Introduction

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Hallmarks of chronic airway inflammation of patients with severe or persistent asthma include the accumulation of activated eosinophils (1) and of extracellular matrix (ECM) components in the airways (2). Activated eosinophils have been shown to contribute to the pathogenesis of asthma by degranulation and release of highly cytotoxic proteins and lipid mediators, and production and release of a panel of cytokines, including T helper 2-type cytokines, proinflammatory cytokines, growth factors, and chemokines (3). In addition, in bronchial tissues of patients with asthma, activated eosinophils have been identified as a major source of transforming growth factor (TGF)- by in situ hybridization (4) and immunohistochemistry (5), suggesting that eosinophils may be involved in the development of airway remodeling by stimulating the synthesis of ECM through TGF- production. The activation of eosinophils and their accumulation in tissue sites are believed to be mediated by mechanisms that involve T cell-derived cytokines, including interleukin (IL)-3, IL-5, and granulocyte macrophage colony-stimulating factor (GM-CSF), which influence eosinophil growth, maturation, and differentiation, and appear to be critical in prolonging the survival of eosinophils in tissues and allowing their movement into the tissues. However, the precise mechanisms of eosinophil activation as well as TGF- production by eosinophils at sites of chronic inflammation remain to be elucidated.

Recently, ECM has been shown to participate in the attachment of cells, tissue growth and repair (6), proliferation and differentiation (7), cell migration and activation (8), cell survival/delay of apoptosis (9), and chemotaxis (10), indicating that ECM may play an important role in the development and persistence of inflammation. Among ECM, the glycosaminoglycan (GAG) hyaluronan (HA), which is a nonsulfated GAG polymer made of repeating disaccharide units and a major component of ECM, undergoes dynamic regulation during inflammation. HA is synthesized mainly by mesenchymal cells by membrane-bound HA synthases, whose complementary DNA have been recently identified and characterized (11), and exists as a high molecular weight (mol wt) polymer, usually in excess of 10⁶ D in its native form (12, 13). At the site of inflammation and tissue injury, HA has been shown to be more polydisperse with an accumulation of lower mol wt forms (14, 15). The accumulation of lower weight forms of HA has been postulated to occur by a variety of mechanisms, including depolymerization by reactive oxygen species, enzymatic cleavage, and de novo synthesis of lower mol wt species (12, 16, 17). More recently, several studies have demonstrated that low mol wt HA, but not high mol wt HA, exhibits pronounced biologic effects on cells and in tissues. HA fragments as small as a hexamer or low mol wt HA (< 5 × 10⁵ D), but not high mol wt HA (> 1 × 10⁶ D), have been shown to stimulate the murine alveolar macrophage cell line MH-S and macrophages recruited to sites of inflammation to produce chemokines and cytokines such as regulated on activation, normal T cells expressed and secreted (RANTES), macrophage inflammatory peptide (MIP)-1, MIP-1, IL-1, tumor necrosis factor (TNF)-, and IL-12 (18, 19), and to induce nitric oxide synthase through a nuclear factor B (NF-B)-dependent mechanism (20), suggesting that low mol wt HA may be an important regulator of inflammatory cell activation at sites of chronic inflammation.

The cell surface glycoprotein CD44, a receptor for HA, is an adhesion receptor for ECM molecules that has been implicated in lymphocyte recirculation, cell migration, T-cell signaling, cell-cell and cell-ECM interactions, and metastasis. Recently, CD44 expression on eosinophils and its upregulation by IL-5 or GM-CSF have been reported (21). Furthermore, increased expression of CD44 on eosinophils from late-phase bronchoalveolar lavage fluid (BALF) of patients with asthma and on hypodense eosinophils has been demonstrated (22), suggesting CD44 as an activation marker for eosinophils and involvement of CD44 signaling in the development and persistence of eosinophilic inflammation through cell-cell and cell-ECM interactions.

