Pig Leukocytes Research Articles

Characterization of pig-mink cell hybrids: assignment of the TK1 and UMPH2 genes to pig chromosome 12

By fusion of thymidine kinase-deficient mink cells with pig leukocytes, a new type of cell hybrid was produced. It was demonstrated that pig chromosomes segregate in pig-mink hybrids and that hybrid cells contain no cytologically visible rearrangements between the chromosomes of parental species, or chromosome fragmentation. With a set of subclones of two primary hybrid clones, the genes for thymidine kinase-1 (TK1) and uridine 5'-monophosphate hydrolase-2 (UMPH2) were assigned to pig Chromosome (Chr) 12. A cell line with a single pig Chr 8 on the background of mink chromosomes was established. This clone could serve as a source of DNA for building a chromosome-specific library of pig Chr 8. The data obtained suggest that pig-mink cell hybrids can be used for mapping of pig chromosomes.

Infection of leucocyte cell cultures derived from different species with pig circovirus

Cultures of leucocyte cells were prepared from pig bone marrow, peripheral blood, lung washings, thymus and lymph nodes. Cell cultures were also prepared from peripheral blood from sheep, cattle and a human. Immunofluorescent (IF) staining of all these cultures, following inoculation with pig circovirus (PCV), detected virus replication in all the cell cultures derived from pigs and in the cell cultures derived from cattle. Virus replication in pig leucocyte cell cultures was confirmed by demonstrating the production of infectious virus. Double immunostaining of PCV infected cells using monoclonal antibodies specific for cell membrane markers indicated infection was confined to monocyte/macrophage cell types. No PCV antigen was detected in T or B cells in infected cell cultures.

Studies on the distribution of immune cells in the uteri of prepubertal and cycling gilts

To establish the cellular basis for the local immune response in the porcine uterus, immunohistochemical studies using a panel of monoclonal antibodies to pig leukocytes were conducted on uterine tissues from prepubertal and cycling gilts. In prepubertal uteri, neutrophils were the most predominant cell type, while MHC class II + cells and CD2 + T lymphocytes were also common. At the early-stage of the oestrous cycle, CD2 + T cells were numerous in the endometrium, particularly in the uterine epithelium and subepithelial regions. However, by the mid-stage of the cycle there was a significant and dramatic fall in CD2 + T cells and other lymphocytes expressing the CD4, CD8 and CD1 phenotypes, MHC class II + cells were predominant throughout the endometrium. During late oestrus there was a dramatic infiltration of neutrophils into the subepithelial stroma. A distinct increase in the CD2 + intraepithelial T lymphocyte population was also observed at this stage of the cycle. It was concluded that in the healthy, non-pregnant pig uterus T lymphocytes, macrophages and neutrophils were the prominent leukocyte cell types and their migration and distribution in the uterus was strongly influenced by the oestrous cycle. These immune cells may play an important interactive role in the cyclic cellular changes in both the structure and function of the endometrium. Furthermore, the leukocyte phenotypes found in the porcine endometrium indicate that a local cellular immune response could be elicited.

Oxygen-induced lung injury in the guinea pig proceeds through CD18-independent mechanisms.

The pathogenesis of pulmonary oxygen toxicity is postulated to be related in part to neutrophil-mediated injury. This study examined the effect of a monoclonal antibody directed against the CD11a,b,c/CD18 glycoprotein complex (beta 2 leukocyte integrins) on oxygen-induced lung injury. M8, a monoclonal antibody that binds to the beta chain of the guinea pig leukocyte integrins that facilitate neutrophil adherence to vascular endothelium, was injected into adult guinea pigs prior to and during exposure to > 98% oxygen. Control oxygen-exposed animals were injected with a noninhibitory antibody to the CD18 complex or with saline. Survival in oxygen was similar for animals treated with M8 when compared with those treated with saline (102 versus 105 h, respectively, NS). Pulmonary edema as assessed by protein in the supernatant of bronchoalveolar lavage fluid (BALF) was higher in the three groups of oxygen-exposed animals than in the air-exposed groups (p < 0.01), but it did not differ between the M8 antibody treatment group and the other oxygen-exposed groups. M8 antibody treatment did not decrease hyperoxia-induced neutrophil accumulation into the lung as assessed by myeloperoxidase activity (MPO) in lung homogenates or by neutrophil counts in histologic specimens. M8 antibody also did not decrease neutrophil counts or MPO in alveolar lavage fluid, both of which were significantly elevated in all oxygen-exposed groups. These results suggest that hyperoxia-induced neutrophil migration into the lung and acute lung injury occurs by CD18-independent processes in the guinea pig model of pulmonary oxygen toxicity.

