Chicken Thymus Research Articles

Spectrin Localization in Bursa of Fabricius and Thymus in Chickens.

Spectrin localizations were immunohistochemically observed by light and electron microscopies in bursa of Fabricius and thymus in chickens. Many bursal lymphocytes were detected as spectrin-positive. These spectrin-positivecells (SPC) were located in the follicular medulla in bursa and the cortical lymphocytes were detected as spectrinnegative. Interestingly, SPC was negligible in thymus. When tissue sections were pretreated with heating at 90°C for antigenicity retrieval of cytoskeletal proteins in aldehyde-fixed-sections before incubation with anti-spectrin, an enhancement on the stain intensity was noted and the degree of enhancement was dramatic. Strong stainings of spectrin were present in the epithelium of the mucosal fold, but was not apparent in follicle-associated epithelium. Furthermore, heat-induced antigenicity retrieval was not assessed in the interfollicular surface epithelium. Although the stain intensity was still weak, thymic lymphocytes were observed as spectrin-positive after heating at 90°C. Among the thymic SPC, epithelial cells were more strongly stained than the lymphocytes after the same pretreatment. Therefore, heating had an enhancing effect on stain intensity of spectrin that is partially susceptible to aldehyde fixation. Ultrastructurally, spectrin was mainly detected as aggregated or diffusive type in the cytoplasm after heating at 90°C. Any difference of subcellular localizations of Spectrin was not seen between the bursal and thymic SPC.

Presence of immunoreactive corticotropin-releasing hormone and cortisol molecules in invertebrate haemocytes and lower and higher vertebrate thymus.

Corticotropin-releasing hormone- and cortisol-like molecules are present in the haemocytes of different molluscan species and in the epithelial cells, interdigitating cells and macrophages - but not in the lymphocytes - of fish, frog, chicken and rat thymus. Taking into account the fact that other pro-opiomelanocortin-derived peptides, such as adrenocorticotropin hormone, are present in the haemocytes and thymus of the same species, these results complete the list of stress mediators present in molluscan haemocytes and further support the hypothesis that, although the prototype stress response we have demonstrated in invertebrates is concentrated in a single cell, i.e. the haemocyte, it is similar to the response seen in vertebrates. Moreover, the data presented here are compatible with the hypothesis that an evolutionary, conserved stress response can occur locally with a single organ, e.g. the thymus, in which all the main mediators of this biological response, such as corticotropin-releasing hormone, adrenocorticotropin hormone and glucocorticoids, are present. The implications of these findings for the physiology of thymus and stress response may be far reaching.

Analysis of splenic and thymic lymphocyte subpopulations in chickens infected with Salmonella enteritidis

Lymphocytes expressing CD3, CD4, CD8, pan lymphocyte, IgA, IgG and IgM cell surface antigens were assessed by in the spleen and thymus of chickens following infection with Salmonella enteritidis using flow cytometric analysis. At 6 days post primary infection and 2 days post secondary infection with S. enteritidis, the percentages of IgA + and IgM + lymphocytes in the spleen were significantly increased ( P < 0.05). At 2 days post secondary infection with S. enteritidis, the percentage of CD4 + T lymphocyte in the spleen and CD8 + T lymphocyte percentage in the thymus were significantly increased ( P < 0.05). These results indicate that S. enteritidis infection induces changes in the spleen and thymus that reflect the dynamics of the host protective immune response.

Localization of neuropeptides in endocrine cells of the chicken thymus.

Interactions between endocrine cells and epithelial cells, mediated by neurotensin, have been proposed in the chicken thymus. In this study, other neuropeptide candidates acting as mediators in the chicken thymus were examined immunohistochemically. Endocrine cells being oval, elongated or triangular in shape were immunoreactive with antibodies against methionine-enkephalin, neuropeptide Y, substance P, and vasoactive intestinal peptide. These findings suggest that 4 neuropeptides may be involved in cell-to-cell interactions in the chicken thymus.

Spectrin Expression in Thymus of Embryonic and Young Chickens.

