2-integrin Expression Research Articles

Immunohistochemical localization of adhesion molecules (CD62 and CD18) in the mammary gland of dairy cows

Localization of the L-selectin (CD62L) and β2-integrin (CD18) bearing cells in different tissues of the bovine mammary gland was examined. Five dairy cows of Holstein-Friesian breed in the middle of their second and third lactation cycle were used in the study. Blood, milk and udder tissue samples were collected from each cow to estimate the milk somatic cell count (SCC) and bacteriological infection of the mammary gland. The expression of CD62L and CD18 on blood cells, milk cells and parenchymal tissues of udder, Fürstenberg’s rosette and the transverse section of the central part of the teat was tested. In the mammary gland quarters the value of SCC in milk secretion was also reflected in the presence of CD18<sup>+</sup> and CD62L<sup>+</sup> leukocytes in mammary tissues. In the quarters where SCC was higher than 10<sup>5</sup> a frequent incidence of L-selectin and β2-integrin cells was observed in the parenchyma and Fürstenberg’s rosette region, while in the quarters with low SCC, none or only a few reactive cells were found. In the mammary parenchymal tissue CD18 positive cells were present in both the epithelial and the connective tissue. In Fürstenberg’s rosette the cells were concentrated in the connective collagenous and loose tissue. None or only scattered L-selectin (CD62L) and β2-integrin (CD18) bearing cells were identified in the transverse section from the central part of the teat. When we compared the L-selectin and β2-integrin expression, the study revealed the down-regulation of L-selectin on the cells of mammary tissue.

Membrane proteinase 3 expression and ANCA-induced neutrophil activation

Proteinase 3 is the major autoantigen in Wegener's granulomatosis (WG). Membrane PR3 expression is bimodal; low expressing cells (mPR3(low)) can be distinguished from cells with high expression (mPR3(high)) within a given individual. High mPR3 expression is a WG risk factor and is associated with relapse. However, no mechanisms for this important clinical observation have been provided. We tested the hypothesis that mPR3 expression, rather than the expression of other membrane molecules implicated in anti-neutrophil cytoplasmic autoantibodies (ANCA) activation, determines the robustness of the PR3-ANCA-mediated response. mPR3(low) and mPR3(high) neutrophils from a given individual were separated by magnetic cell sorting. Superoxide was measured by the ferricytochrome assay, and Akt phosphorylation by Western blotting. Double staining and flow cytometry were used to assay Fc gamma-receptor and beta 2-integrin expression with respect to the mPR3 phenotype. Degranulation was measured via beta-glucuronidase activity, migration with fibronectin-coated transwells, and cell quantification by the myeloperoxidase (MPO) assay. PR3-ANCA-treated mPR3(high) versus mPR3(low) neutrophils showed more superoxide generation (33.7 +/- 15.2 nmol O(2) (-) to 14.6 +/- 8.4, P < 0.01), more degranulation (29%+/- 5 to 22%+/- 3, P < 0.05), and more PI3-K/Akt activation. In contrast, all responses in both mPR3 subsets were similar after other stimuli. We observed no differences in the beta 2-integrin, Fc gamma R IIa, and III expression with respect to the mPR3 subtype. Furthermore, we found no differences in the mobilization of PR3-containing granules and no differences in migration through fibronectin. The degree of neutrophil mPR3 expression has definitive functional consequences.

Celecoxib simulates respiratory burst through pertussis toxin-sensitive G-protein, a possible signal for β 2-integrin expression on human neutrophils

The superoxide anion-generating effect of celecoxib (4-[5-(4-methylpheny)-3-(trifluoromethyl)-1 H-pyrazol-1-yl]benzenesulfonamide); SC58633), a selective cyclooxygenase-2 inhibitor, on human neutrophils was evaluated in this study. Celecoxib induced superoxide anion generation in a concentration-dependent manner in human neutrophils. The EC 50 value of celecoxib on superoxide anion generation was 15.5±2.5 μM. A NADPH oxidase inhibitor, diphenyliodonium (20 μM), and superoxide dismutase (150 U/ml) completely inhibited the free radical generation caused by celecoxib, indicating that the respiratory burst was activated by celecoxib. 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM;10 μM) and staurosporine (200 nM) completely inhibited the superoxide anion release caused by celecoxib, respectively. These data indicated that celecoxib increased superoxide anion release by increasing intracellular calcium and protein kinase C activation. Moreover, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5 H-indolo(2,3-a)pyrrolo(3,4-C)-carbazole (Go-6976; 1 μM) and 3-[1-[3-(amidinothio)propyl-1 H-indol-3-yl]-3-(1-methyl-1 H-indol-3-yl)maleimide, methane sulfate (Ro-31-8220; 0.5 μM), specific inhibitors of conventional protein kinase C isotypes (α, β I and β II), significantly inhibited superoxide anion release caused by celecoxib. Rottlerin (5 μM), a protein kinase C δ inhibitor, did not affect the free radical generation caused by celecoxib. Celecoxib caused translocation of protein kinase C α, β I and β II from the cytosol to the cellular membrane. 2-[2-amino-3-methoxyphenyl]-4 H-1-benzopyran-4-one (PD98059; 20 μM) and wortmannin (100 nM) did not decrease the superoxide anion generation caused by celecoxib, indicating that Mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3 kinase) were not involved in the respiratory burst induced by celecoxib. Pertussis toxin (2 μg/ml), a Gi-protein sensitive inhibitor, significantly inhibited superoxide anion release. Moreover, pertussis toxin significantly inhibited intracellular calcium mobilization and protein kinase C α, β I and β II translocation from the cytosol to the membrane. Celecoxib increased β 2-integrin expression on human neutrophils and this effect was inhibited by BAPTA/AM (10 μM), superoxide dismutase (150 U/ml), genistein (25 μM) and PD98059 (20 μM). This information indicated that intracellular calcium, superoxide anion, tyrosine kinase and MAP kinase are involved in β 2-integrin expression. Furthermore, BAPTA/AM, superoxide dismutase and genistein inhibited celecoxib-increased MAP kinase activity, indicating that MAP kinase is a downstream signal for β 2-integrin expression. In conclusion, celecoxib stimulates superoxide anion release from human neutrophils by activating pertussis toxin sensitive G-protein. An increase in intracellular calcium and protein kinase C α, β I and β II is involved in this process. Celecoxib also regulates β 2-integrin expression through superoxide anion release, tyrosine kinase and p42/p44 MAP kinase on human neutrophils.

The effects of live Streptococcus pneumoniae and tumor necrosis factor-α on neutrophil oxidative burst and β2-integrin expression

To study the effects of TNF-alpha and live Streptococcus pneumoniae on human neutrophil oxidative burst and beta 2-integrin expression using flow cytometry.Six clinical isolates of S. pneumoniae (serotypes 3, 19A, 22F, 6A, 33F and 9N) from patients with bacteremic pneumonia or upper respiratory tract infections were studied. Whole blood was incubated either alone, with TNF-alpha or with S. pneumoniae or with both TNF-alpha and pneumococci at a ratio of one neutrophil per 1--5 bacteria. After 30 min of incubation, the tubes were put into ice, fixed and analysed.S. pneumoniae caused an increase in oxidative burst but not greater than that caused by TNF-alpha alone. When whole blood was preincubated with TNF-alpha for 30 min before the addition of pneumococci, a further increase in the oxidative burst response was seen. The variation in CD11b expression was not significant. Both S. pneumoniae and TNF-alpha caused increases in CD18 expression. The addition of TNF-alpha directly with the bacteria caused no further increase, but preincubation of blood with TNF-alpha 30 min before the addition of the bacteria caused a significant increase in CD18 expression.Live S. pneumoniae stimulates polymorphonuclear leukocytes to produce an oxidative burst and increases expression of CD18, and these effects are enhanced by TNF-alpha.

T lymphocytes from Sézary syndrome patients express β1 integrins whose β(1–6)-branched N-linked oligosaccharides reflect their adhesive capacity