In this study, we investigated the hypothesis that low mol wt HA generated at sites of inflammation may serve to activate eosinophils and contribute to ECM accumulation by mediating TGF- production by eosinophils at the site of chronic inflammation. To this end, peripheral blood eosinophils were isolated from 12 patients with asthma or six healthy subjects, and stimulated in the presence or absence of low mol wt or high mol wt HA. We found that low mol wt HA has a pronounced effect on eosinophil survival in both patients with asthma and healthy subjects in a dose-dependent fashion on Days 2 and 4 when compared with high mol wt HA. The HA-mediated eosinophil survival is partially, but significantly, inhibited ( 50% inhibition) by a blocking monoclonal antibody (mAb) for CD44, a specific receptor of HA, and largely inhibited by an anti-GM-CSF neutralizing antibody but not by an anti-IL-3 or anti-IL-5 neutralizing antibody. In addition, the HA stimulates eosinophils to upregulate GM-CSF messenger RNA (mRNA) expression and to release GM-CSF protein, suggesting that the HA-mediated eosinophil survival is due mainly to induction of GM-CSF release through partial CD44 signaling. We also demonstrate that low mol wt HA results in morphologic changes in eosinophils such as transforming from round to spindle shape and in homotypic aggregation, and upregulates intercellular adhesion molecule (ICAM)-1 expression. In addition, we found that the HA increases TGF- mRNA expression and protein secretion by eosinophils. These observations suggest previously unforeseen interactions between eosinophils and low mol wt HA, and thus, novel pathways by which eosinophils may contribute to the regulation of chronic airway inflammation and airway remodeling in asthma.

	Materials and Methods

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Reagents

Low and high mol wt HA from human umbilical cord were purchased from ICN Biomedicals, Inc. (Costa Mesa, CA) (18) and Sigma Chemical Co. (St. Louis, MO), respectively. Low mol wt HA (ICN) is free of protein (< 2%) and free of chondroitin sulfate (< 3%), and it has been reported that its peak molecular size is approximately 0.2 × 10⁶ D (20, 23). High mol wt HA (Sigma) has been shown to have a molecular size of 3.0 to  5.8 × 10⁶ D. Recombinant human IL-5, specific mouse monoclonal neutralizing antibodies against human GM-CSF, IL-3, and IL-5 were purchased from Genzyme (Cambridge, MA). A rat antihuman CD44 blocking mAb (immunoglobulin [Ig]G_2a, preservative free and low endotoxin) was purchased from Endogen (Cambridge, MA). Control mouse IgG₁ and rat IgG_2a (no azide, low endotoxin) were purchased from PharMingen (San Diego, CA). Fluorescein isothiocyanate (FITC)-conjugated antihuman major histocompatibility complex (MHC) class II (human leukocyte-associated antigen [HLA]-DP, DQ, and DR) and anti-CD54 (ICAM-1) mAbs were purchased from Ancell (Bayport, MN), and FITC-conjugated mouse IgG₁ control mAb was purchased from Southern Biotechnology Associates, Inc. (Birmingham, AL). Micromagnetic beads bound to anti-CD16 mAb and magnetic-activated cell sorter (MACS) columns were supplied by Miltenyi Biotec GmbH (Gergisch-Gladbach, Germany).

Purification and Culture of Human Peripheral Eosinophils

A volume of 50 to 100 ml of blood was obtained from 12 atopic (skin test positive) subjects with mild to moderate symptoms of asthma or six healthy subjects. Eosinophils were purified by negative immunomagnetic selection as previously described (24). In brief, whole blood was subjected to dextran sedimentation (Amersham Pharmacia Biotech AB, Uppsala, Sweden), centrifugation through Percoll (Pharmacia, Uppsala, Sweden), and hypotonic lysis of erythrocytes. Eosinophils were enriched by granulocytes and passage via the MACS system by sequential incubation at 4°C with anti-CD16 mAb magnetic beads to deplete CD16⁺ neutrophils. These procedures consistently resulted in highly purified eosinophils (> 99%). These eosinophils (> 98.5% viable by trypan blue exclusion) were cultured in RPMI supplemented with 10% fetal calf serum (FCS; GIBCO BRL, Grand Island, NY), 100 U/ml penicillin, and 100 mg/ml streptomycin with or without the addition of HA, cytokines, and specific mAbs in 96-well or 24-well, flat-bottom, culture plates (Becton Dickinson, Lincoln Park, NJ).

Eosinophil Survival

Eosinophil survival was assessed as previously described (24). Briefly, eosinophils (2 × 10⁵/well in 0.2 ml RPMI) were cultured in 96-well, flat-bottom plates (Becton Dickinson). Cells were removed from each well after gentle pipetting, and eosinophil survival was assessed by counting viable cells at Days 2 and 4 by trypan blue (GIBCO BRL) exclusion using a hemocytometer.

