Ontogeny of reactive nitrogen species production by blood phagocytes in pigs.

The aim of this work was to evaluate ontogeny of reactive nitrogen species (RNS) production by peripheral blood phagocytes in pig. Pig fetuses (55 and 92 days of gestation) and postnatal piglets (1, 3, 8, 17, 31 and 41 days after birth) were used. RNS production was measured by fluorescent probes diaminofluorescein-diacetate (DAF-FMDA) and dichloro-fluorescein-diacetate (H2DCFDA). Levels of nitration of cell proteins were established by immunofluorescent detection of nitrotyrosine. Levels of plasma nitrites/nitrates were detected spectrophotometrically by Griess reaction. Nitric oxide production measured by DAF-FMDA in neutrophils decreased during postnatal life. Spontaneous RNS measured by H2DCFDA decreased from 55th day of gestation to the 41st day of life. Phorbol-12-myristate-13-acetate activated production decreased during postnatal life. Production of NO measured by DAF-FMDA in macrophages decreased from the first to 41st day after birth. RNS production measured by H2DCFDA in monocytes did not show any significant changes during ontogeny. The level of nitrotyrosine significantly decreased from the third to 17th day. Levels of plasma nitrites/nitrates gradually decreased from the 55th day of gestation to the 41st day after birth. A temporary increase in all parameters occurred after weaning, but without any significance. In conclusion, RNS production has a decreasing trend during ontogeny and is transiently upregulated after weaning.

Caspase 3 activation in the primary enamel knot of developing molar tooth.

Mammalian teeth develop during embryogenesis as epithelio-mesenchymal organs. The primary enamel knot is considered as a signaling center in tooth morphogenesis. After tooth bell formation, this epithelial structure undergoes apoptosis. Activation of caspase 3 represents a crucial step in the intracellular death machinery. Procaspase 3 and caspase 3 molecules were localized in the primary enamel knot of the field vole using immunohistochemistry. Different fixation procedures in cryopreserved and paraffin-embedded tissues and detection systems based on peroxidase and alkaline phosphatase mediated color reactions were applied. Apoptosis was detected using morphological criteria and the TUNEL assay. Procaspase 3 was found in both the epithelial and mesenchymal part of the tooth germ. Active caspase 3 was localized particularly in the primary enamel knot, its distribution correlated with dental apoptosis and showed a similar pattern in the field vole as in the mouse.

Identification of T and B lymphocytes in pigs by combined E-rosette test and surface Ig labelling.

A procedure for combining labelling of B lymphocytes with [125I]anti-Ig-antibodies and E-rosetting is described which permits simultaneous detection of B lymphocytes and T lymphocytes. Pig lymphocytes were incubated with iodinated anti-Ig antibodies and the E-rosette test with SRBC was performed. E-rosettes appeared stable after methanol fixation of cell smears on agarose-coated slides and treatment for autoradiography on stripping films. The percentage of cells classified as B or T lymphocytes in pig peripheral blood closely resembles that in man. No doubly marked cells were demonstrated provided cells were classified as E-rosettes only when 5 or more red cells were attached.

Skin response to tuberculin in pig foetuses and germ-free piglets.

Cellular response to intradermally administered PPD (2 TU) was demonstrated in pig foetuses of various ages and in germ-free piglets. CD2+, CD4+, CD8-, 86D-, SLA-D- T lymphocytes were the predominant cells in the skin tuberculin reaction in both foetuses and germ-free animals. Reactive T cells were observed as early as in mid-gestation, whereas SLA-D+ (porcine MHC class II) cells appeared only in older foetuses. Polymorphonuclear leukocytes were never observed in the PPD reaction.

Alpha-fetoprotein and phagocytosis in athymic nude mice.

Alpha-fetoprotein (AFP) was found to suppress the phagocytic activity of the blood monocytes and neutrophils in vitro. The amounts of AFP detectable by immunofluorescence in the livers of nu/nu, nu/+ and +/+ mice were quite comparable, and thus could not have been responsible for the alterations in phagocytosis found in leukocytes of athymic nude mice during their ontogenetic development.

Development of immune responses in early pig ontogeny.

Low amounts of immunoglobulins, produced without any known cause of stimulation, can be detected in sera and cells of fetal and colostrum deprived newborn pigs. These immunoglobulins are believed to represent the preimmune antibody repertoire on the basis of their polyspecificity and reactivity against self antigens. In vitro activation of liver and spleen cells with various polyclonal B cell activators (PBA) results in pronounced immunoglobulins synthesis as measured in the culture media by enzyme-linked immunosorbent assay. Intrauterine injection of fetal and germfree pigs with PBA led to increased IgM, IgG and IgA levels in sera. Specific responses during fetal development were studied after intrauterine immunization. Antibodies to the heapten and its carrier flagellin, could be detected 7 days after the immunization of 55-day-old fetuses. Fetal and colostrum germfree pigs may be useful experimental models in which developmental immunity can be studied in the absence of maternal antibodies and environmental antigens.

