an essential oligosaccharide present on L-selectin counterreceptors, in cultured endothelial cells.

Sialyl Lewis x (sLex) oligosaccharides have been shown to be present in counterreceptors for L-selectin. We and others have previously shown that high endothelial cells in lymph nodes and at sites of inflammation express sLex. Here we show that also cultured human umbilical vein endothelial cells (HUVEC) express sLex on their cell surface. This oligosaccharide is formed by sequential action of alpha 2,3-sialyl- (alpha 2,3-ST) and alpha 1,3-fucosyltransferases (alpha 1,3-FT) on N-acetyllactosamine. At least two of the several alpha 2,3-ST and four of the several alpha 1,3-FT are present in HUVEC. In functional assays both alpha 2,3-ST and alpha 1,3-FT activities were observed in HUVEC lysates with exogenous lactosamine and sialyllactosamine acceptors, leading to the generation of the sialyllactosamine and sLex sequences, respectively. TNF stimulation increased the level of mRNA expression of FT VI, and the alpha 1,3-FT activity in HUVEC. Taken together these data show that endothelial cells express sLex and that they possess mRNA as well as enzyme activities of several alpha 2,3-ST and alpha 1,3-FT necessary in the final steps of sLex synthesis. Furthermore, inflammatory cytokines such as TNF can enhance transferase activities relevant in generating putative L-selectin counterreceptors.

Genistein enhances the ICAM-mediated adhesion by inducing the expression of ICAM-1 and its counter-receptors.

Binding of circulating cells to endothelium is mediated by recognition between endothelial adhesion molecules and their counter-receptors. The beta 2 integrins are a group of adhesion molecules, mainly expressed on leukocytes, that mediate intercellular binding by recognizing their counterparts on endothelial cells, among others ICAM-1. In this study we have studied the regulation of this interaction in myelomonocytic cells treated with genistein, a tyrosine kinase inhibitor with several other biological functions. We show that genistein upregulates the surface expression of the beta 2-integrins in the monoblastic THP-1 and to a lesser extent in the promyelocytic HL-60 leukemia cell lines. This upregulation leads to an increase in the adherence of THP-1 cells to ICAM-1. Genistein also modulates the expression of ICAM-1 on endothelial cells by potentiating the upregulating effect of TNF and IFN-gamma. Genistein may thus enhance intercellular binding by affecting both the endothelium and the circulating cells.

In vivo evidence of the role of alpha 4 beta 1-VCAM-1 interaction in sarcoma, but not in carcinoma extravasation.

Tumor-cell invasion can occur via either lymphatics or blood vessels. When in the blood circulation, tumor cells have to adhere to endothelium lining the blood vessels before they can extravasate. Several families of adhesion molecules have been recognized: selectins and their oligosaccharide-containing ligands and integrins and their counter-receptors belonging to the immunoglobulin superfamily. Besides their essential role in leukocyte extravasation, these adhesion molecules have been proposed by vitro experiments to be involved in tumor-cell invasion by facilitating the adhesion of malignant cells to endothelium leading to extravasation and metastasis. We have previously shown that, in vitro, several sarcoma cell lines adhere strongly to cultured endothelial cells via alpha 4 beta 1-VCAM-1 interaction. Here we show that sarcoma cells, especially in the metastatic lesions, were strongly alpha 4 beta 1 positive but did not express alpha 4 beta 7, which is another receptor for VCAM-I. Furthermore, we demonstrate that the capillary endothelium within metastatic sarcoma lesions reacted strongly with anti-VCAM-I antibody and very often the alpha 4 beta 1-expressing sarcoma cells were localized in the close vicinity of VCAM-I-expressing vessels. As control material we analyzed carcinoma specimens, but could not detect any alpha 4-integrin expression on malignant cells even though the endothelial cells were often VCAM-I positive. These results suggest that carcinomas do not use alpha 4 beta 1-VCAM-I in extravasation and, taken together, provide circumstantial evidence that in vitro findings of alpha 4 beta1-VCAM-I-dependent sarcoma cell adhesion to endothelium can be extended to in vivo situations.

