PMC:2800179 / 21910-25093 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2800179","sourcedb":"PMC","sourceid":"2800179","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2800179","text":"ICAM-1 Expression Is Induced by Degraded CGN and Is Responsible for Monocytes Aggregation In Vitro \nIn order to study the effect of dCGN on the expression of cell surface antigens, PBM and THP-1 cells were incubated for 36 h in the presence and absence of dCGN. The expression of various cell surface molecules was analyzed by flow cytometry as described in materials and methods. Both forms of dCGN clearly stimulated expression of ICAM-1 (CD54) on PBM and THP-1 cells (Fig. 4A). The increase in ICAM-1 expression was higher on THP-1 cells treated with 40 kDa dCGN (Fig. 4B). Another surface antigen, the lymphocyte function-associated antigen 3 (CD58) was slightly reduced on PBM after treatment with 40 kDa dCGN (Fig. 4B). Interestingly, expression of major histocompatibility complex molecules of class I (HLA-ABC) and of class II (HLA-DR), as well as the monocyte marker CD14, seemed to be reduced by treatment with dCGN (Fig. 4B). However, these differences were not statistically significant.\n10.1371/journal.pone.0008666.g004 Figure 4 Degraded CGN stimulated ICAM-1 expression in monocytes.\nPeripheral blood monocytes (PBM) or THP-1 cells were incubated in the presence or absence of carrageenan for 24 h before being stained for various cell surface antigens. Antigen expression was then analyzed by flow cytometry. A: Histograms of ICAM-1 expression in cells treated with medium only (control), 10 kDa dCGN (C10), or 40 kDa dCGN (C40). B: Fluorescence intensity for expression of the antigens HLA-ABC, HLA-DR, CD14, ICAM-1, and CD58 in cells treated with medium only (control), 10 kDa dCGN (C10), or 40 kDa dCGN (C40). Data are mean +/− SEM. Treatment with dCGN also induced a strong aggregation of monocytes, detected by phase contrast inverse microscopy (Fig. 5). Although this effect was easily observed in monocytes incubated with the 10 kDa dCGN (Fig. 5B), a more robust cell aggregation was observed in monocytes incubated with the 40 kDa dCGN (Fig. 5C). ICAM-1 has been proposed to be the main adhesion molecule responsible for monocyte aggregation. To confirm this, monocytes were incubated with both types of dCGN in the presence of an anti-ICAM-1 antibody, an anti-CD58 antibody and an isotype control IgG1 antibody. The anti-ICAM-1 antibody effectively blocked the cell aggregates induced by dCGN (Fig. 5D, 5E), strongly suggesting that indeed ICAM-1 is responsible for monocyte aggregation. Both the control IgG1 and the anti-CD58 antibody did not modify monocyte aggregation (data not shown).\n10.1371/journal.pone.0008666.g005 Figure 5 Degraded CGN induced monocytes aggregation in vitro.\nMonocytes were incubated in the absence (A) or the presence of 1 mg/ml 10 kDa dCGN (B), 1 mg/ml 40 kDa dCGN (C), or 1 mg/ml 40 kDa dCGN plus 2.5 µg/ml anti-ICAM-1 antibody (D). Cells were observed by phase contrast inverse microscopy at 150X magnification. Inserts in A and B show a close up of cells at 300X magnification. E: Number of monocyte aggregates in 24 wells plate of PBM cell cultured with nothing (control), with 10 kDa degraded CGN (C10), or with 40 kDa degraded CGN (C40). Some cultures had also 2.5 µg/ml anti-ICAM-1 antibody. 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