Polyinosine-polycytidylic acid stimulates versican accumulation in the extracellular matrix promoting monocyte adhesion

Abstract

Viral infections are known to exacerbate asthma and other lung diseases in which chronic inflammatory processes are implicated, but the mechanism is not well understood. The viral mimetic, polyinosine-polycytidylic acid, causes accumulation of a versican- and hyaluronan-enriched extracellular matrix (ECM) by human lung fibroblasts with increased capacity for monocyte adhesion. The fivefold increase in versican retention in this ECM is due to altered compartmentalization, with decreased degradation of cell layer-associated versican, rather than an increase in total accumulation in the culture. This is consistent with decreased mRNA levels for all of the versican splice variants. Reduced versican degradation is further supported by low levels of the epitope, DPEAAE, a product of versican digestion by a disintegrin-like and metallopeptidase with thrombospondin type 1 motif enzymes, in the ECM. The distribution of hyaluronan is similarly altered with a 3.5-fold increase in the cell layer. Pulse-chase studies of radiolabeled hyaluronan show a 50% reduction in the rate of loss from the cell layer over 24 hours. Formation of monocyte-retaining, hyaluronidase-sensitive ECMs can be blocked by the presence of anti-versican antibodies. In comparison, human lung fibroblasts treated with the cytokines, IL-1beta plus TNF-alpha, synthesize increased amounts of hyaluronan, but do not retain it or versican in the ECM, which, in turn, does not retain monocytes. These results highlight an important role for versican in the hyaluronan-dependent binding of monocytes to the ECM of lung fibroblasts stimulated with polyinosine-polycytidylic acid.

Figure 1.

Polyinosine-polycytidylic acid (Poly I:C) alters the distribution of versican and hyaluronan between medium and cell layer in human lung fibroblast (HLF) cultures. (A and B) Versican, (C) hyaluronan. (A) Cell layers from HLFs treated for 24 hours with medium containing 10% FBS with poly I:C, IL-1β plus TNF-α, or no addition were harvested for Western blot analysis after digestion with chondroitin ABC lyase. Material from an equal number of cells was used in each lane, and represent a separate 100-mm dish. Lanes 1–3, control; lanes 4–6, IL-1 β plus TNF-α; lanes 7–9, poly I:C. (B) Western blots showing versican retention in the cell layer of HLFs treated with poly I:C or IL-1β plus TNF-α in 10% FBS medium (shown in A) or total culture extracts (medium plus cell layer; data not shown) were scanned and quantitated with NIH Image-J. Results were normalized to controls. Closed bars, poly I:C; open bars, IL-1β plus TNF-α. (C) After 24-hour exposure to medium containing 10% FBS with poly I:C or IL-1β plus TNF-α, total hyaluronan was increased in both treatments above control, but the amount of hyaluronan in cell layers was significantly increased only in poly I:C–treated cultures. Closed bars, medium; open bars, cell layer. *P < 0.05, **P < 0.01 compared with control. All of the experiments represented in this figure were repeated three or more times with similar results.

Figure 2.

Particle exclusion assays show increased cell coat area in cells treated with poly I:C in comparison with control cells or those treated with IL-1β plus TNF-α. HLFs were treated for 24 hours with 0.1% FBS medium alone, or 10% FBS alone, or 10% FBS containing either poly I:C, or IL-1β plus TNF-α. (A) 0.1% FBS, (B) 10% FBS, (C) 10% FBS and IL-1β plus TNF-α, (D) 10% FBS plus poly I:C. (E) Extracellular matrix (ECM) area in each condition, as defined by exclusion of fixed red blood cells, was quantitated. **P < 0.01 compared with poly I:C. This experiment was performed twice with similar results.

Figure 3.

Estimated copy numbers for the versican splice variants, V0 and V1 are not increased by poly I:C. mRNA for each splice variant was normalized to abundance of 18S RNA in HLFs treated for 12 hours with control, IL-1β plus TNF-α, and poly I:C. Values for both treatments were reduced in comparison with controls. **P < 0.01 compared with control. This experiment was performed twice with similar results. In addition, a significant reduction in the mRNA level for total versican was found in both IL-1β plus TNF-α, and poly I:C–treated cells at 6 and 12 hours after treatment in three different experiments (data not shown).

Figure 4.

