The phagocytic activity of polymorphonuclear leucocytes isolated from normal uterus and that with experimentally induced inflammation in cows.

The purpose of the study was the determination of the activity of the neutrophils isolated from a physiologically sound (non-inflamed) uterus 2 days after ovulation, and comparison of this activity with that of the polymorphonuclear leucocytes (PMNs) obtained from an uterus which had undergone experimentally induced specific or non-specific inflammatory reaction. The study was carried out on 20 cows between the 2nd and the 8th day after ovulation. Activity of PMNs was determined on the basis of the per cent of these cells with Fc receptors for IgG on their surface, and on the basis of assessment of the phagocytic activity. The Fc receptors were determined by the rosette test. The phagocytic activity of PMNs was determined in relation to two standard strains of S. aureus Smith which were phagocytized by mediation of Fc receptors and S. aureus 305 phagocytized by mediation of the "non-immunological" receptors. The study showed that the inflammatory process in the uterus caused intensive migration of large numbers of PMNs into the lumen. These PMNs have their properties altered: there is a significantly lower per cent of cells with Fc receptors in this cell population and lower Fc-dependent phagocytic activity. A significant rise in the phagocytic activity mediated by non-immunological receptors occurs at the same time in this cell population.

Immunological induction of endometritis-model investigations in cows.

The course of the inflammatory reaction in the uterus after local or systemic administration of specific or non-specific antigens was studied. The study was carried out on 40 cows, 10-11 weeks after labour and between 6 and 8 days after ovulation. A specific cell-mediated type of reaction was induced by intrauterine challenge with PPD in cows previously vaccinated with M. bovis subcutaneously or by intrauterine administration. A specific inflammatory process of the type of Arthus reaction was induced by intrauterine challenge with C. fetus ssp. veneralis in animals immunized with these bacteria subcutaneously or by intrauterine instillation of these bacteria. A non-specific inflammatory process in the uterus was initiated by one instillation of lipopolysaccharide (LPS) into the left uterine horn. The cells were washed out from the uterus before and 6, 24, 48 and 72 hours after initiation of the specific or non-specific inflammatory process. The per cent proportions of various cell types were determined. It was demonstrated that intrauterine instillation of specific or non-specific antigens in cows caused a significant rise in PMNs per cent in relation to the control group.

Phagocytosis and intracellular killing of Staphylococcus aureus by bovine blood polymorphonuclear leukocytes after migration in vitro.

Phagocytic activity and intracellular killing of opsonized staphylococci (Smith strain) by bovine blood polymorphonuclear leukocytes (PMNL) was studied after their migration in vitro in blind well chambers. The results indicate that migration of PMNL to RPMI-1640 medium without chemotactic factor significantly (P less than 0.01) increased the percentage of rosette-forming PMNL (from 11 +/- 2 to 49 +/- 4%), as well as phagocytic activity mediated through Fc receptors (FcRs) (from 25 +/- 3 to 81 +/- 4% phagocytizing PMNL and from 151 +/- 22 to 942 +/- 54 number of phagocytized staphylococci/100 PMNL), and intracellular killing of bacteria (from 13 +/- 2 to 59 +/- 7%). On the other hand, PMNL migration to RPMI-1640 medium with the chemotactic factor (serum activated with zymosan [AS] or lipopolysaccharide [LPS] or formyl-L-methionyl-L-leucyl-L-phenylalanine [fMLP]) did not significantly (p greater than 0.05) increase either the FcRs number on the PMNL surface or the phagocytic and bactericidal activity connected with these receptors. The possible mechanisms are discussed.

Phagocytic activity of milk leukocytes during chronic staphylococcal mastitis.

Receptors for IgG on milk leukocytes were detected by rosette formation using sensitized erythrocytes. The percentage (41) of milk leukocytes from uninfected glands forming sensitized erythrocyte rosettes was significantly greater than the percentage (13) of leukocytes from glands with chronic staphylococcal mastitis. A greater percentage of polymorphonuclear leukocytes than macrophages formed sensitized erythrocyte rosettes, regardless of the infection status of the gland from which they were obtained. Both IgG-receptor and nonimmunologic receptor-mediated phagocytosis were greater for milk leukocytes from uninfected glands than for milk leukocytes from chronically infected glands. Preincubation of normal milk leukocytes in whey prepared from mastitic milk resulted in a decrease in their capacity to form sensitized erythrocyte rosettes as well as a reduction in their phagocytic capacity. Immune complexes prepared in vitro also reduced the phagocytic capacity of normal milk leukocytes and inhibited their capacity to form sensitized erythrocyte rosettes. These data indicate a factor, possibly consisting of immune complexes, was present in secretions from glands chronically infected with staphylococci. This factor reduced the phagocytic capacity of milk leukocytes.

Inhibition of lacteal leukocyte phagocytosis by colostrum, nonlactating secretion, and mastitic milk.

The Fc receptors on leukocytes obtained from normal milk, colostrum, nonlactating gland secretion, and mastitic milk were detected by rosette formation, using sensitized erythrocytes. The percentage of leukocytes bearing Fc receptors from normal milk was significantly (P less than 0.01) greater than that from other secretions. Fc receptors were found primarily on polymorphonuclear leukocytes from normal milk, mastitic milk, and colostrum. However, in nonlactating gland secretion obtained 6 weeks after milking was completed, Fc receptors were predominantly on macrophages. The low percentage of leukocytes bearing Fc receptors obtained from colostrum, nonlactating gland secretion obtained 7 days after milking was completed, and mastitic milk was associated with the presence of a blocking factor in these secretions, which specifically attached to Fc receptors. These secretions significantly (P less than 0.01) blocked the Fc receptors on leukocytes from normal milk and on other cells bearing FC receptors. The presence of Fc receptors on leukocytes obtained from normal milk was related to a high percentage of phagocytizing leukocytes through Fc receptors and a large number of phagocytized bacteria (phagocytic activities). In contrast, the low percentage of leukocytes bearing Fc receptors from colostrum, from nonlactating gland secretion (7 days after milking), and from mastitic milk was associated with depressed phagocytic activities. Preincubation of leukocytes from normal milk with whey from colostrum, nonlactating gland secretion (7 days after milking), and mastitic milk significantly (P less than 0.01) inhibited phagocytosis. This effect was associated with the blocking of Fc receptors by these secretions. Possible mechanisms for and implications of these findings are discussed.

Appearance of Fc receptors on polymorphonuclear leukocytes after migration and their role in phagocytosis.

The effect of the migration of bovine blood polymorphonuclear leukocytes (PMNs) in vitro on their phagocytic activity was studied. PMNs were examined before and after migration through various membranes for rosette formation with sensitized sheep erythrocytes to detect Fc receptors (FcRs), phagocytic activity mediated through FcRs with opsonized staphylococci (Smith strain), and phagocytic activity mediated through nonimmunological receptors with unopsonized staphylococci (strain 305). Migration of PMNs was observed from the upper to the lower compartment of the blind well chamber through Millipore and Nuclepore membranes; through Millipore, Nuclepore, and nylon membranes coated with collagen; and through collagen-coated Millipore, Nuclepore, and nylon membranes overlaid with MA-104, BHK-21, MDBK-99, TB, or FBHE cells. Random migration of PMNs toward the plain medium (the same medium in the upper and lower compartments) through the membranes with and without a monolayer of cells increased the percentage of PMNs forming rosettes. In contrast, migration toward the medium containing lipopolysaccharide (LPS), formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), or zymosan-activated serum (Act. serum) did not change the percentage of PMNs forming rosettes. The increased percentage of PMNs forming rosettes was associated with the enhanced phagocytosis of opsonized staphylococci (mediated by FcRs). In contrast, migration of PMNs toward LPS, FMLP, or Act. serum did not enhance phagocytosis mediated through FcRs. However, PMNs after migration toward LPS, FMLP, Act. serum, and plain medium enhanced phagocytosis of unopsonized staphylococci (mediated through the nonimmunological receptors).

Suppression of mitogenic response of bovine lymphocytes during experimental ketosis in calves.

Experimental ketosis was induced by feeding calves a diet containing 1,3 butanediol for 9 or 10 days. Blood beta-hydroxybutyrate was significantly (P less than 0.05) elevated during clinical ketosis. The mitogenic response of the lymphocytes collected during ketosis was significantly (P less than 0.01) suppressed, and the suppression persisted for 2 weeks. During ketosis, all calves fed a 1,3 butanediol diet had clinical signs of an upper respiratory tract infection. Possible relationships between the suppressed function of lymphocytes, the increased concentration of 1,3 butanediol, and the increased susceptibility of the calves to infections during ketosis were considered.

Effect of immune complexes from mastitic milk on blocking of Fc receptors and phagocytosis.

Fc receptors on the surface of milk leukocytes from normal glands, bronchial leukocytes, mastocytoma P-815 cells, and murine leukemia L1210 cells were blocked significantly (P less than 0.01) by cavian and bovine milk collected from inflamed glands (mastitic milk), their wheys, and in vitro-prepared immune complexes composed of the whey from normal milk and serum. Blocking of Fc receptors indicated the presence of immune complexes in the mastitic milk and was detected by inhibition of rosette formation with sensitized erythrocytes or attachment of the aggregated immunoglobulin G. The binding of immune complexes to these cells was also determined by staining with fluorescein isothiocyanate-labeled protein A. As the mastitis subsided, the blocking effect of the mastitic milk also declined markedly. There was no significant difference in blocking capacity between mastitic milk and its whey. The blocking capacity of normal cavian or bovine milk and their wheys was insignificant. Whey from mastitic milk also inhibited phagocytosis of opsonized staphylococci by alveolar macrophages. We suggest that the blocking of Fc receptors on phagocytic cells adversely affects phagocytosis.

Immune-mediated migration of neutrophils into the uterine lumen of guinea pigs.

Metritis was elicited by intrauterine infusion of tuberculin or killed Campylobacter fetus ssp. venerealis into vaccinated guinea pigs and lipopolysaccharide or immune complexes into normal animals. The local inflammatory response to intrauterine infusion of antigens, lipopolysaccharide, and immune complexes was determined by changes in differential cell counts in the uterine lavage fluid and by histopathological examination of uterine tissue. The percentage of neutrophils was significantly (p less than 0.01) greater in uterine lavage fluid collected at 4 hr after infusion of tuberculin into animals vaccinated locally (intrauterine) with M. tuberculosis than in animals vaccinated parenterally (subcutaneously). In contrast, the local response to infusion with C. fetus ssp. venerealis was approximately the same in animals vaccinated intrauterine and subcutaneously with Campylobacter. The systemic response, measured by the delayed type hypersensitivity cutaneous reaction to intradermal injection of tuberculin, was significantly (p less than 0.01) greater in animals vaccinated subcutaneously than intrauterine. Similarly, the concentration of Campylobacter antibody in the serum of animals vaccinated subcutaneously was significantly (p less than 0.01) greater than in guinea pigs vaccinated intrauterine. The intrauterine infusion of immune complexes, composed of C. fetus ssp. venerealis and corresponding antibody, into the uterus of normal guinea pigs stimulated neutrophil migration into the uterine lumen. Infusion of lipopolysaccharide also stimulated neutrophil migration into the uterine lumen. A correlation between an increased percentage of neutrophils in uterine lavage fluid and infiltration of the uterine epithelium with neutrophils was observed.

The effect of local and parenteral vaccination on the response of the guinea pig mammary gland to staphylococcal challenge.

Preparturient guinea pigs vaccinated locally (orally and intramammarily) or parenterally (intradermally) with killed Staphylococcus aureus (KS), were challenged intramammarily (IMM) with KS or viable S. aureus during the next lactation. The number and types of leukocytes emigrating into the milk were determined before and after IMM challenge. The milk leukocytosis after challenge with KS was the greatest in the intradermally (ID) vaccinated animals, moderate in the IMM vaccinated animals, and insignificant in the unvaccinated or orally vaccinated animals. The polymorphonuclear (PMN) leukocyte predominated in the milk of the IMM and ID vaccinated animals during the initial 30 h after challenge with KS. Later (30-102 h postchallenge), the mononuclear leukocyte (macrophage and lymphocytes) was the major cell-type. No significant changes in the number or types of leukocytes occurred in the milk of the unvaccinated or orally vaccinated animals after challenge. Intramammary challenge with viable S. aureus caused a large increase in the number of leukocytes in the milk of all animals. The milk leukocytosis occurred more rapidly in locally vaccinated guinea pigs than unvaccinated or ID vaccinated guinea pigs. The PMN leukocyte predominated in the milk of all animals during the period of maximum response. The incidence and severity of staphylococcal mastitis were less in guinea pigs vaccinated locally than ID vaccinated or unvaccinated animals.

Ammonia toxicity for mammalian and avian lymphocytes from blood.

The effect of addition of ammonia to cultures of avian, bovine, cavian, equine, human, leporinae, murine, ovine and porcine blood lymphocytes was studied. The concentrations of ammonia in the lymphocyte cultures represented normal (0.01-0.5 mg/dl), subtoxic (0.5-1 mg/dl), and toxic (1-10 mg/dl) concentrations of ammonia in blood. Viability of the lymphocytes and their mitogenic reactivity were measured. In general, 1.0 and 10 mg/dl of ammonia (toxic concentrations) affected viability and mitogenic responsiveness of all lymphocytes from tested species. The lower concentrations of ammonia affected only avian, bovine and equine lymphocytes.

Effect of ammonia on viability and blastogenesis of bovine lymphocytes.