Morphologic Study of Eosinophils

Phase microscopy was performed with an inverted-phase microscope using standard techniques. Photomicrographs were taken at 24 h after incubation.

Reverse Transcriptase/Polymerase Chain Reaction

The primers for human GM-CSF were purchased from Stratagene (La Jolla, CA). Oligonucleotides for polymerase chain reaction (PCR) of human TGF-₁ and -actin were synthesized at GIBCO BRL (Tokyo, Japan). The sequences of antisense and sense primers for TGF-₁ and -actin were 5'-TTT CGC CTT AGC GCC CAC TG-3' and 5'-GAA GTT GGC ATG GTA GCC CTT-3', and 5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3' and 5'-CTA GAA GCA TTG CGG TGG ACG ATG GAG GG-3' (24), respectively. Total RNA was extracted from freshly isolated eosinophils or eosinophils cultured with or without HA for 24 h from three patients with asthma using the RNAzol B method (Tel-Test, Inc., Friendswood, TX) and stored at 70°C until use. RNA was reverse-transcribed with SuperScript II reverse transcriptase (RT) (GIBCO BRL) according to the manufacturer's protocol. RNA samples (1 µg of total RNA), 0.5 µg of random primer (Promega, Madison, WI), 10 mM of each deoxynucleotide triphosphate (Promega), and 200 U of reverse transcriptase were incubated in a total of 20 µl of reaction mixture containing the enzyme buffer as supplied by the manufacturer. The reaction mixture was incubated for 10 min at 25°C, for 50 min at 42°C, and for 15 min at 70°C, respectively. The reverse-transcribed products were then amplified with Taq DNA polymerase (GIBCO BRL) following the manufacturer's protocol. A total of 100 µl of PCR mixture consisted of the PCR buffer (20 mM Tris-HCl, 50 mM KCl, and 1.5 mM MgCl₂; GIBCO BRL), 0.2 mM of each deoxynucleotide triphosphate, 4 µl of the reverse-transcribed product, 0.5 µM of both antisense and sense primers, and 2.5 U of Taq DNA polymerase, with 50 µl of mineral oil (Sigma) layered on the surface of the reaction. Thirty cycles of PCR were performed using a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). Each cycle consisted of 1 min of denaturation at 94°C, 2 min of annealing at 55°C, and 2 min for enzymatic primer extension at 72°C. Densitometric analysis of TGF- mRNA expression was performed using NIHImage software (National Institutes of Health, Bethesda, MD) and results are expressed as a ratio of TGF-₁ mRNA expression to -actin mRNA expression.

Cytokine Assay

Eosinophils (1 × 10⁶/well) were cultured with RPMI containing 10% FCS in 24-well plates (Becton Dickinson) to measure GM-CSF. Supernatants were collected at 24 h and stored at 20°C until assayed. GM-CSF and TGF-₁ proteins released into culture supernatants were assessed using commercially available enzyme-linked immunosorbent assay (ELISA) kits from Genzyme and R&D Systems (Minneapolis, MN), respectively. The sensitivities of detection were 2.5 pg/ml and 7 pg/ml, respectively.

Flow Cytometric Analysis of MHC Class II and ICAM-1 (CD54) Expressions on Eosinophils

Twenty-four hours after incubation with or without 10 µg/ml of HA, eosinophils were harvested and washed once in cold-wash buffer (phosphate-buffered saline/0.1% NaN3/2% FCS). The eosinophils (5 × 10⁵ cells) were stained with FITC-conjugated antihuman MHC class II (HLA-DP, DQ, and DR) mAb or anti-ICAM-1 (CD54) mAb or control mAb (IgG₁) for 45 min on ice, and washed twice in cold-wash buffer. Stained eosinophils were analyzed on a FACSort (Becton Dickinson) using CELLQuest software (Becton Dickinson).

Data Analysis

Data were expressed as mean ± standard error of the mean (SEM). Wherever suitable, interpretation of results was done by analysis of variance. The difference was considered statistically significant when P < 0.05.

	Results

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Low Molecular Weight HA Prolongs Eosinophil Survival

To investigate whether HA would regulate an eosinophil activation state, human peripheral blood eosinophils (purity > 99%) from patients with mild to moderate asthma or healthy subjects were incubated with increasing concentrations of low mol wt HA ( 0.2 × 10⁶ D) or high mol wt HA (3.0 to  5.8 × 10⁶ D), and eosinophil survival was examined on Days 2 and 4 by the trypan blue exclusion method as previously described (24). As shown in Figure 1A, low mol wt HA significantly prolonged eosinophil survival in patients with asthma in a dose-dependent fashion. Eosinophil survival rates were 40.0 ± 12.8, 56.8 ± 9.8, and 71.1 ± 6.3% (mean ± SEM, n = 6) on Day 2, and 39.0 ± 13.2, 48.8 ± 12.1, and 57.3 ± 10.2% (mean ± SEM, n = 6) on Day 4, when eosinophils were stimulated with 1, 10, and 100 µg/ml of HA, respectively. High mol wt HA also had a significant effect on eosinophil survival in patients with asthma on Days 2 and 4 when compared with medium alone; however, the effect was significantly smaller than that of low mol wt HA (Figure 1B), suggesting that low mol wt HA has a more pronounced effect on eosinophil function than does high mol wt HA. In addition, there was no significant difference in the effect of low mol wt HA on eosinophil survival between patients with asthma and healthy subjects (56.8 ± 9.4 versus 71.6 ± 20.4% on Day 2 and 48.8 ± 12.1 versus 58.7 ± 31.7% on Day 4, n = 4). Thus, we used eosinophils from patients with asthma to investigate mechanisms of HA-induced eosinophil activation and cytokine production in the following experiments.

View larger version (25K): [in this window] [in a new window]   Figure 1.   Effect of HA on eosinophil survival. Freshly isolated peripheral blood eosinophils (1 × 106/ml, purity > 99%) from patients with asthma were cultured with increasing concentrations of low mol wt HA (A), high mol wt HA (B), or IL-5 (1 ng/ml) as a positive control, and eosinophil survival was assessed at Days 2 and 4 by trypan blue dye exclusion. Results are expressed as survival rate (%) and shown as mean ± SEM of six experiments. *P < 0.05.

Mechanism of HA-Induced Eosinophil Survival

To ascertain that prolonged eosinophil survival induced by low mol wt HA is mediated by CD44, a specific receptor for HA, peripheral blood eosinophils were stimulated with 10 µg/ml of low mol wt HA after incubation with antihuman CD44 blocking mAb or control mAb (IgG_2a). As shown in Figure 2, the anti-CD44 mAb treatment partially, but significantly, reduced the HA-induced eosinophil survival ( 50% reduction), when compared with control mAb (rat IgG_2a) treatment. Although the mechanism responsible for this significant but incomplete reduction by the anti-CD44 mAb treatment is not clear, potential reasons for the observed finding include the blocking ability of the mAb and the involvement of other receptors for HA such as ICAM-1 or RHAMM (receptor for hyaluronic acid-mediated motility).

View larger version (19K): [in this window] [in a new window]   Figure 2.   Role of CD44 in HA-induced eosinophil survival. Freshly isolated peripheral blood eosinophils (1 × 106/ml) from patients with asthma were preincubated with 10 µg/ml of anti-CD44 blocking mAb or control mAb (rat IgG2a) for 45 min at 4°C before stimulation with 10 µg/ml of the low mol wt HA. Eosinophil survival was assessed at Day 4 by trypan blue dye exclusion. Results are expressed as percent of survival to HA-induced eosinophil survival rate in the absence of any mAb and shown as mean ± SEM of four experiments. *P < 0.05.

The eosinophil activation and survival in tissue sites are believed to be mediated by T cell-derived cytokines, including IL-3, IL-5, and GM-CSF (25). Recently, we and others have shown that eosinophils themselves can synthesize a wide array of cytokines, including these cytokines (4, 24, 26). Thus, to investigate whether endogenous cyto-kines are involved in the HA-induced eosinophil survival, peripheral blood eosinophils were cultured with 10 µg/ml of low mol wt HA in the presence of 10 µg/ml of neutralizing mAbs for IL-3, IL-5, GM-CSF, or control mAb (mouse IgG₁), and eosinophil survival was examined on Day 4. The survival was significantly reduced by concurrent incubation with a specific anti-GM-CSF mAb ( 70% reduction) but not with either anti-IL-3 mAb or anti-IL-5 mAb (Figure 3). A further reduction was observed by combining anti-IL-3 mAb or anti-IL-5 mAb with anti-GM-CSF mAb or combining all three antibodies; however, there was no significant difference in the reduction between anti-GM-CSF mAb alone and these combining antibodies. To further confirm the involvement of endogenous GM-CSF in the HA-induced eosinophil survival, RT-PCR and ELISA for GM-CSF were performed to detect GM-CSF de novo synthesis and secretion by eosinophils. As shown in Figure 4A, the expected size of the GM-CSF mRNA band was detected 24 h after HA stimulation using specific primers for human GM-CSF, and the increase in GM-CSF mRNA expression was observed in a dose-dependent fashion by densitometric analysis (Figure 4B). In addition, an increase in GM-CSF secretion was observed in the culture supernatants in a dose-dependent fashion 24 h after HA stimulation (Figure 4C). The concentration of GM-CSF (7.0 ± 2.5 pg/ml, mean ± SEM, n = 6) in the supernatants of eosinophils stimulated with 10 µg/ml of HA was small but has been shown to be enough to enhance eosinophil survival to levels between 50 and 75% (24), i.e., similar to those concentrations induced by exposure to 10 µg/ml of HA. Together, these results indicate that the low mol wt HA increases eosinophil survival mainly via induction of GM-CSF release through partial CD44 signaling.

View larger version (54K): [in this window] [in a new window]   Figure 3.   Effect of neutralizing mAb for IL-3, IL-5, or GM-CSF on low mol wt HA-induced eosinophil survival. Freshly isolated peripheral blood eosinophils (1 × 106/ml) were cultured with the low mol wt HA (10 µg/ml) in the presence of neutralizing mAbs (10 µg/ml each) for IL-3 and/or IL-5 and/or GM-CSF, and eosinophil survival was assessed at Day 4. Results are expressed as % survival to HA-induced eosinophil survival rate in the absence of any mAb and shown as mean ± SEM of six experiments. *P < 0.05 compared with control IgG1 (10 µg/ml); **P < 0.01 compared with control IgG1 (20 µg/ml); +P < 0.01 compared with control IgG1 (30 µg/ml).

View larger version (15K): [in this window] [in a new window]   Figure 4.   HA-induced GM-CSF mRNA expression and protein release by peripheral blood eosinophils. (A) Representative RT-PCR analysis of GM-CSF mRNA expression by eosinophils. Peripheral blood eosinophils (1 × 106/ml) from patients with asthma were incubated with increasing concentrations of low mol wt HA or with medium alone for 24 h, and total RNA was extracted from eosinophils. One microgram of total RNA was reverse transcribed and amplified with antisense and sense primers for human GM-CSF or -actin. The result of RT-PCR for -actin is shown to confirm that equal amounts of RNA were used. (B) Densitometric analysis of GM-CSF mRNA expression by eosinophils. Results are expressed as a ratio of GM-CSF mRNA expression to -actin mRNA expression as determined by densitometric analysis using NIHImage software. Results are expressed as mean ± SEM of three experiments. (C) GM-CSF protein release by eosinophils. Eosinophils (1 × 106/ml) were incubated with increasing concentrations of low mol wt HA or with medium alone for 24 h, and GM-CSF protein release was measured by ELISA. Results are expressed as mean ± SEM of six experiments.

HA-Induced Morphologic Changes in Eosinophils

To investigate another eosinophil activation state, we exam-ined eosinophil morphologic changes in response to HA 24 h after stimulation. As shown in Figure 5, low mol wt HA resulted in eosinophil morphologic changes of transforming from round to spindle shape (Figure 5B) and causing homotypic aggregation in a dose-dependent fashion (Figures 5B and 5C). However, eosinophils remained round in shape in the absence of HA 24 h after incubation (Figure 5A). Although the mechanism responsible for eosinophil aggregation is not examined in this study, as in the case of some macrophages and lymphocyte lines, HA may induce their aggregation by cross-linking cell surface CD44 (27).