Specrtrin, as a plasmalemmal undercoat protein in cytoskeleton, was observed immunohistochemically in thymus of embryonic and young chickens. Spectrin was negligible in thymic lymphocytes until middle stage of embryo and was then detected later stage of embryonic development. However, thymic lymphocytes expressed the spectirn weakly and the cells staining heavily with ant-spectrin could not find to be dominant before hatching. In young chickens, an apparent increase was not seen in the number of cells stained heavily with anti-spectrin. Ultrastructurally, cytoplasmic spectrins, together with the spectrin localization coinciding with plasma membrane, were detected in aggregated or diffusive form in embryonic lymphocytes. New type of spectrin localization was not seen in young chickens. These results may reflect the spectrin heterogeneity in thymic lymphocytes. In these spectrin-positive-cells (SPC), changes of spectrin characteristics, such as staining intensity or ultrastructural localization could not seem to parallel with acquisition of maturiy of thymic lymphocytes.

Evolution of neuroendocrine thymus: studies on POMC-derived peptides, cytokines and apoptosis in lower and higher vertebrates

In previous papers, we showed that neuroendocrine cells reactive to anti-POMC-derived peptides and cytokines are present in the thymus of a fish and an anuran amphibian. Here we report that this phenomenon is general, as neuroendocrine cells positive to anti-POMC-derived peptides (ACTH, β-endorphin, α-MSH) and cytokines (IL-1 α, IL-2, IL-6, TNF- α) are also present in the thymus of chicken and rat. However, the number and the intrathymic localization and distribution of these cells varies in the different species examined. An analysis of apoptotic cells or cells involved in apoptosis, such as interdigitating cells and macrophages, in fish, frog, chicken and rat thymus, using an immunocytochemical method and anti-DNA mAb conjugated with peroxidase (anti-DNA-POD), showed that cells positive to anti-DNA-POD mAb are present in the same thymic areas in which POMC-derived peptides and cytokines were found. In conclusion, these data on apoptotic cells in the thymus of lower and higher vertebrates are compatible with the hypothesis that neuroendocrine cells might play a role in the selection and apoptosis of thymic lymphocytes, a phenomenon which could vary slightly in different species and taxa.

Apoptosis and CD8-down-regulation in the thymus of chickens infected with Marek's disease virus.

Marek's disease virus (MDV)-infected chickens show thymic atrophy during the acute phase of infection. We examined whether the thymic atrophy by MDV-infection was mediated by apoptosis. Apoptosis-specific DNA ladderings were clearly observed in thymocytes one week after MDV-infection. Histological and flow cytometry studies revealed that immature CD4+ CD8+ thymocytes underwent apototic cell death. In addition, the expression level of CD8 molecules on both CD4(-)CD8+ and CD4+ CD8+ thymocyte populations was down-regulated in the infected chickens. These thymic changes might be involved in the pathogenesis of Marek's disease.

Structural Organization, Sequence, and Expression of the Chicken NRAMP1 Gene Encoding the Natural Resistance-Associated Macrophage Protein 1

One of the most common causes of food poisoning in humans is salmonellosis, which is frequently caused by ingestion with Salmonella-contaminated poultry products. Several lines of evidence suggest that genetic factors control resistance and susceptibility of chickens to infection with Salmonellae. In the mouse, innate resistance to infection with intracellular pathogens such as Salmonella typhimurium, several species of Mycobacteria, and Leishmania donovani is controlled by the mouse chromosome 1 Nramp1Bcg gene. To investigate the role of NRAMP1 in the differential resistance and susceptibility of chickens to infections with S. typhimurium, we have cloned and characterized cDNA clones corresponding to the chicken NRAMP1 gene. Nucleotide and predicted amino acid sequence analyses indicate that the chicken NRAMP1 polypeptide encodes a 555-amino-acid residue membrane protein with 12 putative transmembrane domains, two N-linked glycosylation sites, and an evolutionary conserved consensus transport motif. The peptide sequence identity among chicken, mouse, and human NRAMP1 is 68%. The chicken NRAMP1 gene contains 15 exons and spans 5 kb of genomic DNA. One major and two minor transcription initiation sites were detected using primer extension. Nucleotide sequencing of the promoter region revealed the presence of a classical TATAA element and consensus sequences for binding the myeloid specific PU.1 factor and several lipopolysaccharide (LPS) (NF-IL6 and NF-kappa B) and interferon-gamma (IFN-gamma)-inducible response elements. Similar regulatory elements are found in the promoters of mouse and human NRAMP1. Northern blot analyses revealed NRAMP1 expression in reticuloendothelial organs (spleen and liver), lung, and thymus. As demonstrated in mice and humans, the macrophage is also a major site of NRAMP1 mRNA expression in chickens. However, the high levels of expression detected in chicken thymus contrast with the absence of expression of the mammalian Nramp1 gene in this tissue.

