Lymphocyte adhesion to Candida albicans

Abstract

Adherence of lymphocytes to the fungus is the first step in the direct lymphocyte-mediated antifungal effect against Candida albicans. In this study we identified macrophage-1 antigen (Mac-1) (CD11b/CD18, alpha(M)/beta(2)) as the lymphocyte surface structure responsible for the adhesion of activated lymphocytes to the hyphal form of the fungus. Antibodies specific for epitopes of the alpha-subunit (CD11b) and the beta(2)-subunit (CD18) of Mac-1 were shown to completely eliminate lymphocyte adhesion to C. albicans hyphae. Lymphocyte adhesion to C. albicans was also inhibited significantly by known ligands of Mac-1, including the extracellular matrix proteins laminin and fibrinogen, as well as engineered peptides containing arginine-glycine-aspartic acid sequences and the disintegrin echistatin. N-Acetyl-D-glucosamine and beta-glucan, which inhibit Mac-1-mediated adhesion to the yeast, blocked lymphocyte adhesion to hyphae. NIH 3T3 fibroblast transfectants expressing human CD11b/CD18 bound to C. albicans, and their binding was inhibited by antibodies specific for CD11b/CD18. Finally, antibodies specific for CD11b/CD18 effectively inhibited the capacity of activated lymphocytes to have an antifungal effect against hyphae. Our results clearly identify Mac-1 (CD11b/CD18) as the lymphocyte surface structure that mediates activated lymphocyte adhesion to C. albicans and the resultant antifungal effect of the lymphocytes.

FIG. 1.

ECM proteins and RGD-mimetic peptides inhibit adhesion of mIAL to C. albicans. Adhesion of mIAL to C. albicans was assessed by measuring the retention of ⁵¹Cr-labeled lymphocytes in the presence of the following proteins and RGD-mimetic peptides: GRGDSPK (•), GRGDSPK plus 30 μg of anti-CD11b mAb M1/70.15 (▴), FEP (⧫), fibrinogen (▪), EHS laminin (◊), factor X (▾), GRGDSPCA_c (▿), H-2K^b MHC peptide TCVEWLRRYLKN (▵). The conditions and adhesion assessment protocol used are described in Materials and Methods. The data are means ± standard deviations based on two or more experiments. The vertical lines intersecting the abscissa indicate the IC₅₀ for the molecules evaluated.

FIG. 2.

mAbs to CD11b/CD18 inhibit adhesion of mIAL to C. albicans. Adhesion of mIAL to C. albicans was assessed by measuring the retention of ⁵¹Cr-labeled lymphocytes in the presence of the following mAbs to CD antigens: for CD11b, OKM1 (mouse anti-human, IgG2b), M1/70.15 (rat anti-mouse, IgG2b), and 5C6 (hamster anti-mouse, IgG); for CD18, M18/2.A (rat anti-mouse, IgG2a [κ]), and 2E6 (hamster anti-mouse, IgG); for CD11a, M17/4.4 (rat anti-mouse, IgG2b[κ]); and for CD11c, N418 (hamster anti-mouse, IgG). The conditions and adhesion assessment protocol used are described in Materials and Methods.

FIG. 3.

mAbs to CD11b/CD18 and RGD-mimetic proteins inhibit adhesion of hIAL to C. albicans. Adhesion of hIAL to C. albicans was assessed by measuring the retention of ⁵¹Cr-labeled lymphocytes in the presence of the following mAbs to CD antigens and RGD-mimetic proteins: for CD11b, OKM1 (mouse anti-human, IgG2b) and M1/70.15 (rat anti-mouse, IgG2b); for CD18, TS1/18 (mouse anti-human, IgG1); for CD58, TS2/9 (mouse anti-human LFA-3, IgG1); for CD11a, TS1/22 (mouse anti-human LFA-1, IgG1); FBIP; echistatin, a disintegrin RGD-specific integrin inhibitor; heparin, an RGD-containing ECM protein; and GRGDSP_c, a cyclical molecule with the sequence GPenGRGDSPCA (where Pen is penicillamine). The conditions and adhesion assessments protocol used are described in Materials and Methods. The data are the data from 12 separate preparations obtained with human peripheral blood mononuclear cells and are means ± standard deviations.

FIG. 4.

mAbs to CD11b/CD18 inhibit adhesion of 3T3-19 (Mac-1⁺) transfectants to C. albicans. Adhesion to C. albicans of NIH 3T3 fibroblasts expressing transfected human CD11b/CD18 (3T3-19 fibroblasts) and of NIH 3T3 fibroblasts not expressing CD11b/CD18 (3T3-1 fibroblasts) was assessed by measuring the retention of ⁵¹Cr-labeled transfectants in the presence of the following mAbs to CD antigens: for CD11b, OKM1 (mouse anti-human, IgG2b); for CD18, TS1/18 (mouse anti-human, IgG1); and for CD29, clone 551125 (rat anti-mouse β₁ integrin, IgG1). The conditions and adhesion assessment protocol used are described in Materials and Methods. The typical maximum amount of radioactivity for 5 × 10⁴ NIH 3T3 fibroblasts, either 3T3-19 (Mac-1⁺) or (3T3-1), was approximately 5 × 10⁴ cpm.

FIG. 5.

RGD-mimetic peptides in combination with mAbs to CD11b/CD18 inhibit adhesion of 3T3-19 (Mac-1⁺) transfectants to C. albicans. Adhesion to C. albicans of NIH 3T3 fibroblast transfectants expressing human CD11b/CD18 (3T3-19 fibroblasts) was assessed by measuring the retention of ⁵¹Cr-labeled transfectants in the presence of the following mAbs to CD antigens and RGD-mimetic peptides: for CD11b, OKM1 (mouse anti-human, IgG2b) and M1/70.15 (rat anti-mouse, IgG2b); for CD18, TS1/18 (mouse anti-human, IgG1); GRGDSPK; and FBIP. The conditions and adhesion assessment protocol used are described in Materials and Methods. The typical maximum amount of radioactivity for 5 × 10⁴ NIH 3T3 fibroblasts, either 3T3-19 (Mac-1⁺) or (3T3-1), was approximately 5 × 10⁴ cpm.

FIG. 6.

mAbs to CD11b/CD18 block inhibition of growth of C. albicans hyphae by mIAL. Inhibition of C. albicans growth was assessed by measuring the incorporation of [³H]uridine after treatment with mIAL. mAbs either were added to hyphae alone or were preincubated with mIAL as shown in Fig. 1. The following mAbs to CD antigens were used: for CD11b, OKM1 (mouse anti-human, IgG2b) and M1/70.15 (rat anti-mouse, IgG2b); for CD11a, M17/4.4 (rat anti-mouse, IgG2b [κ]); and for CD11c, N418 (hamster anti-mouse, IgG). An asterisk indicates that data are statistically significantly different (P < 0.05), as determined by Student’s independent t test. An asterisk with a superscript a indicates that there was statistically significant blocking of mIAL-mediated growth inhibition by 45 μg of mAb M1/70 (bar 4) or OKM1 (bar 7). An asterisk with a superscript b indicates that there was statistically significant growth inhibition by mIAL treated with 25 μg of M1/70 (bar 5 versus bar 2 or 3) or 25 μg of OKM1 (bar 8 versus bar 2 or 6). Note that the baseline mean growth inhibition value, 58% (bar 2), was compared to four values (bars 9, 10, 11, and 12) to test significance, and the differences were found to be not significant (P > 0.05). The data are means ± standard deviations based on two or more experiments.