Pig leukocyte cysteine proteinase inhibitor (PLCPI), a new member of the stefin family

A new stefin type low- M r, cysteine proteinase inhibitor (PLCPI) was isolated from pig polymorphonuclear leukocytes as a contaminant of the cathelin sample. The inhibitor consists of 103 amino acids, and its M r, was calculated to be 11,768. The inhibitor exhibits considerable sequence identity with inhibitors from the stefin family, particularly with human stefin A. The PLCPI is a fast acting inhibitor of papain and cathepsins L and S ( k ass ⩾ 1 × 10 6 M −1 · s −1) and forms very tight complexes with these enzymes ( K i, ⩽ 190 pM). The affinity for cathepsins B and H ( K i ⩾ 125 nM) was lower. These results also show that the inhibitory activity previously ascribed to cathelin was due to the presence of PLCPI.

A Novel cDNA Sequence Encoding a Pig Leukocyte Antimicrobial Peptide with a Cathelin-like Pro-sequence

It has recently been shown that the precursors of various structurally unrelated leukocyte antimicrobial peptides share similar pro-regions. These, in turn, are highly identical to a cysteine proteinase inhibitor named cathelin, or PLCPI. In this paper we report a novel cDNA sequence of porcine bone marrow origin, encoding a protein characterized by a cathelin-like domain. The putative protein is 147 amino acid residue long, with a calculated mass of 16479 Da and appears to be the precursor of a recently isolated antimicrobial peptide named protegrin PG-2. The unique sequence of the mature PG-2 is located at the C-terminus of the precursor. Similar to the previously reported precursors, both the signal peptide and the pro-sequence of pre-proPG-2 appear highly conserved.

Molecular characterization of a leukotoxin gene from a Pasteurella haemolytica-like organism, encoding a new member of the RTX toxin family

A Pasteurella haemolytica-like organism, a new species of bacterium isolated from piglets with diarrhea, secretes a leukotoxin into the culture media. Western blot (immunoblot) analysis indicated that this leukotoxin cross-reacted with antileukotoxin antibody derived from cattle immunized with P. haemolytica. Five overlapping recombinant bacteriophages carrying the gene for this 105-kDa polypeptide were identified with a DNA probe containing sequences from the P. haemolytica lktCA genes from a P. haemolytica-like organism strain 5943 genomic library. Sequence analysis of a region of the cloned DNA revealed two open reading frames encoding proteins with predicted masses of 19.4 and 101.6 kDa. These genes, which we designate pllktC (P. haemolytica-like organism leukotoxin C gene) and pllktA (A gene), respectively, are similar in sequence to the RTX (repeat of toxin) toxin family. The structure of the 101.6-kDa protein derived from the DNA sequence shows three transmembrane domains in the N-terminal part of the protein, 13 glycine-rich repeat domains in the second half of the protein, and a hydrophobic C-terminal part. pllktC and pllktA are strongly homologous to P. haemolytica lktC and lktA genes. However, this leukotoxin kills both BL-3 and pig leukocytes and is not hemolytic.

Monoclonal antibodies specific for guinea pig eosinophil major basic protein: their use in an ELISA, immunocytochemistry and flow cytometry

Eosinophil major basic protein (MBP), purified from guinea pig eosinophil granules was used to raise five monoclonal antibodies (MoAbs). Their reactivity with MBP was confirmed by immunoblotting and indirect ELISA. Two of the MoAbs were used to develop a sensitive and specific antigen capture (sandwich) ELISA for guinea pig eosinophil MBP which gives an accurate and reproducible standard curve over the range of 10-10,000 ng/ml. The specificity of the ELISA for MBP was confirmed and its suitability for testing biological samples ascertained by measurement of MBP in bronchoalveolar lavage fluid (BALF) and plasma from guinea pigs sensitized and challenged with ovalbumin. The ELISA was also capable of detecting MBP in culture supernatants from purified eosinophil preparations challenged with calcium ionophore in vitro. One of the monoclonals could be used to strongly and specifically stain guinea pig eosinophils in immunocytochemistry, whilst all five could be used to visualize eosinophils in suspension in BALF or peritoneal lavage fluid by flow cytometry. There was no staining of other guinea pig leucocyte types, nor crossreactivity with human eosinophils by immunocytochemistry or flow cytometry.

Participation of cathepsin L on bone resorption

The proteinase responsible for bone collagen degradation in osteo-resorption was examined. The bone pit formation induced by parathyroid hormone (PTH) was markedly suppressed by leupeptin, E-64 and cystatin A, while no inhibition was observed by CA-074, a specific inhibitor of cathepsin B. Pig leucocyte cysteine proteinase inhibitor (PLCPI), a specific inhibitor of cathepsin L, and chymostatin, a selective inhibitor of cathepsin L, completely inhibited the pit formation. Cathepsin L activity in osteoclasts was much higher than the other cathepsin activities. Serum calcium in rats placed on a low calcium diet was decreased by treatment of E-64 or cystatin A, but not by CA-074. These findings suggest that cathepsin L is the main proteinase responsible for bone collagen degradation.

Enrichment of coronavirus-induced interferon-producing blood leukocytes increases the interferon yield per cell: A study with pig leukocytes

SummaryPorcine peripheral blood mononuclear cells, which secrete IFNα in response to a coronavirus, transmissible gastroenteritis virus, were detected by a filter immunoplaque assay (ELISPOT). IFNα-producing cells (IPC), which are present at a low frequency in the blood, could be enriched up to 100-fold by sequential depletion of plastic-adherent cells and cell fractionation on metrizamide density gradients. IPC were present in the non-adherent low-density cell subpopulation. Cell selection experiments using antibody (Ab)-coated immunomagnetic beads revealed that porcine IPC could be positively selected by anti-CD4 or -SLA-class-II Ab, but not by anti-CD2 or -CD8 Ab. The estimated IFN yield per IPC was found to increase when IPC were assayed at higher concentrations. These data suggest that IPC represent a unique and distinct cell population in the blood, which could secrete higher amounts of IFN following its accumulation at a site of viral infection.

Induction of superoxide by 12-O-tetradecanoylphorbol-13-acetate and thapsigargin, a non-phorbol-ester-type tumor promoter, in peritoneal macrophages elicited from SENCAR and B6C3F1 mice: a permissive role for the arachidonic acid cascade in signal transduction.

Local production of reactive oxygen intermediates, e.g., superoxide anion, by tumor promoter-stimulated inflammatory macrophages (MPs) may contribute significantly to tumor development in classical models of two-stage chemical-induced carcinogenesis in murine skin. In the studies reported herein, peritoneal MPs elicited from phorbol-ester-sensitive SENCAR mice demonstrated a time- and dose-dependent release of superoxide anion (4-6 nmol/10(6) cells) when stimulated by 200 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro; MP superoxide response was significantly inhibited (50-70%) by preincubation with 40 microM 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), a protein-kinase inhibitor. Alternatively, TPA-stimulated MPs derived from relatively resistant B6C3F1 mice generated negligible superoxide under the same conditions. A similar strain-dependent induction of superoxide was observed when MPs were stimulated with thapsigargin (TG), a tumor promoter previously shown to act independently of protein kinase C (PKC). TG-stimulated SENCAR MPs released a significant amount of superoxide (2-3 nmol/10(6) cells) that was not inhibited by H-7; MPs from B6C3F1 mice demonstrated negligible stimulation by TG. Preincubation of SENCAR MPs with 100 microM dibromoacetophenone, an inhibitor of phospholipase A2, completely suppressed the superoxide induced by TPA and TG stimulation. Like TPA, 50 microM 1-oleoyl-2-acetylglycerol, a diacylglycerol analogue and PKC activator, also induced a significant amount of superoxide from SENCAR MPs only. In parallel with the superoxide findings, TPA and TG stimulated significantly greater [3H]arachidonic acid release from prelabeled SENCAR MPs (a 32% and 48% increase, respectively, over unstimulated controls) relative to MPs from B6C3F1 mice. Two-dimensional gel-electrophoretic analysis indicated that TPA-induced phosphorylation of a 47-kDa protein (a presumed substrate for PKC previously linked to NADPH oxidase activation in guinea pig and human polymorphonuclear leukocytes) occurred in MPs from both SENCAR and B6C3F1 mice. Therefore, arachidonic acid production may be a common biochemical pathway by which phorbol-ester--and non-phorbol-ester--type tumor promoters activate MPs in SENCAR mice; such a response may be "permissive" for additive (or synergistic) interactions with PKC-driven signal pathways.

Adhesion of Salmonella typhimurium to porcine intestinal epithelial surfaces: identification and characterization of two phenotypes.

Salmonella typhimurium 798 is known to persistently colonize swine. A key step required to initiate colonization of intestines is adhesion of the organism to the intestinal epithelium. However, S. typhimurium 798 initially failed to attach to porcine enterocytes in vitro. An enrichment procedure was used to select adhesive S. typhimurium, and when cells of one colony type were grown in tryptone phosphate broth they were adhesive. Cells from a colony with a different morphology were not adhesive. Adhesion was time dependent, with maximal adhesion occurring at 1 h. As determined by electron microscopy, cells of the adhesive phenotype had pili while none of the cells with the nonadhesive phenotype produced pili. The pili on the adhesive cells were morphologically similar to type 1 pili. Mannose (0.5%) did not affect adhesion, suggesting that the adhesin on strain 798 did not recognize mannose as a receptor. An analysis of envelope proteins from cells of both phenotypes showed that the adhesive-phenotype cells expressed at least 10 unique proteins ranging in size from 20 to 60 kDa. Absorbed antiserum against cells of the adhesive phenotype agglutinated adhesive cells and was used to detect unique surface antigens on the cells of the adhesive phenotype by Western blots (immunoblots). These antigens were in the range of 30 kDa in size. An envelope extract competitively inhibited the binding of S. typhimurium to enterocytes, as did Fab fragments prepared from the absorbed serum. Cells of both phenotypes contained two plasmids, and each had identical restriction digestion patterns. Cells of the adhesive phenotype consistently were found to be more readily phagocytosed by pig leukocytes, and once in the phagocytes they survived better than cells of the nonadhesive phenotype.

Role of histamine receptors of mouse and guinea pig peritoneal leukocytes in the pathogenetic action ofYersinia pestis adenylate cyclase

The effect of purified preparation of adenylate cyclase isolated from Y. pestis on peritoneal leukocytes of white mice and guinea pigs was investigated. Y. pestis adenylate cyclase was shown to accomplish its pathogenic action via histamine-specific receptors on the surface of eukaryotic cells. The involvement of H1 and H2 histamine receptors on target cells in the adenylate cyclase action leading to development of plague infection is discussed.

Multidrog transport in human autoimmune T line cells and peripheral blood lymphocytes

We studied multidrug transport in human autoimmune T line cells and peripheral blood leukocytes, because muitidrug transport is pleiotropically limiting the intracellular accumulation of immunosuppressive and chsmotherapeutie agents. We observed that a subpopulation of peripheral blood leukocytes containing CD4+. CD8+ and Ig+ lymphocytes expressed a muitidrug transport system. Lymphocytic muitidrug transport was seen with all peripheral blood samples analyzed, but showed considerable variations between individuals. In further studies with human lymphocytic cell lines multidrug transport was seen with 1823 human CD4+ T cell lines, interestingly, expression of multidrug transport was independent of T cell activation. No significant transport was observed with EBV-transformed human B lymphocytes, rat T line cells or rat, mouse, or guinea pig leukocytes.