Sézary syndrome (Sz), characterized by slowly progressing clonal proliferation of CD4 +, CD45 RO + T cells, has several forms that are distinguished according to the epidermotropic properties of the pathological cells. In a recent paper (Derappe C, Haentjens G, Lemaire S, Feugeas JP, Lebbe C, Pasqualetto V, Bussel A, Aubery M, Néel D. Leukemia 1996;10:138), we observed that T lymphocytes from most of the Sézary patients [Sz β(1–6) +] expressed high levels of β(1–6)-GlcNAc-branched N-linked oligosaccharides while T lymphocytes from other patients [Sz β(1–6) −] did not. Because this observation suggests the possibility of two forms of Sz, distinguished according to the expression rate of these glycans, we looked for a possible relationship between this expression rate and T-cell adhesiveness. Using an original protocol (Braut-Boucher F, Pichon J, Rat P, Adolphe M, Aubery M, Font J. J Immunol Methods 1995;178:41), we observed that T lymphocytes obtained from the Sz β(1–6) + patients adhered less to normal keratinocyte monolayers than T lymphocytes from Sz β(1–6) − patients and normal donors. As assessed by FACS analysis, all the integrin-subunits studied were more expressed on Sz β(1–6) −, especially α 4, α 5, β 1 and β 2, than on Sz β(1–6) + and normal lymphocytes. Although these results suggest that β 1- and β 2-integrin expression is involved in the adhesive properties of these T-cells, other factors, such as glycosylation, may also contribute. To demonstrate this possibility, we sought the presence of β(1–6)-GlcNAc-branched N-linked oligosaccharides on β 1 integrins expressed by T lymphocytes from Sz patients. Immunoblot experiments, performed using the specific lectin from Phaseolus vulgaris (Leukoagglutinin form), showed that only the β 1 integrin subunit expressed by T lymphocytes from Sz β(1–6) + patients carried these glycans, supporting the concept of the involvement of T-cell glycosylation in the evolution of Sz.

The correlation between β 2-integrin expression and corticotherapy in asthmatic patients

The correlation between β 2-integrin expression and corticotherapy in asthmatic patients

Neutrophil and monocyte 2-integrin expression in maternal and fetal blood: Thilaganthan B.; Makrydimas G.; Plachouras N.; Nicolaides K.H. GBR Am J Obstet Gynecol 1995 172/1 (58–620)

Neutrophil and monocyte 2-integrin expression in maternal and fetal blood: Thilaganthan B.; Makrydimas G.; Plachouras N.; Nicolaides K.H. GBR Am J Obstet Gynecol 1995 172/1 (58–620)

Neutrophil and monocyte β2‐integrin expression in trisomic fetuses

Flow cytometry was used to measure neutrophil and monocyte beta 2-integrin expression in fetuses with trisomy 18 (n = 7) and trisomy 21 (n = 7) at 20-25 weeks' gestation. The values were compared with those of 112 chromosomally normal fetuses. There were no significant differences in beta 2-integrin expression between normal and aneuploid fetuses. These findings demonstrate that in trisomies 21 and 18, alteration in beta 2-integrin expression is unlikely to contribute to the pathogenesis of immunological deficiencies that have been observed in these aneuploidies both prenatally and postnatally.

Neutrophil and monocyte β 2-integrin expression in maternal and fetal blood

OBJECTIVE: The aim of this study was to investigate fetal and maternal leukocyte β 2-integrin expression throughout pregnancy. STUDY DESIGN: In a cross-sectional study of 173 fetuses at 18 to 40 weeks' gestation, 114 women with normal singleton pregnancies at 6 to 40 weeks, and 25 adult volunteers, flow cytometry was used to determine neutrophil and monocyte expression of β 2-integrins. RESULTS: The surface density of β 2-integrins on fetal neutrophils and monocytes increased significantly with gestation, except for the expression of fetal monocyte CD11b, which did not change. There were no significant changes in maternal β 2-integrin expression with gestation. During the third trimester fetal expression of CD18, CD11b, and CD11c on neutrophils and CD11b on monocytes was lower, and maternal expression of CD11a on neutrophils and CD18, CD11a and CD11c on monocytes was higher than were the control adult values. CONCLUSIONS: These data demonstrate that pregnancy is associated with changes in the expression of maternal β 2-integrins and illustrate the pattern of immunophenotypic maturation of fetal β 2-integrins on neutrophils and monocytes.

Induction of beta 2 integrin expression on an eosinophilic cell line (EoL-1) by the supernatant of mononuclear cells stimulated with specific allergen from asthmatic patients.