Apoptotic DNA alterations in pig leukocytes after phagocytosis of bacteria are linked to maturation of the immune system.

The effect of phagocytosis of living bacteria on apoptotic DNA changes was examined in pig leukocytes in relation to immune system maturation. Blood samples of pigs (aged 6, 12 and 18 weeks) were cultivated with a suspension of bacterial cells Salmonella typhimurium LB 5000 at 37 (o)C. In the experimental groups, killed bacteria and microspheric particles were used to detect the influence of the phagocytic process. Phagocytic activity and index were determined in each sample by means of microspheric particles. The ability to kill engulfed microbes (bactericidal capacity) was estimated from the decrease in bacterial colony-forming units (CFU). Samples of cultured cells were taken for DNA analysis at given intervals. DNA ladder assay was used for qualitative apoptotic DNA break detection and the TUNEL AP test was employed for quantification of apoptosis. In 18-week-old animals, spontaneous DNA degradation was observed in the control group without phagocytosis after 8 h. In contrast, cells cultivated with microspheric particles or killed bacteria became apoptotic after 4 h. The rate of apoptotic DNA degradation was decreased in the group exposed to living bacteria. This prolonged survival of phagocytes was also detected in 12-week-old animals, but not at 6 weeks of age. These findings were supported by the ability of phagocytes in 6-week-old animals to engulf microbes, but their killing (bactericidal) ability was significantly decreased in comparison with other stages of immune system maturation. These results suggest that the process of phagocytosis itself is accompanied by activation of the apoptotic program in phagocytic cells of the pig immune system, but the presence of phagocyted living bacteria can delay this activation. The prolonged survival of short-lived cells was only observed in later phases of immune system maturation.

Cell surface enzymes of brain cortex cells or lymphocytes during early allogeneic reaction.

The aim was to study the role of major histocompatibility complex (MHC), in mice named H-2, during early allogeneic reactions (AR) of brain cortex cells or lymphocytes. We used neuronal and glial enriched perikarya, spleen and thymus lymphocytes or their subpopulations. Rat AR was also assayed between C-6 astrocytoma cells and spleen lymphocytes. We demonstrated that: 1) H-2 dependent stimulation of Na+,K+-ATPase and ouabain-sensitive K+-dependent p-nitrophenylphosphatase (K+-pNPPase) activities represented specific response in both AR of unseparated brain cells or lymphocytes. On the other hand, non-specific AR-induced stimulation of Ca2+-ATPase activity was observed. 2) Allogeneic enriched glial fractions reacted similarly by the same enzyme activation in contrast to no change in AR between enriched neuronal fractions. Allorecognition ability of glial cells was confirmed by AR between C-6 astrocytoma cells and lymphocytes. 3) Mature thymus lymphocytes exerted alloreactivity by specific activation of Na+,K+-ATPase or K+-pNPPase, in contrast to no change in AR between immature lymphocyte subpopulations. 4) MHC Class II monoclonal antibody inhibited Na+,K+-ATPase and K+-pNPPase activities in brain cells as well as in thymus and spleen lymphocytes in a dose-dependent manner. Results support former studies about alloantigen-induced uncoupling in brain oxidative cortex metabolism (Kováru Med. Biol. 58: 273, 1980) via Na+,K+-ATPase and K+-pNPPase inhibition by mechanism which can mimic MHC restriction.

Peptide cytokines in CNS and the immune system.

This study describes the effects of cytokine peptides released into the supernatant during an early allogeneic reaction (AR) of mouse spleen lymphocytes or brain cortex cells which differ in their major histocompatibility complex (MHC). The peptides were isolated by ultrafiltration, liquid chromatography and HPLC. We found that both peptides stimulated the cell surface Na+,K+-ATPase and Ca2+-ATPase activities of quiescent spleen lymphocytes in vitro and mimicked early allogeneic cell interactions. Both brain and spleen AR peptides inhibited Concanavalin A-stimulated spleen lymphocyte proliferation, whereas 3H-TdR incorporation into DNA of the E7 neuroblastoma cell line was stimulated by these peptides. The peptide isolated from the supernatant of the allogeneic brain cell reaction inhibited phagocytosis in phorbol myristate-stimulated LA5-9/8 mouse macrophage cell line. Immunosuppressive activity of spleen AR peptide is supported by inhibition of spontaneous E rosette formation by lymphocytes. The immunosuppressive effect of isolated peptide cytokines on lectin-activated lymphocytes was comparable with the serum thymic factor (FTS, Lenfant et al. 1983). These changes demonstrate the pleiotropic cytokine actions mediated by plasma membrane of immune system and brain cells.

