CD44 ligation on peripheral blood polymorphonuclear cells induces interleukin-6 production by Giuseppe Sconocchia, Laura Campagnano, Domenico Adorno, Angela Iacona, Nella Y. Cococcetta, Vittorio Boffo, Sergio Amadori, and Carlo U. Casciani Blood Volume 97(11):3621-3627 June 1, 2001 ©2001 by American Society of Hematology

Analysis of freshly purified PMN purity by FcγRI gene expression Analysis of freshly purified PMN purity by FcγRI gene expression.PMNs were isolated from a buffy coat and incubated overnight in the presence or absence of IFNγ as indicated. Analysis of freshly purified PMN purity by FcγRI gene expression.PMNs were isolated from a buffy coat and incubated overnight in the presence or absence of IFNγ as indicated. NK and monocytes were purified from freshly isolated PBMCs by magnetic cell sorting. With the use of specific primers, we analyzed FcγRI, FcγRII, and β-actin mRNA expression by RT-PCR as described in “Materials and methods.” The present data are given in the following order: Markers (M), β-actin (lanes 1, 4, 7, and 10), FcγRI (lanes 2, 5, 8, and 11), and FcγRII (lanes 3, 6, 9, and 12). Giuseppe Sconocchia et al. Blood 2001;97:3621-3627 ©2001 by American Society of Hematology

CD44 ligation with mAbs induces IL-6 gene expression in peripheral blood PMNs.PMNs were isolated from a buffy coat from a normal donor and incubated for 18 hours in complete medium in the absence or presence of different anti-FcγRs mAbs. CD44 ligation with mAbs induces IL-6 gene expression in peripheral blood PMNs.PMNs were isolated from a buffy coat from a normal donor and incubated for 18 hours in complete medium in the absence or presence of different anti-FcγRs mAbs. RT-PCR, using specific primers, assessed IL-6 and β-actin mRNA expression, as described in “Materials and methods.” The present data are expressed in the following order: unstimulated PMNs (lane 1), anti FcγRI, 32.2 (lane 2), anti FcγRII, IV-3 (lanes 3), and NIH44.1 F(ab)2 (lane 4). This figure shows a representative experiment of 10 performed with similar results. Giuseppe Sconocchia et al. Blood 2001;97:3621-3627 ©2001 by American Society of Hematology

IL-6 gene expression on peripheral blood PMNs on in vitro stimulation with HA.(A) Normal freshly isolated PMNs were stained without (dash line) or with FITC-conjugated HA (solid line). IL-6 gene expression on peripheral blood PMNs on in vitro stimulation with HA.(A) Normal freshly isolated PMNs were stained without (dash line) or with FITC-conjugated HA (solid line). (B) PMNs were stained with a PE-conjugated anti-CD44 in the absence (thick, solid, line) or in the presence of 25 μg (dotted line) and 50 μg (thin, solid line) unlabeled HA. The dashed line represents the red fluorescence of PMNs stained with a PE-conjugated, matching, isotype mAb. (C) PMNs were isolated from a buffy coat from a normal donor and stimulated for 18 hours in the absence or presence of logarithmic dose concentrations of soluble HA: 0 μg/mL (lane 1), 1 μg/mL (lane 2), 10 μg/mL (lane 3), and 100 μg/mL (lane 4), as well as linear dose concentrations of immobilized HA: 0 μg/mL (lane 5), 250 μg/mL (lane 6), and 1000 μg/mL (lane 7). Using specific sense and antisense primers, RT-PCR analyzed IL-6 or β-actin gene expression. The figure shows 1 of 3 significant experiments performed with similar results. FL1-H and FL2-H indicate the logarithmic numbers of green and red fluorescence, respectively. Giuseppe Sconocchia et al. Blood 2001;97:3621-3627 ©2001 by American Society of Hematology

Kinetics of IL-6 gene expression and protein production after CD44 ligation on normal peripheral blood PMNs.Freshly purified PMNs were isolated from a buffy coat from a normal donor and stimulated in complete medium. Kinetics of IL-6 gene expression and protein production after CD44 ligation on normal peripheral blood PMNs.Freshly purified PMNs were isolated from a buffy coat from a normal donor and stimulated in complete medium. At the indicated times after stimulation, PMNs were harvested as source of total RNA, and the supernatants were collected for ELISA IL-6 protein assays. (A) This panel shows that, using sense and antisense specific primers, RT-PCR analyzed IL-6 and β-actin gene expression: lanes 1, 5, 9, 13, and 17, unstimulated PMNs; lanes 2, 6, 10, 14, and 18, NIH44.1 F(ab)2; lanes 3, 7, 11, 15, and 19, HA 100 μg/mL; and lanes 4, 8, 12, 16, and 20, LPS 12.5 ng/mL. (B) Left side shows IL-6 production measured in the supernatants collected at the indicated times: unstimulated PMNs (open squares), PMNs stimulated with NIH44.1 F(ab)2 (closed circles), HA 100 μg/mL (open circles), and LPS 12.5 ng/mL (closed triangles). Right side shows the IL-6 production measured in the supernatants of human PMNs stimulated overnight with immobilized or soluble HA at the indicated doses. Giuseppe Sconocchia et al. Blood 2001;97:3621-3627 ©2001 by American Society of Hematology

CD44-dependent IL-6 production requires protein transcription and tyrosine kinase activity.Freshly purified PMNs isolated from a buffy coat from a normal donor were stimulated as indicated in the presence (closed bars) or absence (open bars) of metabolic in... CD44-dependent IL-6 production requires protein transcription and tyrosine kinase activity.Freshly purified PMNs isolated from a buffy coat from a normal donor were stimulated as indicated in the presence (closed bars) or absence (open bars) of metabolic inhibitors: actinomycin D (A), genistein (B), and staurosporine (C). The figure shows 1 of 3 experiments performed with similar results. Giuseppe Sconocchia et al. Blood 2001;97:3621-3627 ©2001 by American Society of Hematology

Synergistic effect between IFNγ and CD44 stimulation on IL-6 production in human peripheral blood PMNs.Freshly purified PMNs isolated from buffy coats from normal donors were stimulated for 18 hours in the absence or presence of the indicated stimuli. Synergistic effect between IFNγ and CD44 stimulation on IL-6 production in human peripheral blood PMNs.Freshly purified PMNs isolated from buffy coats from normal donors were stimulated for 18 hours in the absence or presence of the indicated stimuli. In experiment 1 and 2, PMNs were treated as follows: rabbit anti-CD44 (Fab)2, 0.25 μg/mL; IFNγ, 200 IU/mL; IL-2, 300 IU/mL; and G-CSF, 100 ng/mL. The figure shows 2 of 4 experiments performed with similar results. Giuseppe Sconocchia et al. Blood 2001;97:3621-3627 ©2001 by American Society of Hematology