Activation-Dependent Modulation of Hyaluronate-Receptor Expression and of Hyaluronate-Avidity by Human Monocytes Johannes M. Weiss, Andreas C. Renkl,

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Activation-Dependent Modulation of Hyaluronate-Receptor Expression and of Hyaluronate-Avidity by Human Monocytes Johannes M. Weiss, Andreas C. Renkl, Thomas Ahrens, Brigitte H. Mai, Ralf W. Denfeld, Erwin Schöpf, Jan C. Simon Journal of Investigative Dermatology Volume 111, Issue 2, Pages 227-232 (August 1998) DOI: 10.1046/j.1523-1747.1998.00286.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Monocytes infiltrating at sites of cutaneous inflammation upregulate HA binding and HA-receptor expression. Cryosections of skin samples from allergic contact dermatitis were stained with the following. (A) IgG1 control MoAb, secondary cy3-conjugated goat anti-mouse MoAb, and HA-FITC. (B, D–L) cy3-conjugated anti-CD14 MoAb (red) and the indicated FITC-labeled reagents (green). Double positive cells appear yellow-orange (B). Arrows: CD14+– HA-binding cells around the subpapillary plexus. (C) cy3-conjugated anti-CD3 MoAb (red) and HA-FITC (green). *CD3+ cells do not bind HA. (D) ICAM-1, (E) panCD44 MoAb (SFF-2), (F) panCD44 MoAb (MEM-85), (G) CD44v3 MoAb (VFF23), (H) CD44v4 MoAb (VFF11), (I) CD44v5 MoAb (VFF8), (J) CD44v6 MoAb (VFF18), (K) CD44v7 MoAb (VFF9), (L) CD44v9 MoAb (11.24). Arrows: double positive cells. In (E)–(L) epidermal keratinocytes appear green because they express CD44 epitopes. The dotted line indicates the dermo–epidermal junction. Scale bars, (A, C–L) 12 μm, (B) 36 μm. Journal of Investigative Dermatology 1998 111, 227-232DOI: (10.1046/j.1523-1747.1998.00286.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Activation by plastic adhesion, IFN-γ, and LPS upregulates monocyte HA-binding capacity. Purified monocytes (A) were analyzed either fresh or following stimulation with IFN-γ or LPS for 48h and stained with HA-FITC (FL-1). Binding of HA to CD3+ lymphocytes sorted from the same PBMC preparation was performed accordingly (B). Representative results of five independent experiments. Journal of Investigative Dermatology 1998 111, 227-232DOI: (10.1046/j.1523-1747.1998.00286.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 CD44 isoforms and ICAM-1, but not RHAMM, are upregulated during monocyte activation. PBMC from a single donor were used fresh or following culture in the presence or absence of IFN-γ or LPS for 48h or 96h. Cells were gated for CD14 expression and stained with either HA-FITC, antibody against the N-terminal domain of CD44 (CD44s), antibodies recognizing epitopes encoded by CD44 exons v3, v4, v5, v6, v7, and v9, ICAM-1, or RHAMM. 7-Aminoactinomycin was added to exclude dead cells. cMFI was calculated by subtracting MFI of control from MFI of specifically stained cells, as detailed in Materials and Methods. Journal of Investigative Dermatology 1998 111, 227-232DOI: (10.1046/j.1523-1747.1998.00286.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 CD44 mRNA and protein expression is upregulated during Mo activation. Ninety-five per cent enriched Mo were used fresh or following stimulation as indicated by plastic adherence, IFN-γ, LPS. (A) For northern blot analysis, 5 μg of total RNA were size fractionated, transferred to nylon N-plus membranes, followed by hybridization with a 810bp human CD44s probe containing the exons of the invariant extracellular domain of CD44 and with a human β-actin probe. (B) For western blot analysis, 15 μg of total cellular protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. CD44 protein was detected by MoAb Hermes-3. Journal of Investigative Dermatology 1998 111, 227-232DOI: (10.1046/j.1523-1747.1998.00286.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 MoAb against panCD44 block HA binding on activated monocytes. Monocytes were purified as described above, cultured in the presence of IFN-γ for 48h. and subsequently analyzed by FACS. Indicated MoAb were added to cell suspensions 15min prior to addition of HA-FITC. To test for specificity, HA-FITC-labeled cells were incubated with hyaluronidase or heparinase for 30min at 4°C. Percentage inhibition was calculated by the formula: 1 – (cMFI MoAb blocked/cMFI control). Positive percentage indicates blocking of HA-FITC binding, negative percentage indicates increased binding. The values shown are averaged from five experiments ±SD. Asterisks indicate statistically significant inhibition (one way ANOVA, Dunnett’s test, p < 0.0001). Journal of Investigative Dermatology 1998 111, 227-232DOI: (10.1046/j.1523-1747.1998.00286.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions