David H. Townson

Expression of Monocyte Chemoattractant Protein-1 and Distribution of Immune Cell Populations in the Bovine Corpus Luteum Throughout the Estrous Cycle

Abstract This study characterizes the expression of monocyte chemoattractant protein-1 (MCP-1) and the relative distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle. Immunodetectable MCP-1 was evident in corpora lutea of cows at Days 6, 12, and 18 postovulation (Day 0 = ovulation, n = 4 cows/stage). Day 6 corpora lutea contained minimal MCP-1 that was confined primarily to blood vessels. In contrast, relatively intense staining for MCP-1 was observed in corpora lutea from Days 12 and 18 postovulation. MCP-1 was again most evident in the cells of the vasculature, but it was also observed surrounding individual luteal cells, particularly by Day 18. An increase in immunohistochemical expression of MCP-1 on Days 12 and 18 postovulation corresponded with increases in MCP-1 mRNA and protein in corpora lutea as determined by Northern blot analysis and ELISA. Monocytes and macrophages were the most abundant immune cells detected in the bovine corpus luteum, followed by CD8+ and CD4+ T lymphocytes. In all instances, Day 6 corpora lutea contained fewer immune cells than corpora lutea from Days 12 and 18. In conclusion, increased expression of MCP-1 was accompanied by the accumulation of immune cells in the corpora lutea of cows during the latter half of the estrous cycle (Days 12–18 postovulation). These results support the hypothesis that MCP-1 promotes immune cell recruitment into the corpus luteum to facilitate luteal regression. These results also raise a provocative issue, however, concerning the recruitment of immune cells several days in advance of the onset of luteal regression.

Chemokines in the corpus luteum: Implications of leukocyte chemotaxis

Chemokines are small molecular weight peptides responsible for adhesion, activation, and recruitment of leukocytes into tissues. Leukocytes are thought to influence follicular atresia, ovulation, and luteal function. Many studies in recent years have focused attention on the characterization of leukocyte populations within the ovary, the importance of leukocyte-ovarian cell interactions, and more recently, the mechanisms of ovarian leukocyte recruitment. Information about the role of chemokines and leukocyte trafficking (chemotaxis) during ovarian function is important to understanding paracrine-autocrine relationships shared between reproductive and immune systems. Recent advances regarding chemokine expression and leukocyte accumulation within the ovulatory follicle and the corpus luteum are the subject of this mini-review.

Cooperative Expression of Monocyte Chemoattractant Protein 1 Within the Bovine Corpus Luteum: Evidence of Immune Cell-Endothelial Cell Interactions in a Coculture System

Abstract Endothelial cells (EC) of the bovine corpus luteum (CL) are a known source of proinflammatory mediators, including monocyte chemoattractant protein 1 (CCL2) and endothelin 1 (EDN1). Here, a coculture system was devised to determine if immune cells and PGF2α together affect CCL2 and EDN1 secretion by EC. Luteal EC were cultured either alone or together with peripheral blood mononuclear cells (PBMC), and treated without or with PGF2α for 48 h (n = 6 experiments). Coculture of EC with PBMC increased CCL2 secretion an average of 5-fold higher compared with either cell type alone (P < 0.05). Basal secretion of EDN1 by EC was substantial (∼2 ng/ml), but was not affected by coculture with PBMC (P > 0.05). EC cocultured with concanavalin A-activated PBMC (ActPBMC) increased CCL2 secretion an average of 12-fold higher compared with controls (P < 0.05), but again, EDN1 secretion was unchanged (P > 0.05). Interestingly, PGF2α did not alter either CCL2 or EDN1 secretion, regardless of culture conditions (P > 0.05). In a second series of experiments (n = 3 experiments), mixed luteal cells (MLC) were cultured alone or with PBMC as described above. Secretion of CCL2 and EDN1 was not affected by coculture or by PGF2α (P > 0.05), but MLC produced less progesterone in the presence of ActPBMC (P < 0.05). Collectively, these results suggest that immune cells and EC can interact cooperatively to increase CCL2 secretion in the CL, but this interaction does not affect EDN1 secretion nor is it influenced by PGF2α. Additionally, activated immune cells appear to produce a factor that impairs progesterone production by luteal steroidogenic cells.