Expression of MHC class II antigens and immunoglobulins in immunized pig foetuses.

Pig foetal spleen, liver and thymus cells were examined by using polyclonal antibody to pig immunoglobulins and monoclonal antibody reacting with a light chain determinant of pig MHC class II antigens. Pig foetuses were immunized with flagellin on the 72nd day of prenatal life. On the 14th day following antigen administration, large numbers of class II antigen-bearing cells and Ig-containing cells were demonstrated in the spleen using the immunofluorescence technique. Topographical localization of these cells in cryostat sections was very similar.

Immunoglobulins on the surface of lymphocytes in ontogeny.

Immunoglobulins on the surface of lymphocytes from pig foetuses and newborn precolostral piglets were studied using iodinated monospecific anti-pig gamma- or mu-antisera. Labelling of cells was determined by autoradiography in light and electron microscope. Beginning with the 80th day of gestation the lymphocytes with surface IgM were detected in significant amounts in the spleen, whereas at earlier stages of prenatal development the IgM-bearing lymphocytes were detected rarely. The cells which carry surface immunoglobulins were classified as small lymphocytes with typical ultrastructural morphology.

Autoimmunity: from physiology to pathology. Natural antibodies, mucosal immunity and development of B cell repertoire.

Presence of spontaneously produced immunoglobulins bearing a broad spectrum of "natural" antibody specificities (including autoantibodies) in sera and other body fluids results mainly from inapparent immunization and polyclonal B cell activation by microflora and food antigens occurring mostly on mucosal surfaces. Early postnatal ontogeny in external environment is characterized by rapid growth and functional maturation of secondary lymphatic tissues as a consequence of this "natural" mucosal immunization. Under normal circumstances a state of "oral" tolerance to intestinal antigens is actively established after this period. Studies performed in germ-free, antigen-free and maternal antibody-deprived animals showed that low amounts of natural antibodies (mainly of IgM isotype) are formed without any known cause of stimulation. These "nonstimulated" antibodies, similarly as hybridomas originating from nonimmunized newborns, correspond to the preimmune repertoire of antibodies characterized by poly-specificity, high connectivity and reactivity against self antigens. Together with other innate humoral and cellular factors, they probably represent the first line of anti-infectious resistance. Moreover, due to their connectivity they are supposed to play an important role in B cell repertoire shaping (forming an idiotypic network), through interaction with a broad spectrum of immunological components they act as regulatory molecules, and through their participation in catabolic events they can promote morphogenetic changes during fetal development. Beneficial therapeutic effects of nonspecific gammaglobulin (IVIG) application observed recently in patients with autoimmune diseases suggest that they can influence autoimmune reactivity by a not yet analyzed mechanism. Other functions of natural autoantibodies can be suggested and expected to be found in the near future.(ABSTRACT TRUNCATED AT 250 WORDS)

Precursors of B-lymphocytes with surface IgM and their possible migration from bone marrow to peripheral lymphatic tissues.

The lymphocytes with surface immunoglobulins (sIg) represent precursors of B-lymphocytes and were studied in ontogeny of pigs by autoradiography using iodinated monospecific anti-mu, anti-gamma or anti-alpha antibodies. The earliest detection of sIgM+ small lymphocytes was at 44 days of gestation in liver, followed at 51 days in spleen and at 60 days in bone marrow, the thymocytes being sIg-negative. The first circulating small lymphocytes were detected at 38--40 days and are most probably thymus-derived.

Inhibitory action of pig alpha-fetoprotein through early activated thymocyte surface Na+, K+-adenosinetriphosphatase.

An immunomodulatory effect of pig alpha-fetoprotein (AFP) on early phytohaemagglutinin (PHA)-stimulated intact thymocytes from piglets was analyzed. The activity of surface-localized Na+, K+-adenosinetriphosphatase was estimated, as the early reaction of mitogen-stimulated T-lymphocytes. A high inhibition of PHA-stimulated enzyme activity was found in the presence of AFP. The reaction was dependent of PHA concentration when using a constant AFP concentration. The highest AFP-induced decrease in PHA-induced Na+, K+-ATPase activity was 72%. AFP was isolated by a new technique based on salting out in ammonium sulphate and acetate buffer solutions. A gentle, simple and rapid procedure makes it possible to obtain AFP in a high degree of purity with preserved biological activity. The presented results can contribute to understanding the molecular aspects of AFP immunoregulation of lymphocyte reactivity in vitro.

Immunoglobulin-producing cells in lymphatic tissues of germfree and conventional rabbits as detected by an immunofluorescence method.