Reduced bactericidal activity in neutrophils from scorbutic animals and the effect of ascorbic acid on these target bacteria in vivo and in vitro

Actinomycetes, involved in oral and periodontal diseases, cause serious infections in immunocompromised hosts. Severely scorbutic guinea pig leukocytes killed only 12% of phagocytosed actinomycetes, had distorted nuclear morphology, had 16 times less ascorbate, and had no chemotactic responses in vitro. Ascorbate reversed these indices and also prevented nitrosamine formation by oral organisms. Degranulating leukocytes release lactoferrin and ascorbate that chelate iron, essential for microorganisms. Ascorbic acid, 2,2'-bipyridine and 1,10-phenanthroline were bactericidal to several bacterial pathogens at millimolar concentrations. Iron alone reversed this effect. In in vivo experiments an Actinomyces viscosus monoflora was implanted in rhesus monkeys. Plaque and serum samples showed decreased (by six orders of magnitude) bacterial counts and decreased actinomycete antibody titers in animals given 1 g ascorbate/d. Removing ascorbate returned counts and titers to preascorbate concentrations. Fifteen marmosets, receiving twice daily topical applications of ascorbate or water, had comparatively lower gingival, calculus, and plaque indices and only slightly lowered actinomycete counts.

CGS 22745: a selective orally active inhibitor of 5-lipoxygenase.

CGS 22745, and aralkyl hydroxamic acid, inhibited 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4) synthesis in guinea pig leukocytes (IC50 = 0.6 microM). The compound did not appreciably affect cyclooxygenase (ram seminal vesicles), 12-lipoxygenase and thromboxane synthase (human platelets) or 15-lipoxygenase (human neutrophils). CGS 22745 inhibited A23187-induced formation of LTB4 in blood (IC50's of 4.3, 0.56 and 3.2 microM for human, dog and rat, respectively). At 1 mg/kg i.v. in dogs, it caused 96% inhibition of A23187-stimulated LTB4 formation ex vivo after 5 min. Its effective biological half-life was greater than 160 min. In dogs at 3 and 10 mg/kg p.o., CGS 22745 inhibited ex vivo A23187-stimulated LTB4 formation at 3 hr by 48% and 97%, respectively. The inhibition persisted up to 6 hr (26% at 3 mg/kg; 49% at 10 mg/kg). CGS 22745 (3, 10 and 30 mg/kg p.o.) inhibited exudate formation, mononuclear cells and PMN accumulation in a dose-dependent manner during the late phase (48 and 72 hr) of carrageenan-induced pleurisy in the rat.

Inhibition of human leukocyte 5-lipoxygenase by a 4-hydroxybenzofuran, L-656,224: Evidence for enzyme reduction and inhibitor degradation

Detailed studies of the interaction of L-656,224 (2-[(4′-methoxyphenyl)methyl]-3-methyl-4-hydroxy-5-propyl-7-chlorobenzofuran) with 5-lipoxygenase were conducted using the enzymes from human and pig leukocytes. L-656,224 was a potent inhibitor of these 5-lipoxygenases although its efficiency varied with enzyme concentration. L-656,224 also stimulated the pseudoperoxidase activity of 5-lipoxygenase as measured by the consumption of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD), indicating that this compound can reduce the enzyme. Furthermore the inhibitor was degraded rapidly by both cell-free leukocyte extracts and purified 5-lipoxygenase after incubation with 13-HPOD, ATP and calcium ions. The degradation of L-656,224 was also observed during inhibition of the lipoxygenase reaction and occurred mainly after the initial lag phase of the reaction when hydroperoxides begin to accumulate. A single major radioactive product was formed after incubation of [ 3H]L-656,224 with purified 5-lipoxygenase in the presence of 13-HPOD. This product was unstable and could not be isolated. During the course of the pseudoperoxidase reaction, [ 3H]L-656,224 covalently labelled the enzyme, suggesting that a chemically reactive species had been formed. These data are consistent with the hypothesis that L-656,224 reduces the oxidized form of the 5-lipoxygenase to an inactive form, with degradation of the inhibitor and regeneration of the active enzyme with hydroperoxides.