(A) Treatment with poly I:C reduces the accumulation of a versican degradation product in the ECM. After 24 hours of treatment with control medium, or poly I:C in medium containing 10% FBS concentrate (hydrodynamic size, >100,000 Da), cell layer proteins were harvested with urea buffer and ethanol precipitated before Western blotting with an 8% PAGE gel with antibody to DPEAAE, the C-terminal epitope on versican fragments produced by degradation by a disintegrin-like and metallopeptidase with thrombospondin type 1 motif (ADAMTS) 1, 4, or 5. Material from an equal number of cells was used in each lane, and represent a separate 100-mm dish. (B) mRNAs for versican-degrading enzymes were altered by both poly I:C and IL-1β plus TNF-α. At 6 hours after treatment, levels of mRNA for ADAMTS 1 and 5 were similarly reduced by both treatments. ADAMTS 4 mRNA was only slightly changed by poly I:C treatment, whereas it was increased 12-fold in IL-1β plus TNF-α–treated cells. Up-regulation of mRNA for ADAMTS 4, a versicanase, may help to explain the absence of versican in IL-1β plus TNF-α–treated cell layers. Closed bars, poly I:C; open bars, IL-1β plus TNF-α. *P < 0.05, **P < 0.01 compared with control. All of the experiments shown in this figure were performed at least three times with similar results. Similar changes in mRNA were obtained at 12 hours after treatment.

Figure 5.

(A) Expression of the HAS genes is differently altered by IL-1β plus TNF-α and poly I:C. Levels of mRNA for the hyaluronan synthases, HAS-1 through -3, were determined by quantitative RT-PCR and expressed for each treatment as fold increase above control at 12 hours after addition of treatments. mRNA levels for HAS-1 were increased in both IL-1β plus TNF-α– and poly I:C–treated cells. HAS-2 mRNA levels were decreased in poly I:C–treated cells. HAS-3 mRNA was increased only in the IL-1β plus TNF-α–treated cells. Open bars, IL-1β plus TNF-α; closed bars, poly I:C. **P < 0.01 compared with control. This experiment was performed four times with similar results, and significant similar changes were found at 6 hours after treatment. (B) Treatment with poly I:C increases the average hydrodynamic size of hyaluronan in the ECM secreted by HLFs. HLFs were metabolically labeled with tritiated glucosamine for 24 hours in the presence of 10% serum–containing medium alone or with addition of 20 μg/ml poly I:C. Radiolabeled cell layer extracts were separated from free glucosamine with a Sephacel G-50 (GE Healthcare) column, and then applied to an S-1,000 column (GE Healthcare) in 4 M guanidine buffer to determine the hydrodynamic size profile. Open circles, poly I:C; closed circles, control. All fractions above a K_av of 0.6 were completely degraded by hyaluronidase (HYAL) (data not shown). Thus, all of the high–molecular weight material eluted from this column is hyaluronan. This experiment was performed twice with similar results.

Figure 6.

(A) Pulse–chase analysis of [³H]-labeled hyaluronan shows reduced turnover in poly I:C–treated cells. HLFs were metabolically labeled with tritiated glucosamine for 24 hours in the presence of 10% serum–containing medium alone or with addition of IL-1β plus TNF-α or 20 μg/ml poly I:C, as in Figure 5B, and then chased with fresh 0.1% FBS medium. Remaining radioactivity was normalized to the amount in the cell layers at time zero of the chase period. Closed circles, controls; open circles, poly I:C; open triangles, IL-1β plus TNF-α. The initial rate of degradation was much greater in controls and in IL-1β plus TNF-α– than in poly I:C–treated cells, whereas a second, more gradual, rate appeared to be similar for both control and poly I:C–treated cells. This experiment was performed twice with similar results. (B) mRNAs associated with hyaluronan degradation are decreased by poly I:C treatment. Cells were arrested in low-serum–containing medium, and then stimulated with IL-1β plus TNF-α, poly I:C, or no addition in 10% serum–containing medium. The cells then were harvested for mRNA analysis after 6-hour incubation. CD44, HYAL1, and HYAL2 mRNAs were significantly decreased in poly I:C–treated cells in comparison with controls. There was also a small, but significant, decrease in HYAL2 mRNA in IL-1β plus TNF-α–treated cells. Reduction in these degradative enzymes and in CD44, the cell surface receptor for hyaluronan, may help to explain the slower release of radiolabeled hyaluronan from poly I:C–treated cells. In contrast, mRNA for CD44 was increased over twofold in IL-1β plus TNF-α–treated cells. Open bars, IL-1β plus TNF-α; closed bars, poly I:C. *P < 0.05, **P < 0.01 compared with control. This experiment was performed three times with similar results.

Figure 7.

