L.M. Sordillo

Mammary Gland Immunity and Mastitis Susceptibility

Lactation is considered the final phase of the mammalian reproductive cycle, and the mammary gland provides milk for nourishment and disease resistance to the newborn. However, the cellular and soluble immune components associated with mammary tissues and secretion also can play an important role in protecting the gland from infectious diseases, such as mastitis. Mastitis can affect essentially all lactating mammals, but is especially problematic for dairy cattle. The most recent estimates from the National Mastitis Council suggest that mastitis affects one third of all dairy cows and will cost the dairy industry over 2 billion dollars annually in the United States in lost profits (National Mastitis Council (1996) Current Concepts in Bovine Mastitis, National Mastitis Council, Madison, WI). The overall impact of mastitis on the quality and quantity of milk produced for human consumption has provided the impetus to better understand the pathophysiology of the mammary gland and develop ways to enhance disease resistance through immunoregulation. As such, the bovine species has played a critical and prominent role in our current understanding of mammary gland immunobiology. This paper provides a comprehensive overview of mammary gland immunity and how the stage of lactation can impact important host defenses. While this review emphasizes the bovine system, comparisons to humans and other domestic mammals will be addressed as well.

Enhanced production of bovine tumor necrosis factor-α during the periparturient period

This study examined the production of tumor necrosis factor (TNF-(u) by mononuclear cells isolated from peripheral blood and supramammary lymph nodes of periparturient and mid to late lactating dairy cows. Monocyte-enriched cell populations were stimulated with lipopolysaccharide (LPS) and analyzed for TNF-(Y concentrations. Flow cytometric analysis was performed to determine the frequencies of relevant cell populations. Isolated mononuclear cells from periparturient dairy cows produced significantly higher levels of TNF-a than mid to late lactating dairy cows regardless of tissue location. A corresponding increase in the frequency of monocytes also was observed in tissue samples obtained from periparturient animals. The higher proportion of monocytes capable of producing TNF-a: in the periparturient dairy cow may account for the increased levels of this potent mediator. Within the periparturient period, peripheral blood mononuclear cells were found to produce significantly less TNF-(r than cells isolated from mammary lymph nodes. However, flow cytometric analysis revealed similar monocyte concentrations in both the peripheral blood and mammary lymph node. This indicates that the differences in cytokine production may be due to variations in monocyte activation state with respect to tissue location. It is possible that greater potential to produce TNF-(Y during the periparturient period may contribute to the severe acute phase response of the mammary gland to coliform infections during this time. Limiting TNF-(Y production by monocytes, particularly within the mammary gland, may reduce the severity of clinical coliform mastitis in periparturient dairy cattle.

Nuclear factor-κB mediates over-expression of cyclooxygenase-2 during activation of RAW 264.7 macrophages in selenium deficiency

Selenium (Se) is an essential micronutrient for all mammalian species and is associated with a variety of physiological functions, notably immune system, in the form of selenoproteins. Inadequate Se nutrition has been linked to various diseases, including rheumatoid arthritis, cardiomyopathy, and cancer. Important to this discussion is that cyclooxygenase-2 (COX-2) is over-expressed in all the aforesaid pathologies; however, a casual relationship between Se status and COX-2 expression remains to be established. The present study is based on the hypothesis that oxidant stress, a consequence of Se deficiency, lowers the activation potential of the redox-sensitive transcription factor, NF-kappaB, and that the activated NF-kappaB is required for the altered expression of COX-2. To test this hypothesis, we have investigated the relationship between Se status and COX-2 expression in response to LPS stimulation in RAW 264.7, a macrophage-like cell line. In Se-deficient cells, the Se-dependent glutathione peroxidase activity (Se-GPx), a measure of Se status, was markedly reduced and the overall oxidative stress was significantly higher than Se-supplemented cells. Upon lipopolysaccharide (LPS) stimulation, we found 2-3-folds higher COX-2 protein expression as well as higher PGE2 levels in Se-deficient cells than Se-supplemented cells. In comparison, COX-1 protein expression was not affected by either LPS stimulation or Se status. Following LPS stimulation, the nuclear localization of NF-kappaB was significantly increased in Se-deficient macrophages, thereby leading to increased expression of COX-2. This is the first report demonstrating an inverse relationship between Se status and the expression of COX-2.

