CD44-HA Regulate in vivo iNOs Expression and Metalloproteinases Activity in a murine model of Arthitris-Like Inflammation

CABRERA PAULA; BLANCO GUILLERMO; GRECZANIK SOFÍA; ALVAREZ ELIDA; HAJOS SILVIA

INFLAMMATION RESEARCH

Birkhäuser Verlag, Basel

Lugar: USA; Año: 2004 vol. 53 p. 1 - 11

1023-3830

Objective: To evaluate the effects of anti-CD44 IM7.8.1 antibody, HMW-HA and LMW-HA on leukocyte migration and adhesion, and the induction of proinflammatory mediators, in mouse air-pouch inflammation induced by zymosan.To evaluate the effects of anti-CD44 IM7.8.1 antibody, HMW-HA and LMW-HA on leukocyte migration and adhesion, and the induction of proinflammatory mediators, in mouse air-pouch inflammation induced by zymosan. Methods: Leukocytes were obtained from zymosan-air pouches after the intra-pouch injection of anti-CD44 IM7.8.1, isotype control, HMW-HA, LMW-HA or PBS. TNF-a, IL-1b and iNOS mRNA were estimated in leukocytes by semi-quantitative RT-PCR. Matrix metalloproteinases (MMPs) from exudates were evaluated by zymography and Western Blot. Adhesion and migration of leukocytes were evaluated in HA-coated plates and Boyden chambers respectively.Leukocytes were obtained from zymosan-air pouches after the intra-pouch injection of anti-CD44 IM7.8.1, isotype control, HMW-HA, LMW-HA or PBS. TNF-a, IL-1b and iNOS mRNA were estimated in leukocytes by semi-quantitative RT-PCR. Matrix metalloproteinases (MMPs) from exudates were evaluated by zymography and Western Blot. Adhesion and migration of leukocytes were evaluated in HA-coated plates and Boyden chambers respectively.a, IL-1b and iNOS mRNA were estimated in leukocytes by semi-quantitative RT-PCR. Matrix metalloproteinases (MMPs) from exudates were evaluated by zymography and Western Blot. Adhesion and migration of leukocytes were evaluated in HA-coated plates and Boyden chambers respectively. Results: IM7.8.1 decreased iNOS mRNA levels and the activity of both MMP-9 and MMP-2 eight h after injection into zymosan air pouch while IM7.8.1, HMW-HA and LMW-HA had no effect on IL1-b or TNF-a mRNA levels. Leukocytes from air pouch adhered to and migrated in vitro against both HMW-HA and LMW-HA. LMW-HA increased the number of leukocytes in the air pouch and iNOS mRNA levels as compared to PBS injection. In contrast, HMW-HA decreased leukocyte count and reduced iNOS mRNA levels. Paradoxically, the activity of both MMP-9 and MMP-2 was increased by HMW-HA and decreased by LMW-HA.IM7.8.1 decreased iNOS mRNA levels and the activity of both MMP-9 and MMP-2 eight h after injection into zymosan air pouch while IM7.8.1, HMW-HA and LMW-HA had no effect on IL1-b or TNF-a mRNA levels. Leukocytes from air pouch adhered to and migrated in vitro against both HMW-HA and LMW-HA. LMW-HA increased the number of leukocytes in the air pouch and iNOS mRNA levels as compared to PBS injection. In contrast, HMW-HA decreased leukocyte count and reduced iNOS mRNA levels. Paradoxically, the activity of both MMP-9 and MMP-2 was increased by HMW-HA and decreased by LMW-HA.b or TNF-a mRNA levels. Leukocytes from air pouch adhered to and migrated in vitro against both HMW-HA and LMW-HA. LMW-HA increased the number of leukocytes in the air pouch and iNOS mRNA levels as compared to PBS injection. In contrast, HMW-HA decreased leukocyte count and reduced iNOS mRNA levels. Paradoxically, the activity of both MMP-9 and MMP-2 was increased by HMW-HA and decreased by LMW-HA. Conclusions: Both CD44 and HA can modulate leukocyte migration and induction of proinflammatory mediators in mouse zymosan air pouch inflammation. IM7.8.1 had consistent anti-inflammatory effects, reducing iNOS, MMP-9 and MMP-2. HMW-HA and LMW-HA were able to modulate both the induction of proinflammatory mediators and leukocyte count in the air pouch.Both CD44 and HA can modulate leukocyte migration and induction of proinflammatory mediators in mouse zymosan air pouch inflammation. IM7.8.1 had consistent anti-inflammatory