Increase in apoptotic polymorphonuclear neutrophils in peripheral blood after intramammary infusion of Escherichia coli lipopolysaccharide

Abstract

A transient increase in apoptotic polymorphonuclear neutrophils (PMNs) as revealed by the terminal deoxynucleotidyl, transferase-mediated dUTP nick end labeling (TUNEL) technique in bovine jugular and milk vein blood was observed 4 h after intramammary infusion of Escherichia coli lipopolysaccharide (LPS) (jugular vein; before infusion 10.1%, 4 h 58.3%: milk vein; before infusion 13.2%, 4 h 76.6%) decrease in PMA-induced oxidative bursts of PMNs was also observed during the same period and continued until 8 h after the infusion. TUNEL-positive cells showed an intention of a Comet tail as detected by a single-cell gel electrophoresis assay (Comet assay) and the morphological apoptotic future, though DNA fragmentation was not clearly detected. A definite decrease in peripheral PMNs and a marked increase in PMNs in the LPS-infused teat cistern were observed during the same period. The migration of milk vein blood-derived PMN and the expression of adhesion receptors (L-selectin and CD18) on PMN were suppressed, accompanied by an increase in apoptotic cells. TUNEL-positive PMN observed in normal animals showed a reduced migration capacity. The increase in apoptotic PMNs observed in the LPS-infused cattle was thought to be due to the remaining intravenous spontaneous apoptotic cells existing under the normal condition (the aging cell), and this increase appeared to lower the expression of adhesion receptors and the migration capacity. Decreased PMA-induced oxidative burst activity in PMN was thought to be derived from these aging cells and immature band cells appearing in the circulation as a subsequent event of leukopenia and/or severe stress associated with mastitis. The results from the present study indicate the possibility that the function of PMN in the circulation at early stages of bovine mastitis is regulated by the kinetics of PMN aging.

Introduction

Mastitis is the most costly disease in dairy cattle and is usually caused by bacterial infections that damage the udder tissues, thus initiating an inflammatory response. Polymorphonuclear neutrophils (PMNs) are known to be the major defense against bacterial infection of the mammary gland (Schalm et al., 1976). PMN exhibit directed migration from the blood across the mammary epithelium into milk in response to chemotactic stimuli produced by invading organisms (Paape et al., 1981). Regulation of PMN migration from the circulation to an inflamed udder gland is of particular importance with regard to the further development of fundamental methods for the treatment of mastitis. Therefore, numerous studies have been carried out to more precisely identify the functions of PMN in the circulation and the inflamed udder. Leukopenia (predominantly neutropenia, Heyneman et al., 1990, Monfardini et al., 1999, Smits et al., 1998, Saad and Ostensson, 1990) and impaired function in blood neutrophils, a decrease in reactive oxygen species (ROS; Heyneman et al., 1990), the dysfunction of diapedesis (Smits et al., 1998), and of the chemotaxic response (Kremer et al., 1993), are commonly observed during the early stages of acute mastitis. The reasons for these phenomena are not fully understood, although some authors have suggested that the immature band PMN appearing during this period as a reaction to leukopenia appears to be due to these impaired functions (Burvenich et al., 1994, Heyneman et al., 1990).

Mature PMN have the shortest life span among leukocytes and die rapidly via apoptosis both in vitro and in vivo (Whyte et al., 1993, Lee et al., 1993). In healthy humans, mature PMN have a half-life of 6–7 h and undergo spontaneous apoptosis within 24 h (Cartwright et al., 1964, Fliedner et al., 1964). Apoptotic PMN can be recognized and ingested by macrophages to be removed from the circulation (Savill et al., 1989). Whyte et al. (1993) have demonstrated a direct relationship between apoptosis and a loss of cytoskeletal function, phagocytosis, degranulation, and respiratory bursts by using counterflow centrifugation of aged PMN. These spontaneous apoptotic PMN are thought to be aged PMN (Fülöp et al., 1997). PMN aging, i.e., the kinetics of aging PMN, is therefore important in determining the function of PMN in bovine mastitis, although little is known regarding this topic. Therefore, in the present study, we investigated the relationships between the functions of PMN and immature and apoptotic PMN in the circulation in the early stages of mastitis introduced by intramammary infusion of E. coli lipopolysaccharide (LPS).