Diminished Mammary Gland Lymphocyte Functions Parallel Shifts in Trafficking Patterns during the Postpartum Period

Abstract Once activated, lymphocytes can regulate both specific and nonspecific immune responses. Alterations in lymphocyte function may increase the hosts vulnerability to bacterial infections such as mastitis. Susceptibility to mastitis as well as diminished leukocyte functional capabilities have been shown to be influenced by lactational stage. Therefore, the present study characterized the phenotypes and functions of several bovine lymphoid populations at two points in the lactational cycle. Mononuclear cells were isolated from peripheral blood, supramammary lymph nodes, and mammary parenchyma of mid-lactating and postpartum dairy cows. The phenotypic composition, proliferative ability, cytokine secretion, and cytotoxic activity of isolated leukocytes were assessed with respect to lactational stage and tissue source. Lower percentages of T lymphocytes were consistent with diminished mitogen-stimulated proliferation and spontaneous cytotoxic activity by lymphocytes isolated from postpartum compared with mid-lactating animals. Stimulation with interleukin-2 did not enhance the cytotoxic activity or proliferative ability of lymphocytes isolated postpartum to similar levels observed for those isolated from mid-lactating animals. These data indicate that certain diminished lymphocyte functions observed during the postpartum period may result from shifts in leukocyte trafficking patterns.

Bovine CD8+ suppressor lymphocytes alter immune responsiveness during the postpartum period

This study examined the immunoregulatory role of CD8+ lymphocytes during the postpartum period. Peripheral blood cells were isolated from postpartum and mid to late lactating animals. Flow cytometric analysis was performed to determine the frequencies of relevant cell populations. Depletion of CD8+ lymphocytes from whole cultures significantly decreased proliferation and cytotoxic ability of cells isolated from mid to late lactating animals. Enrichment of whole cultures with CD8+ lymphocytes further decreased their proliferative ability but pure CD8+ lymphocyte had increased cytotoxic activity. In contrast, neither depletion nor enrichment of whole cultures with CD8+ lymphocytes altered the already diminished proliferative responses of cells isolated from postpartum cows. No cytotoxic activity was observed by cells isolated from postpartum animals. Cultures from mid to late lactating cows mainly expressed IFN-gamma mRNA where as IL-4 mRNA was mainly expressed by cultures isolated from postpartum animals. Flow cytometric analysis revealed that CD8+ lymphocytes have a high level of activation and expression of the beta-chain during the postpartum compared with the mid to late lactating period. These data indicate that CD8+ lymphocytes are of the suppressor compared to the cytotoxic nature immediately following parturition.

Altered eicosanoid biosynthesis in selenium-deficient endothelial cells

Selenium (Se) is an integral part of the Se-dependent glutathione peroxidase (Se-GSH-Px) catalytic domain. By modulating the cellular levels of fatty acid hydroperoxides, Se-GSH-Px can influence key enzymes of arachidonic acid cascade, in this case cyclooxygenase (COX) and lipoxygenase (LOX). To investigate this phenomenon, the effects of cellular Se status on the enzymatic oxidation of arachidonic acid were investigated in bovine mammary endothelial cells (BMEC), which were cultured in either Se-deficient (-Se) or Se-adequate (+Se) media. When stimulated with calcium ionophore A23187, BMEC produced eicosanoids of both COX and LOX pathways. Compared with the Se-adequate cells, the production of prostaglandin I(2) (PGI(2)), prostaglandin F(2) (PGF(2alpha)), and prostaglandin E(2) (PGE(2)) was significantly decreased in Se-deficient cells, whereas the production of thromboxane A(2) (TXA(2)) was markedly increased in the -Se BMEC cultures. Although the enzymatic oxidation of arachidonic acid by the LOX pathway was found to be relatively less than by the COX pathway, the BMEC cultured in -Se media produced significantly more 15-hydroperoxyeicosatetraenoic acid (15-HPETE) than the +Se cells produced. Based on these results, we postulate that cellular Se status plays an important regulatory role in the enzymatic oxidation of arachidonic acid by the COX and LOX pathways. The altered eicosanoid biosynthesis, especially the overproduction of 15-HPETE, in -Se BMEC may be one of the underlying biochemical phenomena responsible for vascular dysfunction during Se deficiency.

Increased neutrophil adherence and adhesion molecule mRNA expression in endothelial cells during selenium deficiency.