View larger version (101K): [in this window] [in a new window]   Figure 5.   Morphologic changes in peripheral blood eosinophils by low mol wt HA. Freshly isolated eosinophils were cultured with 1 µg/ml (B) or 10 µg/ml (C) of low mol wt HA or medium alone (A) for 24 h, and photomicrographs were taken (original magnification: ×200).

HA-Induced ICAM-1 and MHC Class II Expression on Eosinophils

It has been previously established that eosinophils studied ex vivo from the sputum of asthmatics express ICAM-1 and HLA-DR, whereas peripheral blood eosinophils do not express these surface proteins (28). To investigate whether HA affects ICAM-1 and MHC class II expressions on the eosinophil surface, flow cytometric analysis was performed 24 h after administration using FITC-conjugated antihuman ICAM-1 mAb or MHC class II mAb. As shown in Figure 6, the low mol wt HA (10 µg/ml) markedly increased the percentage of ICAM-1-positive eosinophils (from 30.5 to 93.5%), whereas MHC class II was highly expressed on eosinophils cultured with medium alone, and only a small increase in MHC class II-positive cells was observed in the presence of HA (from 88.5 to 97.1%). The mechanism of HA-induced ICAM-1 expression on peripheral blood eosinophils is not clarified in this study. However, HA has been shown to induce ICAM-1 expression through a mechanism involving activation of NF-B and activating protein-1 (AP-1) in murine kidney tubular epithelial cells (29). In addition, synergy between eosinophil survival factors (IL-3, IL-5, and GM-CSF) and TNF- was found mainly responsible for ICAM-1 induction on peripheral blood eosinophils (30). According to these results, the HA-induced, endogenous GM-CSF may be involved in the induction of ICAM-1 on peripheral blood eosinophils through activation of NF-B and AP-1.

View larger version (26K): [in this window] [in a new window]   Figure 6.   HA-induced ICAM-1 and MHC class II expressions on peripheral blood eosinophils. ICAM-1 and MHC class II expressions on eosinophils were measured 24 h after stimulation with 10 µg/ml of low mol wt HA or medium alone using FITC-conjugated mAbs for IgG1 (open histograms), ICAM-1 (lower panels, filled histograms), or MHC class II (upper panels, filled histograms).

HA-Induced TGF- Production in Eosinophils

Recently, we and others have shown that eosinophil is a major source of TGF- in the bronchial tissues of patients with asthma by in situ hybridization (4) and immunohistochemistry (5), indicating that eosinophils may play an important role in the development of airway remodeling by stimulating the synthesis of ECM through TGF- production. However, the precise mechanism of TGF- production by eosinophils recruited to the sites of inflammation has not been clearly elucidated. To investigate whether the ECM-eosinophil interaction would regulate TGF- production by eosinophils, peripheral blood eosinophils were stimulated with increasing concentrations of low mol wt HA, and TGF- mRNA expression and protein synthesis were examined by an RT-PCR using specific primers for human TGF-₁ and ELISA. As shown in Figure 7A, the expected size of band was detected at 24 h using specific primers for human TGF-₁, and the TGF- mRNA expression was markedly increased after incubation with low mol wt HA (Figure 7B). In addition, an increased TGF- protein release by eosinophils was observed 24 h after HA stimulation (Figure 8) in a dose-dependent fashion. These data indicate that low mol wt HA-eosinophil interactions may be involved in TGF- production by eosinophils at the site of chronic inflammation.

View larger version (22K): [in this window] [in a new window]   Figure 7.   HA-induced TGF- mRNA expression by peripheral blood eosinophils. (A) Representative RT-PCR analysis of TGF- mRNA expression by eosinophils. Freshly isolated peripheral blood eosinophils (1 × 106/ml) were incubated with increasing concentrations of low mol wt HA or with medium alone for 24 h, and total RNA was extracted from eosinophils. One microgram of total RNA was reverse transcribed and amplified with antisense and sense primers for human TGF-1 or -actin. (B) Densitometric analysis of TGF- mRNA expression. Results are expressed as a ratio of TGF-1 mRNA expression to -actin mRNA expression as determined by densitometric analysis using NIHImage software. Results are expressed as mean ± SEM of three experiments.