Sources of Antibody-Dependent Cellular Cytotoxicity Deficiency in Young Pig Leukocytes

Antibody-dependent cellular cytotoxicity (ADCC) activity against pseudorabies virus-infected target cells has been found to be lower in young pig peripheral blood leukocytes (PBLs) than in adults. Experiments were designed to investigate the reason(s) for low activity in the young, which are more at risk of fatal infection than adults. The percentage of polymorphonuclear leukocytes (PMNs), the major ADCC effector cell, in the whole leukocyte population did not have a bearing on the deficiency. Enrichment for PMNs did not alleviate differences in activity between young and adult pigs. Additionally, no suppressor cell(s) or factor(s) could be demonstrated to account for the ADCC deficiency. The source of the ADCC deficiency in the young was found to be related to the decreased ability of young pig effector cells to bind antibody-sensitized targets. This deficiency relative to adults was associated with decreased antibody binding to high affinity Fc receptors on young pig neutrophils.

Killing of oral, gram‐negative, facultative bacteria by the rabbit defensin, NP‐1

Oral, gram-negative, facultative bacteria, including Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. have been associated with destructive periodontal infection. Neutrophils play a critical role in defending the periodontium against destructive infection. Defensins are antimicrobial peptides that have been isolated in human, rabbit, guinea pig, and rat leukocytes that may constitute an important nonoxidative mechanism of killing. The purpose of this study was to examine the sensitivity of a battery of oral, gram-negative, facultative bacteria to the bactericidal effects of the isolated rabbit peptide NP-1. All species tested were killed by NP-1; however, there was strain-to-strain variation in sensitivity. The bactericidal effect was not dependent on net bacterial growth, although metabolic activity was evident as assessed by bacterial oxygen consumption. We conclude that bacteria are sensitive to the cidal mechanism involved in defensin-mediated bacterial killing and that the conditions of this assay system support the killing of bacteria by the defensin peptides.

Major-histocompatibility-complex-class-II-positive cells and interleukin-2-dependent proliferation of immune T cells are required to reject carcinoma cells in the guinea pig.

Tumor immunity induced by bacillus Calmette-Guérin was studied in the line 10 hepatocellular carcinoma (line 10) in the strain-2 guinea pig. Line 10 immunity was investigated in vitro with a lymphocyte proliferation assay using line 10 tumor protein extracted with 3 M KCl and in vivo by adoptive transfer of line-10-immune spleen cells. Monoclonal antibodies against guinea pig leucocyte markers were used to block functional properties of the immune cells in order to determine which cell types or cell markers are involved in the immune response to the line 10 tumor. In vitro cells from the spleen, peripheral blood and regional lymph node of immune animals reacted with a proliferative response to line 10 protein. This antigen-specific response was caused by T cells and was regulated by major histocompatibility complex (MHC) class II molecules. In blocking experiments it was found that CT5 (anti-PanT), or MSgp4 [anti-(MHC class I antigen)] monoclonal antibodies did not block but sometimes stimulated the proliferative response. The effect of H159 (anti-PanT) was irregular, while H155 [anti-(T helper)], and 5C3 [anti-(IL-2 receptor)] monoclonal antibodies blocked the response almost completely. We studied the relevance of the results in vitro obtained and found that mAb 5C3 [anti-(IL-2 receptor)] inhibited the adoptive transfer of line 10 immunity, suggesting that the rejection of line 10 cells is caused by a mechanism that is interleukin-2 (IL-2)-dependent. Moreover, complement lysis of MHC-class-II-antigen-positive immune spleen cells inhibited completely the rejection of the line 10 tumor cell challenge in the adoptive-transfer experiments. In conclusion, our data show that MHC class II molecules or cells possessing these molecules are involved in immunity against line 10 tumor cells, as (a) monoclonal antibodies against MHC class II antigens inhibited the in vitro proliferative response of T cells to tumor antigens and (b) removal of MHC-class-II-positive immune spleen cells abrogated the antitumor effect in the adoptive-transfer experiments. Interleukin-2-dependent proliferation of immune T cells is required for the rejection of line 10 tumor cells.