Leukocyte aggregation and activation on endothelial cells (EC) are important preliminary events in leukocyte migration into tissue and subsequent inflammation. Thus, an increase in leukocyte adherence has the potential to affect inflammatory disease outcome. Selenium (Se) is an integral part of the antioxidant enzyme glutathione peroxidase (GSH‐Px) and plays an important role in the maintenance of the redox state of a cell. Se supplementation in the bovine has been shown to improve the outcome of acute mastitis caused by coliform bacteria, in part by enhancing the speed of neutrophil migration into the affected mammary gland. However, the mechanisms by which Se modulates neutrophil migration have not been elucidated. Therefore, an in vitro model of Se deficiency in primary bovine mammary artery EC was used to examine the impact of Se status on the adhesive properties of EC. The effect of Se on functional activities was examined by measuring neutrophil adherence to Se‐deficient and Se‐supplemented EC. Se‐deficient EC showed significantly enhanced neutrophil adherence when stimulated with tumor necrosis factor α (TNF‐α) for 4 or 24 h, interleukin‐1 for 12 h, or H2O2 for 20 min (P < 0.05). To determine the mechanisms underlying these changes in neutrophil adherence, the expression of EC adhesion molecules, ICAM‐1, E‐selectin, and P‐selectin were examined at the molecular level by a competitive reverse transcription‐polymerase chain reaction. Results revealed higher mRNA expression for E‐selectin and ICAM‐1 in Se‐deficient EC stimulated with TNF‐α for 3 and 6 h, and greater expression of P‐selectin mRNA in Se‐supplemented EC with 3‐h TNF‐α stimulation. These studies provide new information to establish the role of Se nutrition in the initiation of leukocyte adherence to endothelium. J. Leukoc. Biol. 65: 658–664; 1999.

Increased 15-HPETE production decreases prostacyclin synthase activity during oxidant stress in aortic endothelial cells

Selenium (Se) is an integral component of glutathione peroxidase and is able to detoxify peroxides that can affect arachidonic acid (AA) metabolism, thereby influencing eicosanoid biosynthesis. This study investigated the effects of oxidant stress, a consequence of Se deficiency, on eicosanoid formation and important key enzyme expression in bovine aortic endothelial cells (BAEC). Bovine aortic endothelial cells cultured in Se-deficient media and stimulated with tumor necrosis factor alpha or H2O2 produced significantly less prostacyclin (PGI(2)) and more 15-hydroxyeicosatetraenoic acid, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and thromboxane than Se-supplemented BAEC. Additionally, reverse transcription polymerase chain reaction and immunoblotting determined that the mRNA and protein levels of the eicosanoid forming enzymes cyclooxygenase-1 (COX1), cyclooxygenase-2 (COX2), and PGI synthase were not significantly changed. The addition of 15-HPETE to Se-supplemented BAEC inhibited the production of PGI(2) suggesting that the accumulation of lipid hydroperoxides during Se-deficiency may be the underlying factor in the altered eicosanoid production during Se deficiency. Furthermore, inhibition of COX and addition of PGH(2) to Se-deficient or Se-supplemented BAEC still resulted in lower PGI(2) formation by Se-deficient cells. Together, these results suggest that Se deficiency modifies eicosanoid production by affecting the activity of key enzymes, particularly PGI synthase, rather than their transcription or translation.

ALTERED INTERLEUKIN-2 PRODUCTION BY LYMPHOCYTE POPULATIONS FROM BOVINE LEUKEMIA VIRUS-INFECTED CATTLE

Abstract The effects of bovine leukosis virus (BLV) on the phenotypic and functional characteristics of peripheral blood mononuclear cells were investigated. Whole blood differentials showed that persistently lymphocytotic (BLV+PL) dairy cattle had more lymphocytes and fewer neutrophils than the aleukemic seropositive (BLV+AL) or seronegative (BLV–) animals. Flow cytometric analyses of peripheral blood mononuclear cells indicated that the BLV+PL animals had more B lymphocytes, with a concomitant decrease in CD2 positive cells when compared with the BLV– group. Mononuclear cells from the BLV+AL animals also had fewer CD2 positive cells, but no differences in B lymphocytes were observed when compared with BLV– cattle. Peripheral blood mononuclear cells were used in blastogenesis assays to assess the functional ability of lymphocytes. Lymphocytes from BLV+PL animals had lower proliferative responses to concanavalin A and pokeweed mitogen when compared with cells from the BLV– or BLV+AL groups. The level of spontaneous blastogenesis in the absence of mitogenic stimulation was high for lymphocytes obtained from BLV+AL cattle. Cultures of lymphocytes obtained from BLV+PL animals produced greater amounts of interleukin-2 (IL-2) than BLV+AL and BLV– groups, although no differences were observed in the expression of IL-2 receptors. The development of uncontrolled lymphocytosis in BLV-infected cattle may result from an altered responsiveness to IL-2-regulated B-lymphocyte proliferation.

Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization.

A 25 kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na(2)CO(3), indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73-83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3 Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243-248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87-101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H(2)O(2). Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.