Neurotensin-containing endocrine cells and neurotensin receptor mRNA-expressing epithelial cells in the chicken thymus.

Distribution of neurotensin-containing cells and neurotensin receptor-expressing cells was examined in the chicken thymus using a combination of in situ hybridization histochemistry for neurotensin receptor mRNA and immunohistochemistry for neurotensin. Neurotensin receptor mRNA-expressing cells and neurotensin-immunoreactive cells were localized in the medulla. Neurotensin receptor mRNA-expressing cells showed a round or elongated shape, while neurotensin-immunoreactive cells revealed round, ovoid, spindle or reticular shapes. Neurotensin-immunoreactive cells were much more numerous than those displaying labeling for the neurotensin receptor mRNA in males and females. Furthermore, electron microscopic immunohistochemistry for neurotensin showed that immunoreactive cells contained a large number of round or spherical granules which were 250-350 nm in diameter. This evidence indicates that neurotensin is localized in endocrine cells, and that neurotensin receptor mRNA is expressed in epithelial cells. It further suggests the possibility of microenvironmental interaction of neurotensin between endocrine cells and epithelial cells within the chicken thymus.

Intrathymic Distribution of the Two Avian Thymic Parvalbumins

Although parvalbumins are generally viewed as intracellular Ca2+buffers/transporters, avian species express two thymus-specific isoforms that may play alternative biological roles. These proteins, known as avian thymic hormone (ATH) and chicken parvalbumin 3 (CPV3), are conjectured to influence thymopoiesis. In this paper, we compare their intrathymic distributions. Isoform-specific monoclonal antibodies against ATH and CPV3 were labeled with fluorescein and Cy5, respectively, and then used to probe paraffin sections of chicken thymus tissue. Confocal microscopy of the stained sections reveals that ATH and CPV3 are both confined to the thymic cortex and that they are frequently coexpressed by a subset of epithelial cells. However, their expression patterns are not completely superimposible. Cortical epithelial cells are also observed that stain for just one of the two avian parvalbumin isoforms, albeit at lower frequency. Significantly, a subset of cortical thymocytes exhibits peripheral staining for one or both of the proteins. This result may imply the existence of plasma membrane receptors for the two proteins on select T-cell precursors. Alternatively, it may signal low level expression of the thymic parvalbumins by the thymocyte population.

Comparison of Virus Replication Efficiency in Lymphoid Tissues among Three Infectious Bursal Disease Virus Strains

In order to study replication efficiency of infectious bursal disease virus (IBDV) in lymphoid tissues, both the virus titers and the virus antigen titers in four lymphoid tissues were compared among chickens inoculated with Ehime/91 (about 50% mortality), J1 (no mortality), or K (attenuated) IBDV strains during 1-7 days postinoculation (PI). IBDV antigens in tissue homogenates were detected by an enzyme-linked immunosorbent assay. In the bursa of Fabricius, higher virus titers were maintained for 1-3 days PI in chickens inoculated with Ehime/91 or J1 strains, whereas the virus titers increased gradually and reached to the peak on 3 days PI in chickens inoculated with the K strain. There were no clear differences in both the virus and the virus antigen titers in bursae and thymus between chickens inoculated with Ehime/91 and J1 stains. However, the virus and/or the virus antigen titers in spleen and bone marrow of chickens inoculated with Ehime/91 strain were higher than those of the J1-inoculated chickens. Immunohistochemical analyses indicated that larger numbers of IBDV antigen-positive cells were detected in spleen and bone marrow of the Ehime/91 group than in those of the J1 group. There was almost no detectable virus and virus antigens in thymus, spleen, and bone marrow of the K-inoculated chickens throughout the experiment. These results suggest that efficient replication of IBDV not only in the bursa but also in the spleen and the bone marrow may be required for clinical infectious bursal disease.

