Lectin binding studies on murine peritoneal cells: Physicochemical characterization of the binding of lectins from Datura stramonium, Evonymus europaea, and Griffonia simplicifolia to murine peritoneal cells

Abstract

Purified 125I-labeled lectins from Datura stramonium, Evonymus europaea, and Griffonia simplicifolia (I-B4 isolectin) were used to analyze changes in the expression of carbohydrates on the surface of resident (PC) and thioglycollate-stimulated murine (C57B/6J) peritoneal exudate cells (PEC). The lectins from D. stramonium, E. europaea, and G. simplicifolia I-B4 bind specifically to PEC with relatively high affinity (Kd = 5.65 +/- 1.08 X 10(-7) M, 1.08 +/- 0.12 X 10(-8) M, and 1.33 +/- 0.15 X 10(-7) M, respectively). Assuming a single lectin molecule binds to each cell surface saccharide, the number of receptor sites per cell ranged for different cell samples from 22.3 to 50.0 X 10(6), from 3.8 to 4.8 X 10(6), and from 2.0 to 16.8 X 10(6) for D. stramonium, E. europaea, and G. simplicifolia I-B4 lectins, respectively. There were approximately 3- to 7-fold, 16- to 20-fold, and 2- to 20-fold increases in binding capacity for D. stramonium, E. europaea and G. simplicifolia I-B4, respectively, compared to the binding to resident, peritoneal cells. Scatchard plots of the binding of all three lectins to PEC were linear, suggesting that the receptor sites for these lectins are homogeneous and noninteracting. The binding capacity of these lectins to PEC was unchanged after trypsin digestion of cells. The expression of carbohydrates on the surface of PEC was also monitored by an agglutination assay. PEC were agglutinated by all three lectins whereas PC either were not agglutinated or were agglutinated only at high lectin concentrations. On the basis of our knowledge of the carbohydrate binding specificity of the D. stramonium and G. simplicifolia I-B4 lectins, we postulate that, parallel with thioglycolate stimulation, there is an increase in the number of N-acetyllactosamine residues and terminal alpha-D-galactosyl end groups. The blood group B, and H type 1 determinants--DGa1 alpha 1,3[LFuc alpha 1,2]DGa1 beta 1,3(or 4)DGlcNAc and LFuc alpha 1,2DGa1 beta 1,3DG1cNAc, respectively, as well as DGa1 alpha 1,3DGa1 beta 1,3(or 4)DGlcNAc--may be considered to be possible receptors for the E. europaea lectin. These glycoconjugates, present on the surface of peritoneal exudate cells, provide new chemical markers for studying the differentiation of resident peritoneal cells.

Assessment and In Vivo Scoring of Murine Experimental Autoimmune Uveoretinitis Using Optical Coherence Tomography

“…Lectin B4, which labels a subset of the myeloid lineage as well as vascular endothelium [20] was used to stain this structure. As no vessels were observed in the regions of the folds, or when stained with collagen IV (data not shown), lectin B4 expression represents activated myeloid cells [19] and 3D reconstruction further emphasises the encapsulation of inflammatory cells (myeloid and T-cells) in the fold and within the subretinal space (see Movie S1 ). …”

“…Following dissection, the retinae were placed into 100% methanol overnight, then blocked and stained in 1% BSA (Sigma Aldrich, UK), 3% Triton X-100 and 5% non-specific goat serum (AbD serotec, UK). Antibodies were all used at 1∶200 dilution including anti-collagen IV (code: 2150-1470, AbD serotec, UK), an equal mix of anti-CD4 (code: 553043, BD Biosciences, UK) and anti-CD8 (code: 553027, BD Biosciences, UK), anti-Iba1 (code: 019-19741, WAKO, Osaka, Japan) and biotinylated lectin B4 from Bandeiraea simplicifolia (code: L2140, Sigma aldrich, UK) [19]. Alexa fluor 488, 546 and 633 secondary antibodies (Invitrogen-Molecular Probes, Leiden, The Netherlands) were used at 1∶500 dilution.…”

Carbohydrate and peptide antigens in macrophage populations derived from human bone marrow and milk: an immunomorphological and immunochemical analysis

“…In some in vitro studies, an association was detected between the activation of rat and mouse macrophages and the expression of certain lectin binding sites (Maddox et al, 1982;Petryniak et al, 1986;Irimura et al, 1987;Tabor et al, 1989;Krugluger et al, 1990;Warfel et al, 1991;Warfel & Zucker-Franklin, 1992). In this context, little is known about resident macrophages of normal bone marrow, which are important regulators of immune responses and are involved in the complex functional network of normal and pathologically altered haematopoiesis (Dorshkind, 1990).…”

“…For example, in rat bone marrow macrophages, soybean agglutinin (SBA) binding sites were significantly increased after stimulation with colony stimulating factor 1 (Krugluger et al, 1990). GSA-I-B4 binding of murine peritoneal macrophages was induced by specific and unspecific activators like thioglycollate, interferon-~ and lipopolysaccharide (Maddox et al, 1982;Petryniak et al, 1986;Tabor et al, 1989). In response to GSA-I-B4 binding, murine peritoneal macrophages disclosed a reduction of fibronectin and transglutamase release, whereas the secretion of plasminogen activator inhibitor type 2 and collagenase was significantly increased (Warfel & Zucker-Franklin, 1992).…”

“…Various in vitro studies using rat or mouse macrophages are in keeping with the assumption that lectins such as GSA-I-B4 and SBA detect activation-associated antigens (Maddox et al, 1982;Petryniak et al, 1986;Irimura et al, 1987;Tabor et al, 1989;Krugluger et al, 1990;Warfel et al, 1991;Warfel & Zucker-Franklin, 1992). Additionally, carbohydrate antigens substituting mucin-type glycoproteins are known to act as mediators of cell adhesion (Shimizu & Shaw, 1993).…”