Prolactin-Induced Expression of Intercellular Adhesion Molecule-1 and the Accumulation of Monocytes/Macrophages During Regression of the Rat Corpus Luteum

Abstract Intercellular adhesion molecule-1 (ICAM-1) is thought to facilitate the recruitment and migration of monocytes/macrophages to sites of inflammation. Here we investigated whether the luteolytic effect of prolactin in the hypophysectomized rat is associated with the expression of ICAM-1. In addition, we examined the effect of exogenous testosterone (or its potential conversion to estradiol endogenously) on the corpus luteum to address recent speculation that ovarian steroids might augment luteal regression. Immature, 30-day-old rats were ovulated with eCG and hCG and then hypophysectomized; this resulted in a single cohort of persistent corpora lutea. The rats were assigned randomly into four treatment groups: vehicle treatment without or with testosterone (VEH-T4, VEH+T4) and prolactin treatment without or with testosterone (PRL−T4, PRL+T4). Corpora lutea of control rats exhibited minimal ICAM-1 staining and contained relatively few monocytes/macrophages. In contrast, corpora lutea of prolactin-treated rats exhibited prominent ICAM-1 staining and contained numerous monocytes/macrophages. Testosterone did not overtly affect ICAM-1 staining, numbers of monocytes/macrophages, or concentrations of plasma progestins (progesterone and 20α-dihydroprogesterone) in either VEH or prolactin treatment groups; notwithstanding, luteal weights increased significantly in response to testosterone in VEH+T4 rats compared to VEH-T4 rats and prolactin-treated rats. We conclude that ICAM-1 expression and monocyte/macrophage accumulation are associated with prolactin-induced luteal regression in the rat and that these aspects are not influenced by testosterone.

Short communication: Rapid antibiotic screening tests detect antibiotic residues in powdered milk products

Rapid antibiotic screening tests are widely used in the dairy industry to monitor milk for the presence of antibiotic residues above regulated levels. Given the persistent concern over contamination of milk products with antibiotic residues, we investigated the utility of IDEXX Snap test devices (IDEXX Laboratories Inc., Westbrook, ME) as tools for detecting antibiotic residues in powdered milk products. Five powdered milk products were reconstituted according to manufacturer specification with distilled water: Carnation (Nestlé USA Inc., Solon, OH), Nido youth and Nido adult (Nestlé Mexico Inc., Mexico City, Mexico), ELK (Campina, Eindhoven, the Netherlands), and Regilait (Saint-Martin-Belle-Roche, France). Positive samples were generated by spiking reconstituted milk with penicillin G, cephapirin, or tetracycline to either the European Union-regulated maximum residue limit or the FDA-regulated safe/tolerance level, whichever was lower. Control, unspiked negative milk samples and positive samples were tested with appropriate IDEXX Snap test kits (penicillin G and cephapirin with New Beta-Lactam, tetracycline with New Tetracycline). All samples yielded definitive results consistent with expectations, and there were no instances of false-positive or false-negative readings. These results suggest that both the New Beta-Lactam and New Tetracycline IDEXX Snap test kits effectively detect antibiotic residues in commercially available powdered milk samples and are useful tools for monitoring antibiotic residues in reconstituted powdered milk products.

Cytokeratin 18 expression inhibits cytokine-induced death of cervical cancer cells.