Poly I:C increases colocalization of versican and hyaluronan in the cell layer. Cells from HLF cultures treated for 24 hours with 10% FBS–containing medium with poly I:C, IL-1β plus TNF-α, or no addition (control) were fixed and stained for hyaluronan (red), versican (green), or nuclei (blue). Compared with controls, increased amounts of hyaluronan were present in both IL-1β plus TNF-α– and poly I:C–treated cells, whereas versican was increased in only poly I:C–treated cells. When the two stains were merged in a single exposure, colocalization (yellow) was present on control cells and those treated with poly I:C. (A–C) Control, (D–F) IL-1β plus TNF-α, (G–I) poly I:C. (A, D, and G) Hyaluronan, (B, E, and H) versican, (C, F, and I) merged images with both stains (in I, nuclei appear magenta due to the presence of red hyaluronan stain above the plane of the cell). Monochrome images of the blue (4′,6′-diamidino-2-phenylindole [DAPI], nuclear), red (Texas red, hyaluronan), and green (Alexa Fluor 488, versican) emissions were collected at equivalent camera settings (exposure time and γ) for each color with a SPOT cooled CCD digital camera. Images were pseudocolored with the SPOT software, and merged with Adobe Photoshop (San Jose, CA). The brightness and contrast from all images of control and treated samples were adjusted equally for each color. This experiment was performed four times with similar results.

Figure 8.

Inhibition of monocyte retention by interference with ECM formation in poly I:C–treated cells. (A) Poly I:C increases monocyte adhesion to HLF cultures, whereas IL-1β plus TNF-α does not. Fluorescently labeled U937 monocytes were incubated with HLF cultures for 90 minutes at 4°C after 24-hour exposure of the HLFs to 10% FBS–containing medium with poly I:C (PIC), IL-1β plus TNF-α, or no addition. Some wells were treated with HYAL for 20–30 minutes before addition of monocytes. Open bars, plus HYAL. This experiment was performed four times with similar results. **P < 0.01 compared with control. (B) Formation of a poly I:C–induced monocyte-retaining ECM can be blocked by the addition of antibody to versican. HLFs were stimulated with poly I:C in 10% FBS–containing medium for 24 hours in the presence or absence of antibody to the N terminus of versican or a control antibody. The subsequent monocyte-binding assay was performed in the absence of antibody. Significant inhibition of monocyte retention by the ECM was observed. Monocyte binding was measured as arbitrary fluorescence units, reflecting Calcein AM in the cells. Fluorescence was linearly associated with monocyte number (data not shown). **P < 0.01 compared with cells treated with poly I:C alone, ^##P < 0.01 compared with cells treated with the same concentration of anti-versican antibody. Open bars, plus HYAL, gradient bars, anti-versican antibody (12C5), gray bars, mouse antibody against Drosophila frizzled protein (1C11) used as a control. This experiment was performed twice with similar results. “%AB” refers to concentration of monoclonal antibody concentrate by volume.

Figure 9.

Hyaluronan (HA) incorporation into the ECM is required for monocyte adhesion in poly I:C treated cells. (A) Inhibition of poly I:C–induced ECM formation by hyaluronan fragments (F). HLFs were stimulated with poly I:C in 10% FBS–containing medium for 24 hours in the presence or absence of low–molecular weight hylauronan fragments. The subsequent monocyte-binding assay was performed in the absence of hyaluronan fragments. Some wells were treated with HYAL for 20–30 minutes before addition of monocytes. Complete inhibition of HYAL-sensitive monocyte binding was observed in the fragment-treated wells. Open bars, plus HYAL. *P < 0.05 compared with poly I:C, **P < 0.01 compared with controls. Monocyte binding was measured as arbitrary fluorescence units, reflecting Calcein AM in the cells. Fluorescence was linearly associated with monocyte number (data not shown). This experiment was performed three times with similar results. (B) Fluticasone partially inhibits poly I:C–induced hyaluronan synthesis and poly I:C–induced formation of a monocyte-retaining ECM. HLFs were treated with poly I:C for 24 hours in the presence of different concentrations of fluticasone (0, 10⁻⁹, 10⁻⁸, and 10⁻⁷ M), and then assayed for hyaluronan synthesis or for monocyte adhesion. In both cases, the results were significantly reduced from control levels (P < 0.05) at 10⁻⁹ M fluticasone. When the degree of inhibition of hyaluronan synthesis was plotted against the inhibition of monocyte retention, the relationship was linear. This experiment was performed twice with similar results.

Figure 10.

Colocalization of adherent monocytes with versican and hyaluronan in poly I:C–induced ECM. Cells treated as in Figure 7 were also incubated with monocytes for 90 minutes at 4°C before fixation. The monocyte nuclei were also labeled with DAPI (blue). No HLF nuclei are visible in Figures 8A–8D. (A) Hyaluronan, (B) versican, (C) merged, and (D) C plus monocytes. Monocyte aggregates were predominantly located where versican and hyaluronan were colocalized. Fluorescent micrograph images of the blue (DAPI, nuclear), red (Texas Red, hyaluronan), and green (Alexa Fluor 488, versican) emissions were collected with a Nikon Coolpix digital camera (Nikon, Melville, NY). Brightness and contrast were identical for each color in single and merged images. This experiment was performed twice with similar results.