Lymphatic tissues of GF and CV rabbits were observed. No cells producing IgA and IgM antibodies were detected in appendix, sacculus rotundus, ileum terminale and thymus of GF rabbits. IgA cells were found in lymph nodes of GF rabbits.

Lymphatic tissue of the intestinal tract of germfree and conventional rabbits.

The lymphatic tissue of ileum, sacculus rotundus and appendix was poorly developed without germinal centres in germfree rabbits; on the other hand, in conventional rabbits, the lymphatic tissue was abundant with numerous germinal centers. In germfree rabbits, the stroma of villi of the jejunum and ileum possessed low cellularity, in conventional rabbits, the villi were wide with rich cellularity. The basal position of lymphocytes was predominant in the villi of ileum of germfree rabbits, in conventional rabbits a high percentage of lymphocytes was in the apical position.

Ontogenetic analysis of some surface markers on pig lymphocytes using fluorescence-activated cell sorter.

Surface markers were demonstrated on pig lymphocytes using anti-T cell-IgG and anti-Helix pomatia (HP) IgG during prenatal and postnatal development. A fluorescence-activated cell sorter analysis of T-cell surface markers was accompanied by an image analysis to prove the association of T antigenic determinants with the plasma membrane only. We found development-dependent changes in both anti-T cell and HP surface markers in both primary and secondary lymphatic organs. The number of T-positive (T+) cells estimated by anti-T cell-IgG was very similar to the results obtained by spontaneous E-rosette forming tests. At all selected age intervals, changes in the number of T+ cells were not significant in the thymus, but a marked increase in T+ cells was found in both spleen and lymph nodes. The image analysis confirmed the expression of T cell markers on the cell surface. The distribution of T cell markers was uneven, i.e. various degree of fluorescence intensity on whole ring-pattern projection of the cell surface image was estimated. In second lymphatic organs especially, fluorescence intensity of cells, i.e. total number of T cell markers estimated by anti-T cell-IgG, increased with age. On fetal day 73, T cell markers were slightly expressed, but very high fluorescence intensity and heterogeneous distribution of T cell markers on lymphocytes were found on fetal day 107 and postnatal day 56. The results indicate the possibility of functional maturation of various T cell markers on T cell subsets, furthermore a different degree of expression of T cell markers on various T cell subsets can be suggested. The number of HP+ cells increased with age in both primary and secondary lymphatic organs. In the prenatal period, the expression of HP receptors was very weak in both primary and secondary organs in contrast to the marked increase in HP+ cells during the postnatal interval. Differences in fluorescence intensity of cells were found, representing the increase by 22% in thymus cells comparing to cells of secondary lymphatic organs. Heterogeneity of HP+ cell populations in thymus was shown by the Scatchard plot, indicating at least two subpopulations of HP+ cells with different avidity to HP. Cells with low HP avidity could include a subset with cytolytic activity.

Ontogeny of T lymphocytes in the pig.

Mononuclear cells isolated from pig fetal thymus (and thymus region), spleen and cord blood were examined for their reactivity with polyclonal sheep anti-pig T cell antiserum. First immunofluorescence-positive cells were detected after 28 d of gestation in the thymus region, cord blood and the liver.

The effect of an antigenic stimulation on proliferative response in athymic mice.

The proliferation activity of the main cellular categories of bone marrow after infusion of 3H-thymidine was studied in nu/nu and +/+ 1-month- and 3-month-old BALB/c mice in comparison with lymphoid cells in the spleen and mesenteric lymph nodes. The stem cell defect in nu/nu mouse bone marrow is compensated by an increased proliferation in myeloid series and in agranulocytes. The increase of proliferation activity among lymphoid cells in peripheral lymphoid organs was observed only in the 3-month-old mice with a delay in the nudes.

Lysosomal enzyme activities in polymorphonuclear leukocytes, macrophages, serum, and spleen of conventional, germ-free, and antigen-free Minnesota miniature swine.

The activities of lysozyme, myeloperoxidase, and elastase were lower in PMNs and AMs from GF and AF Minnesota miniature piglets than in the leukocytes from their CONV counterparts. In the spleen and serum of gnotobiotic piglets only the levels of lysozyme were slightly reduced. Substantially depressed activities of these LEs were found also in PMNs from precolostral piglets in comparison with PMNs from their CONV mother. The bisassociation of GF piglets with Enterococcus liquefaciens and Escherichia coli caused an increase of LE activities in their AMs, spleens, and sera. Fewer LEs were released after phygocytic stimulation with zymosan from PMNs of GF, AF, and precolostral piglets than from PMNs of CONV animals of the same age. These data suggest that the antigenic-microbial stimulation is important for the development of normal lysosomal enzyme activities in PMNs and AMs from gnotobiotic animals.