View larger version (16K): [in this window] [in a new window]   Figure 8.   HA-induced TGF- protein release by peripheral blood eosinophils. Freshly isolated peripheral blood eosinophils (1 × 106/ml) were incubated with increasing concentrations of low mol wt HA or with medium alone for 24 h, and TGF- release was measured by ELISA. Results are expressed as mean ± SEM of four experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have shown that low mol wt HA, but not high mol wt HA, has a more pronounced effect on eosinophil survival in both patients with asthma and healthy subjects by inducing GM-CSF through partial CD44 signaling. In addition, the low mol wt HA activated eosinophils to change their morphology and to increase ICAM-1 expression, and resulted in an increase of TGF- mRNA expression and protein release by eosinophils. These results are in agreement with previous data in which low mol wt HA (< 5 × 10⁵ D), but not high mol wt HA (> 1 × 10⁶ D), activated the murine alveolar macrophage cell line MH-S or macrophages recruited to sites of inflammation to produce a panel of cytokines (18, 19), although the mechanisms responsible for these different functions of HA based on the size of molecular weight are still unclear. A previous study demonstrated that fluorescein-labeled high mol wt HA binds to macrophages and is displaced by the low mol wt HA, indicating that the two forms can recognize the same receptors (18). One possible explanation for these different functions of HA is that the low mol wt HA may bind more firmly to cells to induce receptor cross-linking than does the high mol wt HA, although this possibility needs further investigation.

It has been shown that eosinophil activation is mediated by mechanisms that involve T cell-derived cytokines in vitro. In particular, IL-4, IL-5, and GM-CSF appear to be critical in eosinophil activation, adhesion molecule expression, and prolongation of survival. Recently, it has been demonstrated that eosinophils themselves can synthesize these cytokines by stimulating their surface receptors with IgA immune complexes or ligands, including adhesion or costimulatory molecules, and be activated by these cytokines in an autocrine fashion (24), suggesting a functional versatility of these cells through interactions with other cells or ECM in vivo. Little is known, however, about the control and regulation of eosinophil activation and of eosinophil-derived cytokine production at the site of inflammation. In this study, we indicate that previously unrecognized interactions between eosinophils and low mol wt HA, whose accumulation has been demonstrated at the site of chronic inflammation (14) and in BALF from patients with persistent asthma (31), activate eosinophils to enhance their survival by inducing GM-CSF and to induce their aggregation and ICAM-1 expression, indicating a possible activation mechanism in eosinophils through ECM-cell modes at sites of chronic inflammation, including asthma, as it has been suggested for macrophages (18).

An important outcome of this study is the increased release of TGF- by eosinophils, which is believed to be crucial in the development of airway fibrosis in asthma, in response to low mol wt HA. Hallmarks of chronic inflammation and tissue fibrosis are the increased synthesis and degradation of components of ECM. As with other ECM components, HA turnover and degradation increase during inflammation, and lower mol wt species of HA accumulate through several mechanisms, including depolymerization by reactive oxygen species and degradation by hyaluronidase. In addition, fibroblasts and smooth muscle cells have been shown to synthesize biologically active low mol wt HA in response to stimulation with cytokines and growth factors (17, 32). Importantly, this accumulation is detected before the influx of inflammatory cells and deposition of collagen (33), suggesting an important role of interaction between low mol wt HA and eosinophil recruited to the site of inflammation for further ECM accumulation and fibrosis in the airway by inducing TGF- production. IL-4 and IL-5 have also been shown to stimulate eosinophils to synthesize TGF- in vitro (34).

The relevance of the interaction between low mol wt HA and eosinophils in allergic inflammation in vivo remains to be determined. However, the fact that low mol wt HA stimulation with macrophages, as we show here, and eosinophils results in the synthesis and release of a number of proinflammatory cytokines and growth factors (4, 24, 26) does support the hypothesis that low mol wt forms of HA generated in the context of the inflammatory milieu may function in eosinophils as important signaling molecules and induce the expression of genes whose functions are critical to the maintenance of allergic inflammatory response and the development of tissue fibrosis. According to the evidences that blockade of CD44-HA interaction by anti-CD44 mAb treatment abrogates tissue edema and leukocyte infiltration in murine arthritis (35) and reduces eosinophil survival in this study, we speculate that interference between eosinophils and low mol wt HA, whether by inhibition or by blockade, may have a profound effect in multiple ECM-cell interactions in the tissue and, hence, in the regulation of allergic inflammatory response and airway fibrosis in asthma.