Expression of the growth hormone gene in immune tissues

It is well established that growth hormone (GH)-like proteins and mRNA are present in immune tissues, but it is not known whether this reflects ectopic transcription of the GH gene or the expression of a closely related gene. This possibility was, therefore, investigated. Immunoreactive (IR) GH-like proteins were readily measured by radioimmunoassay and immunoblotting in the spleen, bursa of Fabricius and thymus of immature White Leghorn chickens, in which IR-GH was similar in size and antigenicity to the major GH moieties present in the pituitary gland. RT-PCR of mRNA from these immune tissues, with oligonucleotide primers spanning the coding region of pituitary GH cDNA, also generated cDNA fragments identical in size (689 bp) to pituitary GH cDNA.BamHI andRsaI cleavage sites were located in these cDNA sequences in the same position as those in pituitary GH cDNA. These amplified cDNA sequences also contained sequences that hybridized, by Southern blotting, with a chicken pituitary GH cDNA probe, thus suggesting a high degree of homology between pituitary and immune GH transcripts. The nucleotide sequence of the PCR products generated from these immune tissues, determined by a modified cycle dideoxy chain termination method, were also identical to pituitary GH cDNA. This homology extended over 593 bp of the spleen cDNA (spanning nucleotides 70-663 of the pituitary GH cDNA and its coding region for amino acids 5-201), 613 bp of the bursa cDNA fragment (spanning nucleotides 63-676 of the pituitary GH cDNA and its coding region for amino acids 3-207) and 607 bp of the thymic cDNA fragment (spanning nucleotides 61-665 of pituitary GH cDNA and its coding region for amino acids 4-203). These results clearly establish that the GH mRNA is present in immune tissues, in which GH-IR proteins are present. The local production of GH within the immune system of the domestic fowl, therefore, suggests it has paracrine or autocrine roles in modulating immune function.

Identification and analysis of the expression of CD8 alpha beta and CD8 alpha alpha isoforms in chickens reveals a major TCR-gamma delta CD8 alpha beta subset of intestinal intraepithelial lymphocytes.

Expression screening has been used to clone cDNAs encoding the alpha- and beta-chains of chicken CD8. Amino acid sequence similarities with the mammalian sequences were about 30%. Many amino acid residues of structural or functional importance were more highly conserved, as were the overall structures of both chains. Like human CD8 alpha, the chicken alpha-chain lacked sites for N-linked glycosylation, but the beta-chain contained three such sites. In COS cells transfected with CD8 beta cDNA, surface expression of the beta-chain was dependent on co-transfection of the alpha-chain cDNA, indicating that, as in mammals, chicken CD8 can be expressed as a CD8 alpha alpha homodimer or as a CD8 alpha beta heterodimer. Immunofluorescence analysis with mAbs that were shown to identify the CD8 alpha- and CD8 beta-chains revealed that the vast majority of the CD8+ cells in the thymus, spleen, and blood of adult chickens express both CD8 alpha- and CD8 beta-chains. However, a relatively large proportion of the CD8+ TCR-gamma delta cells in the spleens of embryos and young chicks express only the alpha-chain of CD8. Among intestinal epithelial lymphocytes the major CD8+ T cell populations present in mice are conserved, but there is a population of TCR-gamma delta CD8 alpha beta cells that is not found in rodents. This observation is important in interpretation of experiments examining the pathways of development of intestinal intraepithelial lymphocytes in chickens.

Ultrastructure of myoid cells in the chick thymus.

1. The ultrastructure of the myoid cells in the thymus of 2-week old chicks was examined by electron microscopy. 2. Myoid cells are round or elongated, distributed singly or in clusters in the medulla and at the corticomedullary junction. They are interposed between reticular epithelial cells to provide support for the meshwork of the microenvironment. They are also inserted into intercellular cysts, or associated with blood vessels and endocrine-like cells. 3. The cytoplasm of the myoid cell is packed with myofibrils arranged in circular bundles around the nucleus, aligned in register to form sarcomeres or randomly distributed. The interfibrillar cytoplasm contains smooth endoplasmic reticulum, mitochondria and free ribosomes. 4. The myoid cells contribute to the formation of the structural framework of the medulla. Their association with intercellular cysts and blood vessels suggests that their contractility may affect cystic and vascular flow.