The effect of addition of ammonia into the tissue culture on viability and functions of bovine lymphocytes was studied. The concentrations of ammonia in the tissue cultures represented toxic, subtoxic, and normal concentrations of ammonia in the bovine blood during clinical and subclinical urea toxicosis. Lymphocytes separated from peripheral bovine blood were incubated in control medium and test medium with various concentrations of ammonia and/or PHA or Con A. Viability of the lymphocytes was measured by trypan blue exclusion test and their mitogenic reactivity by incorporation of 3H thymidine into DNA of lymphocytes. Approximately 30% bovine lymphocytes were killed by ammonia in medium during 72 hours of incubation. Ammonia also affected the response of lymphocytes to stimulation with PHA or Con A as well as mixed lymphocyte culture reaction. The mitogenic response of lymphocytes was also reduced when lymphocytes were preincubated with ammonia for even 1 hour. The mitogenic response was not restored when the number of lymphocytes preincubated with ammonia was reconstituted to the initial concentration to compensate for the killed lymphocytes before stimulation with PHA. Therefore, addition of ammonia to the culture either killed lymphocytes or permanently impaired their functions.

Effect of ammonia on in vivo and in vitro immune responses.

The effects of exposure of animals to ammonia on their delayed type of dermal response, the mitogenic and antigenic responses of their lymphocytes, and the bactericidal and phagocytic activities of their alveolar macrophages were examined. Experimental guinea pigs vaccinated with Mycobacterium bovis BCG were exposed to 3.75 micrograms of ammonia per dl of air (50 ppm) or 6.75 micrograms of ammonia per dl of air (90 ppm), whereas control animals also vaccinated with BCG were maintained in the normal environment. The delayed type of dermal response to tuberculin injected 3 weeks later was significantly (P less than 0.05) less in experimental animals exposed to 6.75 micrograms of ammonia per dl than in control animals. In vitro, the response of blood lymphocytes and bronchial lymphocytes to phytohemagglutinin, concanavalin A, and tuberculin stimulation was significantly (P less than 0.01) less than the response of lymphocytes from control animals. The response of normal blood lymphocytes to phytohemagglutinin incubated in medium containing 1 or 10 mg of ammonia per dl was significantly (P less than 0.01) reduced as compared with the response of lymphocytes incubated without ammonia. The viability of lymphocytes incubated with these concentrations of ammonia was significantly (P less than 0.01) affected. There was no significant difference in the bactericidal or phagocytic activities of alveolar macrophages collected from animals exposed to ammonia and control animals. However, ammonia added to the culture of alveolar macrophages from normal animals significantly inhibited their bactericidal activity.

Reduction in mitogenic response of bovine lymphocytes by ketone bodies.

The effect of toxic or subtoxic concentrations of ketone bodies (acetone, beta-hydroxybutyrate, acetoacetate) in blood on function of bovine lymphocytes was studied in vitro. Lymphocytes separated from peripheral bovine blood were distributed into the wells of Linbro Microtiter plates containing control medium and test medium with various concentrations of ketones and/or phytohemagglutinin. The mitogenic response of lymphocytes was measured by incorporation of [3H]thymidine into DNA of lymphocytes. Toxic and subtoxic concentrations of beta-hydroxybutyrate or the toxic concentration of acetoacetate significantly affected the mitogenic response of bovine lymphocytes. The reduction in the mitogenic response also occurred when lymphocytes were only preincubated for 2 hours or longer with beta-hydroxybutyrate or acetoacetate. The presence of the toxic concentration of acetone in the medium did not affect the mitogenic stimulation of lymphocytes.

Morphogenesis of rabbit small intestinal mucosa.

The sequential morphologic changes of rabbit small intestinal mucosa were studied from primodial stage to birth in 86 fetuses and during the early days of life in 18 rabbit newborns. In 13-day-old fetuses, the epithelium was flat and stratified. By the 17th day, epithelial ridges were formed. The first evidence of rudimentary villus formation was noted in 20-day-old fetuses and true villi appeared by 24 days of the gestation. Intestinal glands (crypts of Lieberkühn) were first observed at 29 days. The intestine of the newborn rabbit did not possess all mucosal constituents present in the adult. The duodenal glands (Brunner's glands) appeared in the first 2 days after birth, and the muscularis mucosa, on the 5th day. The histological maturity of the intestine occurred 20 days after the rabbit was born. At all stages, the morphogenesis followed a cranio-caudal gradient developmental pattern.

Cell-mediated immune response of the mammary gland in guniea pigs. I. Effect of antigen injection into the vaccinated and unvaccinated glands.

The leukocytic response of the mammary gland in the locally vaccinated guniea pigs to challenge with specific antigen during lactation was investigated. The response was measured by enumerating cells in milk collected at various times prior to and following antigenic challenge. A significant leukocytosis was observed in milk from vaccinated animals. The maximum cellular response by vaccinated animals was observed at 16 h to 30 h after challenge. The majority of leukocytes collected at that time did not form EA rosettes. Differential cell counts showed that the polymorphonuclear leukocytes were the major cell type until 30 h postchallenge while the mononuclear leukocytes predominated later. The delayed cellular response to challenge and the predominancy of leukocyte type at various times after challenge are discussed. It is proposed that the leukocyte response of the mammary glands in vaccinated animals was a cell-mediated immune reaction.