Objectives: In cervical cancer, increased cytokeratin 18 (CK18) filament expression is associated with disease progression. However, it may also provide resistance to cytokine-induced apoptosis. The present study tested whether CK18 expression influences susceptibility to cytokine-induced apoptosis. Methods: The cervical cancer cell lines C-4II (high CK18 expression), ME-180 (low CK18 expression), and 2 subtypes of HeLa cells containing or lacking CK18 expression (CK18+ and CK18− cells, respectively) were exposed to vehicle (control), Fas ligand (FasL) (50 ng/mL), or tumor necrosis factor &agr; (TNF-&agr;; 10 ng/mL) without/with cycloheximide (CHX; 2.5 μg/mL) to test the hypothesis that diminished CK18 expression increases susceptibility to cytokine-induced apoptosis. Results: Flow cytometric analysis of cell death via TUNEL staining revealed that cytokine-induced apoptosis was 2-fold greater in ME-180 cells than C-4II cells in response to FasL+CHX or TNF-&agr;+CHX (P < 0.05). Similarly, there was a higher incidence of FasL-induced apoptosis in CK18− HeLa cells (23% and 91% apoptotic for FasL and FasL+CHX, respectively) than CK18+ HeLa cells (1% and 11%, respectively; P < 0.05). Surprisingly, TNF-&agr; had no effect on either CK18+ or CK18− HeLa cells (P > 0.05). Caspase 3 activity was greater in CK18− HeLa cells than in CK18+ HeLa cells at 8 and 18 hours after FasL treatment (P < 0.05), an effect abrogated by the caspase 8 inhibitor IETD-fmk (P < 0.05). Conclusions: Cervical cancer cells with diminished CK18 expression are more susceptible to cytokine-induced apoptosis, particularly in response to FasL treatment. These observations suggest that relative CK18 expression is an important factor when considering therapeutic strategies to enhance immune cell-mediated death of cervical cancer cells.

Immune cell–endothelial cell interactions in the bovine corpus luteum

Early embryonic mortality accounts for a substantial portion of reproductive failure in agriculturally important livestock, including the dairy cow. The maintenance of early pregnancy requires a fully functional corpus luteum (CL) that is not susceptible to regression following fertilization, yet the cellular mechanisms of luteal regression are not clearly understood. Immune-cell accumulation within the CL at the time of regression is a well-documented phenomenon in a variety of species. In the dairy cow, immune-cell accumulation precedes luteal regression by several days and coincides with an increase in expression of the chemokine monocyte chemoattractant protein 1 (CCL2), suggesting that immune-mediated events promote tissue destruction. Recent studies indicate that endothelial cells comprising the CL are a primary source of CCL2 secretion. Moreover, although uterine-derived prostaglandin F(2α) (PGF) initiates luteal regression in the cow, PGF does not directly provoke CCL2 secretion by luteal endothelial cells. Instead, PGF-induced luteal regression is thought to require cooperative interaction among immune cells, endothelial cells, and steroidogenic cells of the CL to further promote CCL2 secretion, enhance immune-cell recruitment, and eliminate luteal tissue. This brief review focuses on putative interactions between immune cells and endothelial cells derived from the bovine CL that result in enhanced CCL2 expression and the elaboration of other inflammatory mediators (for example, cytokines), which perpetuate luteal regression. Fundamental knowledge of immune-endocrine interactions within the reproductive system of cows has relevance to other CL-bearing mammals, including humans and endangered animals, particularly in the development of methods to control and/or improve fertility. Thus, it is a timely topic for this symposium concerning ecological immunology and public health.

Actions of Prostaglandin F2α and Prolactin on Intercellular Adhesion Molecule-1 Expression and Monocyte/Macrophage Accumulation in the Rat Corpus Luteum

Abstract Expression of intercellular adhesion molecule-1 (ICAM-1) and the accumulation of monocytes/macrophages are inflammatory events that occur during PRL (PRL)-induced regression of the rat corpus luteum. Here we have compared the ability of prostaglandin F2α (PGF) and PRL to induce, in rat corpora lutea, inflammatory events thought to perpetuate luteal regression. Immature rats were ovulated with eCG-hCG and then hypophysectomized (Day 0), which resulted in a single cohort of persistent, functional corpora lutea. On Days 9–11, the rats received twice daily injections of saline, PGF (Lutalyse, 250 μg/injection), or PRL (312 μg/injection) to induce luteal regression. Surprisingly, luteal weight and plasma progestin concentrations (progesterone and 20α-dihydroprogesterone) did not differ between PGF-treated rats and controls; whereas both luteal weight and plasma progestins declined significantly in PRL-treated rats. Furthermore, corpora lutea of PGF-treated rats and controls contained relatively minimal ICAM-1 staining and few monocytes/macrophages. In contrast, but as expected, corpora lutea of PRL-treated rats stained intensely for ICAM-1 and contained numerous monocytes/macrophages. In an additional experiment, there was no indication that luteal prostaglandin F2α receptor mRNA diminished as a result of hypophysectomy. These findings suggest that prolactin, not PGF, induces the inflammatory events that accompany regression of the rat corpus luteum.

