Expression of macrophage CD44 receptor in the course of experimental inflammatory response of bovine mammary gland induced by lipopolysaccharide and muramyl dipeptide

Abstract

The aim of this study was to investigate development over time of the surface expression of CD44 on macrophages during an inflammatory response of bovine mammary gland. Intramammary instillation of muramyl dipeptide (MDP) and lipopolysaccharide (LPS) resulted in a significant increase in the total count of CD44+ non-vacuolised macrophages (_NMAC) after 24 h. During resolution of the inflammatory response, there was observed a gradual decrease in the total count CD44+ _NMAC. The lower total count and proportion of CD44 + vacuolised macrophages (_VMAC) was observed as the effect of MDP and LPS at 24 h after induction (P < 0.01). During resolution, the total count and proportion of CD44 + _VMAC increased. We have demonstrated CD44 receptor is expressed during the inflammatory response caused by LPS and MDP and the effect of these components on CD44 expression was particularly evident during initiation of the inflammatory response. High expression of CD44 in resolution of inflammatory response may relate to macrophages´ involvement in the processes leading to restitution of injured tissues.

Introduction

The initial host response to invading pathogenic bacteria includes acute neutrophil inflammatory influx, followed by the recruitment of macrophages. Migration of these cells from blood to the site of inflammation is regulated by adhesion molecules. One of these is CD44, as it been observed using murine models that the recruitment of macrophages and neutrophils into the site of inflammation is CD44 dependent (Alstergren et al., 2004, Hollingsworth et al., 2007).

CD44 is a major cells-surface type I transmembrane receptor for hyaluronan (HA) and is expressed by most cell types: leukocytes, fibroblasts, neurons, and myeloid cells. Thus, CD44 is a broadly distributed receptor that has been implicated in a number of immunological phenomena, such as lymphocyte homing, cell activation, hemopoiesis, and growth factor presentation, as well as in such disease processes as arthritis, allergies, and tumor metastasis (Naor et al., 1997, Pure and Cuff, 2001, Siegelman et al., 1999).

As a cell surface receptor, CD44 is used for extracellular matrix molecules, and it is involved in the binding and metabolism of HA (Vivers et al., 2002). HA assumes a significant role in maintaining tissue integrity as a ubiquitous element of the extracellular matrix. Ligation of CD44 with short fragments of HA can induce a number of pro-inflammatory cytokines (McKee et al., 1996). Therefore, the engagement of CD44 on immune cells with HA is a key event in inflammatory responses (Pure and Cuff, 2001) and inflammation is associated with increased expression of surface CD44 on hematopoietic cells (Cichy and Pure, 2003).

Studies in models of lung injury suggest that CD44 can modify not only recruitment of inflammatory cells, but it plays an important role also during termination of inflammatory responses (Teder et al., 2002, Wang et al., 2002). The termination of inflammation is an important event for successful repair after tissue injury, and it requires resolution of the inflammatory response and removal of extracellular matrix breakdown products and apoptotic neutrophils (Savill and Haslett, 1995).

The role of CD44 on macrophages in relation to resolution of inflammatory response was studied in vitro (Hart et al., 1997, Liang et al., 2007, Vachon et al., 2006), in vivo (Liang et al., 2007, Hollingsworth et al., 2007), and also in situ (Teder et al., 2002). These studies suggest that membrane CD44 is particularly involved in resolution as a tool for clearing of HA. Since fragmented HA accumulates during tissue injury and CD44 is required to clear HA, and impaired clearance of HA, due to insufficient presence of CD44, results in unremitting inflammation (Jiang et al., 2006). Moreover, it has been observed that CD44 efficiently mediates recognition and phagocytosis of neutrophils (Hart et al., 1997, Vivers et al., 2004) and that it is a competent phagocytic receptor of macrophages (Vachon et al., 2006).

The critical role of CD44 in resolution was demonstrated particularly in lung inflammation with a model of bleomycin-induced lung injury (Teder et al., 2002) and in Escherichia coli (E. coli) and Streptococcus pneumoniae (S. pneumoniae) pneumonia in mice (Wang et al., 2002). CD44-deficient mice succumbed to unremitting inflammation following lung injury, characterized by impaired clearance of apoptotic neutrophils and persistent accumulation of HA fragments at the site of tissue injury (Teder et al., 2002). Moreover, CD44 deficiency results in enhanced inflammation in E. coli but not in S. pneumoniae-induced pneumonia (Wang et al., 2002). This suggests a previously unrecognized role for CD44 in limiting the inflammatory response to E. coli and/or Gram-negative bacterial pathogens. This confirmed the fact that lipopolysaccharide (LPS) may modulate CD44-mediated biological effects in monocytes during inflammation (Levesque and Haynes, 1997).

The critical role of CD44 on macrophages in inflammation is supported by studies using anti-CD44 antibodies and CD44-deficient mice, but the regulatory mechanism for CD44 expression on macrophages during inflammatory response in situ is not completely known. Moreover, it is not known if the expression of CD44 on macrophages is modulated by the stage of inflammatory response and/or by various bacterial pathogens.

Therefore, the study was undertaken to investigate the time course of the surface expression of CD44 on macrophages during an inflammatory response. For this purpose we used a modified procedure of Wardley et al. (1976) with intramammary instillation of LPS and muramyl dipeptide (MDP) as a model to discriminate between effects of Gram-negative and Gram-positive bacteria infections. LPS is believed to be the most important marker for Gram-negative bacteria and imitates E. coli infections of the mammary gland (Mehrzad et al., 2001, Paape et al., 1996). On the other hand, peptidoglycan by-product MDP is a key element of the immune response to Gram-positive bacteria, because it serves as a salient stimulus from these bacteria (Dinarello et al., 1978).