Carcinoembryonic antigen is expressed on endothelial cells. A putative mediator of tumor cell extravasation and metastasis.

Carcinoembryonic antigen (CEA) is a highly glycosylated cell surface protein. It is produced in large amounts in essentially all colon and several other adenocarcinomas. It has therefore been widely used as a clinical tumor marker. CEA is also a member of the immunoglobulin superfamily. Members of this family, such as ICAM-1, ICAM-2, VCAM-1 and NCAM, are known to participate in cell-cell adhesion. Similarly, the intercellular adhesion properties of CEA have been documented: it has been shown to mediate homotypic adhesion of cultured human colon adenocarcinoma cell lines. In this study we show for the first time that CEA is expressed on cultured human umbilical vein endothelial cells and on the endothelial cell line Ea.hy926. The expression of CEA on cultured endothelial cells can be enhanced by TNF-alpha or IFN-gamma, and decreased by IL-4. We demonstrate using immunohistochemistry that anti-CEA monoclonal antibody reacted with FVIII-positive endothelium in tissue sections prepared from lymph nodes. Finally, we were able to show that CEA-positive breast carcinoma cells bind to purified CEA protein, whereas CEA-negative breast carcinoma cells do not. These results reveal for the first time that endothelial cells express CEA on the cell surface and suggest that CEA-expressing adenocarcinomas could adhere to endothelial cells via CEA-CEA interaction, thus facilitating tumor cell extravasation and hematogenic metastasis.

Sialyl Lewis(x)- and L-selectin-dependent site-specific lymphocyte extravasation into renal transplants during acute rejection.

Kidney allograft rejection is an inflammatory process dominated by lymphocytes. During rejection lymphocytes preferentially adhere to the peritubular capillary endothelium (PTCE), which acquires morphological features common to high endothelium. These observations indicate that PTCE is the site of lymphocyte entry into the rejecting renal allograft. Of the identified endothelial adhesion molecules, ICAM-1 was already expressed on the endothelium of normal kidneys, and its expression was strongly enhanced during rejection without site-specific restriction. VCAM-1 was not expressed on the endothelium of normal or syngeneic kidneys, but its expression was induced during allograft rejection not only in PTCE, but occasionally also on the endothelium of larger vessels. Sialyl Lewisx (sLex) showed a very restricted pattern of expression; endothelium was sLex-negative both in control and syngeneic kidneys. On the other hand, PTCE reacted strongly with anti-sLex antibody in allografts. When kidney frozen sections were treated with sialidase the binding of lymphocytes decreased by 70%. Low-dose chymotrypsin treatment of lymphocytes, known to remove L-selectin from the lymphocyte surface, decreased their binding to PTCE by 60%. Likewise lymphocyte adhesion to PTCE was inhibited by 70% by anti-sLex- and anti-L-selectin-antibodies and by sLex tetrasaccharide. Finally PTCE in the allografts, but not in syngeneic grafts or normal kidneys, bound an L-selectin-IgG fusion protein, indicating that ligands for L-selectin were induced during rejection.

Characterization of the E-selectin ligand on NK cells.

In this study we demonstrate that human CD56+CD16+/CD3- NK cells adhere to the E-selectin expressed by stimulated HUVEC in a sialidase- and Ca(2+)-dependent manner, and express a silylated Lex adhesion structure. We have characterized this sLe(x) epitope on NK cell in detail and show here that the sLe(x) on NK cells was not recognized by the CSLEX1 Ab, but was readily identified by two anti-di-sLe(x) Abs, KM-93 and FH-6. Furthermore, cleaving sialic acid with a sialidase treatment revealed a pool of Le(x) epitopes on the NK cells surface, providing further proof that NK cells express sLe(x) epitopes. Extensive protease treatments did not cleave the sLe(x) epitope from NK cells, which suggests that it could be linked to a lipid backbone. This di-sLe(x) was able to mediate adhesion to E-selectin, suggesting that it represents an essential part or is closely related to a selectin ligand on NK cells. We were also able to show that NK cells possess several alpha 2,3 sialyltransferases and alpha 1,3 or alpha 1,3/4 fucosyltransferases. These enzymes are crucial in the synthesis of sLe(x) epitopes on cell surfaces. Taken together, we provide evidence that NK cells have a di-sLe(x) oligosaccharide capable of adhesion to E-selectin, and NK cells have the machinery (i.e., relevant transferases) to generate these sialylated Lewis oligosaccharides.