Assessing the research and education needs of the organic dairy industry in the northeastern United States

Demographic and management data about organic dairies have been reported previously, but the current study is the first needs assessment of research and educational priorities of organic dairy farmers in the northeastern United States based directly upon their input. Our objectives were to (1) develop an initial understanding of the emerging research and educational needs of organic dairy farmers in the northeastern United States via focus group interviews, and (2) prioritize the needs identified by the focus groups with a broader population of organic dairy farmers via survey methods. Focus group interviews determined the questions used for the survey questionnaire distributed to 1,200 members of the Northeast Organic Dairy Producers Alliance. The members were asked about demographic information, but more importantly, challenges concerning business management and marketing, organic certification, and animal nutrition, health, and reproduction. The results (183 respondents, 15% response rate) were parsed by region (New England farms compared with New York and Pennsylvania farms), herd size (i.e., 12 to 37, 38 to 59, and >60 cows), and years of organic certification (<4 yr vs. ≥ 4 yr); however, no differences between regions were observed for demographic data. The average farm consisted of 309 acres and 57 milking cows, on which most of the forage was homegrown but grains were purchased (73% of farms). Among the greatest challenges identified by the farmers were obtaining a steady, fair price for milk (85% respondents); determining dry matter intake for animals on pasture (76%); and controlling nuisance flies (89%). Needs for additional research included organic treatments for mastitis (92% respondents), growing forages for organic production (84%), and developing value-added products (84%). Farms with <4 yr of organic certification were concerned with level of knowledge and experience of local certifiers, whereas organic producers with ≥ 4 yr of organic certification were more interested in field testing of new organic products. Opportunities for educational programs included learning about direct marketing possibilities (76% respondents) and providing training to regional veterinarians interested in organic remedies (91%). In conclusion, the information obtained from the current needs assessment provides a foundation for future research proposals and educational outreach programs, germane to stakeholder needs, which could benefit the organic dairy industry within the region and beyond.

Estrous cycle-dependent changes of Fas expression in the bovine corpus luteum: influence of keratin 8/18 intermediate filaments and cytokines

BackgroundFas expression and Fas-induced apoptosis are mechanisms attributed to the selective destruction of cells of the corpus luteum (CL) during luteal regression. In certain cell-types, sensitivity to these death-inducing mechanisms is due to the loss or cleavage of keratin-containing intermediate filaments. Specifically, keratin 8/18 (K8/K18) filaments are hypothesized to influence cell death in part by regulating Fas expression at the cell surface.MethodsHere, Fas expression on bovine luteal cells was quantified by flow cytometry during the early (Day 5, postovulation) and late stages (Days 16–18, postovulation) of CL function, and the relationship between Fas expression, K8/K18 filament expression and cytokine-induced cell death in vitro was evaluated.ResultsBoth total and cell surface expression of Fas on luteal cells was greater for early versus late stage bovine CL (89% vs. 44% of cells for total Fas; 65% vs.18% of cells for cell surface Fas; respectively, P<0.05, n=6-9 CL/stage). A similar increase in the steady-state concentration of mRNA for Fas, as detected by quantitative real-time polymerase chain reaction, however, was not observed. Transient disruption of K8/K18 filaments in the luteal cells with acrylamide (5 mM), however, had no effect on the surface expression of Fas (P>0.05, n=4 CL/stage), despite evidence these conditions increased Fas expression on HepG2 cells (P<0.05, n= 3 expts). Exposure of the luteal cells to cytokines induced cell death (P<0.05) as expected, but there was no effect of K8/K18 filament disruption by acrylamide (P>0.05) or stage of CL (P>0.05, n= 4 CL/stage) on this outcome.ConclusionIn conclusion, we rejected our null hypothesis that the cell surface expression of Fas does not differ between luteal cells of early and late stage CL. The results also did not support the idea that K8/K18 filaments influence the expression of Fas on the surface of bovine luteal cells. Potential downstream effects of these filaments on death signaling, however, remain a possibility. Importantly, the elevated expression of Fas observed on cells of early stage bovine CL compared to late stage bovine CL raises a provocative question concerning the physiological role(s) of Fas in the corpus luteum, particularly during early luteal development.