Adhesion molecules recently have been considered to play an important role in inflammatory processes in bronchial asthma. Our previous study revealed high expression of beta 2-integrin family (CR3, LFA-1 alpha, CD18) on hypodense eosinophils. Thus, from the point of view of cell-to-cell interaction between mononuclear cells and eosinophils, we examined whether the supernatant of mononuclear cells obtained from mite-allergic asthmatic patients cultured with specific allergen mite-allergen is involved in adhesion molecule expression using an eosinophilic cell line (EoL-1). These characteristics of beta 2-integrin family expression (high expression of beta 2 integrin) were induced by the supernatant of mononuclear cells from mite-allergic asthmatic patients stimulated with mite-allergen as well as with a combination of the recombinant eosinophilopoietic growth cytokines (IL-3, GM-CSF and IL-5). Thus, we could conclude that some cytokines produced by specific allergen stimulated mononuclear cells in asthmatics might be involved in allergic inflammation through the induction of adhesion molecule expression on eosinophils in asthma or allergic disorders.

Bovine leukocyte adhesion deficiency ‐clinical course and laboratory findings in eight affected animals

The clinical course of Bovine Leukocyte Adhesion Deficiency (BLAD) in eight Holstein Friesian cattle is described. Affected animals were presented with a history of poor thriving and recurrent bacterial infections. Five of these animals had to be killed because of severe respiratory disease shortly after admittance. Three affected animals survived calfhood only as a result of frequent antibacterial treatments. At one year of age, failure to thrive and stunted growth were still evident, but infections requiring antibiotic treatments occurred only sporadically. Clinical manifestations of BLAD were found in the digestive system (gingivitis, periodontitis, alveolar periostitis, diarrhoea), the respiratory system and the skin (impaired wound healing, chronic dermatitis). A leukocytosis based on a mature neutrophilia, which persisted during infection-free periods, was observed in all animals. Granulocytes were substantially deficient of beta 2-integrin expression on their membranes. Anaemia, which was noted in four animals, may be related to the Anaemia of Inflammatory Disease Complex (AID). The serum total protein content increased with time and was associated with elevated gamma-globulin levels. We suggest that, at a certain age, animals affected with BLAD are able to cope with environmental agents due to compensatory mechanisms of the immune system.

Immunocomplexes stimulate different signalling events to chemoattractants in the neutrophil and regulate L-selectin and beta 2-integrin expression differently.

Neutrophils express receptors for numerous phlogistons which, when occupied, trigger distinct signal-transduction pathways. Previous studies have shown that stimulation of neutrophils with chemoattractants induces shedding of the adhesive molecule L-selectin and increased expression of the beta 2-integrin CD11b/CD18. We determined the effect of ligation of classic, G-protein-linked chemoattractant receptors [C5a, interleukin-8 (IL-8), formylmethionyl-leucylphenylalanine (FMLP) and substance P], receptors for the Fc portion of IgG (Fc gamma receptors) and receptors for transforming growth factor beta (TGF beta) on expression of adhesive molecules by neutrophils and the stimulus-transduction mechanisms thought to mediate these changes. We were surprised to observe that occupancy of Fc gamma receptors by immunocomplexes (BSA-anti-BSA) stimulated increased expression by neutrophils of CD11b/CD18 at concentrations which did not affect L-selectin expression (EC50 9 micrograms/ml versus 350 micrograms/ml respectively, P < 0.00001, n = 5). In contrast, similar to previous studies, recombinant C5a, recombinant IL-8 and FMLP all stimulated increased expression of CD11b/CD18 (170-260% of basal, P < 0.001, n = 5) and shedding of L-selectin (56-75% reduction from basal, P < 0.001, n = 5) at similar concentrations and with similar potencies (EC50 = 2, 5, and 3 nM respectively). In contrast, neither TGF beta 1 nor, surprisingly, substance P affected expression of CD11b/CD18 or L-selectin. The regulation of expression of CD11b/CD18 or L-selectin in response to FMLP or immunocomplexes was unaffected by cytochalasin B (5 micrograms/ml) or the tyrosine kinase inhibitor tyrphostin-25 (25 microM). Although occupancy of both chemoattractant (FMLP) and Fc gamma receptors stimulated increments in the second messenger diacylglycerol, disruption of actin microfilaments by cytochalasin B enhanced diacylglycerol generation in response to FMLP but not in response to ligation of Fc gamma receptors. Moreover, both FMLP and immune aggregates provoked fluxes of intracellular Ca2+ concentration which differed with respect to both magnitude and kinetics and did not correlate well with regulation of adhesive-molecule expression. As upregulation of CD11b/CD18 is tightly linked to exocytosis of specific granules, these results suggest that shedding of L-selectin by activated neutrophils is not linked to exocytosis. These studies provide further evidence that receptors for chemoattractants and immunocomplexes on the neutrophil are linked to multiple signalling pathways.