Enhanced ICAM-1-dependent adhesion of myelomonocytic cells expressing increased levels of beta 2-integrins and CD43.

Interaction of ICAM-1 and its ligands plays an important role in the leukocyte binding to endothelium. The best characterized ICAM-ligands belong to the family of beta 2-integrins (CD11/CD18), but recently it has been suggested that CD43, a molecule with no structural resemblance to integrins binds ICAM-1 also. On the leukocytes the main regulatory pathway for ICAM-mediated binding is believed to be a short-term regulation of the avidity of CD11/CD18. In this study the authors investigated whether a quantitative increase in the surface expression of ICAM-ligands also can lead to enhanced binding to purified ICAM-1. PMA-treatment differentiates myelomonocytic cell lines into macrophages with a concomitant increase in the surface expression and mRNA-levels of the beta 2-integrin alpha- and beta-chains as well as that of CD43, another ICAM-ligand. The binding of the PMA-treated THP-1 cells to ICAM-1 was increased simultaneously compared to non-treated cells. The binding was blocked completely with antibodies to CD18 and ICAM-1. It is concluded that in addition to the transient qualitative regulation, a long-term quantitative regulation of ICAM-1 ligands also plays a role in increasing the adhesiveness of myelomonocytic cells. This may be relevant in chronic inflammation episodes.

Expression of six protein kinase C isotypes in endothelial cells.

Protein kinase C (PKC) family is an important regulatory element in signal transduction, cellular regulation and tumor promotion. The classical PKC isotypes (alpha, beta and gamma) are Ca(2+)-dependent and can be activated by diacylglycerol. The novel isotypes, PKC delta, PKC epsilon, PKC eta (L) and PKC theta, are Ca(2+)-independent, whereas the two atypical PKCs (zeta and lambda) lack the Ca(2+)-binding region and are not activated by diacylglycerol. Here we show that cultured human endothelial cell line EA.hy926 as well as freshly isolated human umbilical vein endothelial cells express members of all PKC subfamilies. No traces of PKC gamma or delta were detected in endothelial cells. On the contrary the classical PKCs (alpha and beta), the novel PKC epsilon, as well as the atypical PKC zeta are present at the mRNA level in human endothelial cells and the corresponding proteins are also detected by immunoblotting.

Down-regulation of monocytic VLA-4 leads to a decreased adhesion to VCAM-1.

The alpha 4 beta 1 integrin VLA-4 is expressed on practically all leukocytes, except on mature granulocytes. Here we show that in vitro treatment of monocytic cells with phorbol-12-myristate-13-acetate (PMA) leads to a selective decrease in the VLA-4 alpha-chain expression, both at the RNA and protein level. Meanwhile the expression of beta 1 and that of alpha 5, another alpha-chain associating with beta 1, was seen to increase. The decrease of alpha 4 expression was restricted to monocytic cells, and was not observed on other VLA-4-positive cells tested (MOLT-4 T cells and HOS sarcoma cells). The down-regulation of the VLA-4 alpha-chain was followed by a decreased binding capacity of the cells to recombinant VCAM-1. This data indicates that while previous findings show that the integrin-dependent adhesion may rapidly be regulated by altering the avidity of the interacting molecules, their quantitative modulation also has a clear impact on adhesion.