effects, reducing iNOS, MMP-9 and MMP-2. HMW-HA and LMW-HA were able to modulate both the induction of proinflammatory mediators and leukocyte count in the air pouch. Key words: CD44 – Hyaluronic acid – Air-pouch – iNOS – MetalloproteinasesCD44 – Hyaluronic acid – Air-pouch – iNOS – Metalloproteinases Inflamm. res. 53 (2004) 556–566 1023-3830/04/100556-11 DOI 10.1007/s00011-004-1295-8 Introduction Adhesive interactions between leukocytes and components of the extracellular matrix constitute an important signaling source to regulate the course of the inflammatory response. CD44 is a multifunctional adhesion molecule, that has been shown to participate in diverse functions including lymphocyte maturation, homing and activation, hematopoesis, embryogenesis, apoptosis as well as primary adhesion during leukocyte migration to sites of inflammation [1–3]. The principal ligand of CD44 is hyaluronic acid (HA) [4–5], a high molecular weight glycosaminoglycan, that is a major component of the extracellular matrix, and is naturally present in cartilage and synovial fluid. High levels of HA are found at sites of inflammation, not only as native high molecular weight HA (HMW-HA), but also as low molecular weight fragments (LMW-HA) that can be generated from native HA by degradative enzymes, or that may result from de novo synthesis [6–7]. HA degradation has been demonstrated in inflammatory diseases such as rheumatoid arthritis and pulmonary fibrosis. Various studies have reported that HA can activate cell motility of tumor cell lines of epithelial and leukocyte origin [8–10]. This property may contribute to tissue invasion and metastasis of tumor cells as well as leukocyte infiltration during inflammation. Several proinflammatory events may be regulated by CD44 upon interaction with HA [11]. It has been demonstrated that HA fragments induce proinflammatory mediators in vitro, including cytokines IL-1b and TNF-a [12], members of the CXC and CC family of chemokines [13–14], matrix metalloproteinases (MMPs) [15] and inducible nitric oxide synthase (iNOS) [16]. HA fragments were able to induce in vitro iNOS production in Kupffer cells, endothelial cells and alveolar macrophage cell lines [17]. In contrast, some beneficial anti-inflammatory effects have been described for HMW-HA, which has been used as a viscous lubricant in patients with osteoarthritis [18]. Moreover, the ability of HMW-HA to reduce the production of nitric oxide has been demonstrated in experimental osteoarthritis in rabbits [19]. The induction of MMPs is a relevant proinflammatory event in the pathogenesis of rheumatic diseases [20]. MMPsb and TNF-a [12], members of the CXC and CC family of chemokines [13–14], matrix metalloproteinases (MMPs) [15] and inducible nitric oxide synthase (iNOS) [16]. HA fragments were able to induce in vitro iNOS production in Kupffer cells, endothelial cells and alveolar macrophage cell lines [17]. In contrast, some beneficial anti-inflammatory effects have been described for HMW-HA, which has been used as a viscous lubricant in patients with osteoarthritis [18]. Moreover, the ability of HMW-HA to reduce the production of nitric oxide has been demonstrated in experimental osteoarthritis in rabbits [19]. The induction of MMPs is a relevant proinflammatory event in the pathogenesis of rheumatic diseases [20]. MMPs © Birkhäuser Verlag, Basel, 2004 Inflammation Research CD44 and hyaluronic acid regulate in vivo iNOS expression and metalloproteinase activity in murine air-pouch inflammation P.V. Cabrera*, G. Blanco*, L. Alaniz, S. Greczanik, M. Garcia, E. Alvarez and S. E. Hajos Cátedra de Inmunología-IDEHU, Facultad de Farmacia y Bioquímica, Universidad Nacional de Buenos Aires (UBA) – CONICET, Junín 956, piso 4, 1113 Buenos Aires, Argentina, Fax: ++54 11 4964 0024, e-mail: gblanco@ffyb.uba.ar Received 8 December 2003; returned for revision 29 January 2004; accepted by M. J. Parnham 18 May 2004 Correspondence to: G. BlancoG. Blanco