A Novel Multilineage Cell-Surface Antigen Expressed on Terminally Differentiated Chicken B Cells in Mucosal Tissues

A murine monoclonal antibody, 5M19, produced to a cell line transformed by an avian myeloblastosis virus, detects an antigen, designated chL5, that is expressed on multiple lineages of hematopoietic cells from noninfected chickens. Immunochemical analysis demonstrated the chL5 antigen to be a homodimer of two disulfide-bonded chains each having an apparent molecular weight of 128,000. The highest level of expression was found on myelomonocytic cells, including granulocytes, monocytes, and macrophages, as well as on activated T lymphocytes and plasma cells in mucosal tissues. A low level of chL5 expression is detectable on T lymphocytes in the thymus and peripheral tissues of juvenile chickens, and mitogen activation of T lymphocytes results in a twofold increase in antigen expression. By Day 15 of embryonic development chL5 is expressed by a small population (12%) of yolk sac cells and by the majority of mononuclear cells in the thymus, bone marrow, and spleen. Also, chL5 + cells populate the embryonic bursa of Fabricius before the appearance of IgM + B lymphocytes. A novel characteristic of this antigen is its expression on about 50% of plasma cells in the mucosal tissues of the Harderian gland and cecal tonsils while present on only 3-11% of circulating B cells and less than 2% of splenic plasma cells. Immature B lymphocytes are negative for chL5 expression. Mitogen-induced activation and differentiation of peripheral B cells does not alter the percentage of chL5 + B cells. This antigen, therefore, is a useful marker for the analysis of terminal differentiation of B lymphocytes. The cell distribution of chL5 and the correlation of expression with cell aggregation in vitro suggests that the antigen may be involved in cell adhesion.

Novel avian thymic parvalbumin displays high degree of sequence homology to oncomodulin.

CPV3 is a novel avian parvalbumin. It displays an isoelectric point of 4.6, intermediate between that of avian thymic hormone (pI = 4.3) and the muscle parvalbumin isoform (pI = 5.2). Expression of CPV3, like that of avian thymic hormone (ATH), is restricted to the thymic stroma. However, the CPV3 content of chicken thymus tissue (120 micrograms/g tissue) is 4 times lower than that of ATH (500 micrograms/g tissue). The polymerase chain reaction (PCR) was used to gain access to the nucleotide sequence of CPV3. A 147-base pair fragment of the coding sequence, corresponding to residues 48-97, was amplified from total chicken cDNA using degenerate PCR primers. A RACE-PCR strategy was then used to extend the known sequence in both the 5' and 3' directions. The cDNA sequence thus obtained includes 671 base pairs. Primer extension analysis suggests that the cloned cDNA corresponds to a full-length transcript. Northern analysis of chicken mRNA indicates that the average CPV3 transcript is approximately 800 nucleotides in length, significantly smaller than the ATH message (approximately 1000 nucleotides). Southern analysis suggests the presence of a single CPV3 gene in the chicken genome. The translated nucleotide sequence, displaying 108 residues between the initiator and termination codons, is that of a beta-parvalbumin. The CPV3 sequence exhibits 58% identity with ATH and 52% identity with the chicken muscle isoform. Interestingly, CPV3 and the mammalian oncodevelopmental parvalbumin called oncomodulin are identical at 73 of 108 residues (68% identity). Correspondingly, flow-dialysis measurements with 45Ca2+ indicate that the Ca(2+)-binding domains are inequivalent, as in oncomodulin. The apparent dissociation constants, at pH 7.4 in 150 mM NaCl, are approximately 10 nM and 80 nM.

An active FK506-binding domain of 17,000 daltons is isolated following limited proteolysis of chicken thymus hsp56.

We have previously identified hsp56, a protein component of steroid receptor complexes, as an FK506 binding protein [Yem et al. (1992) J. Biol. Chem. 267, 2868-2871]. We now report that hsp56 is also found to be a major immunophilin in chicken thymus, by virtue of binding to FK506-Affi-Gel-10 as well as positive cross-reactivity with a polyclonal antiserum directed against human hsp56. Limited digests of purified chicken hsp56 with endoproteinase Lys C result in the production of a unique polypeptide having a mass of about 17 kDa (p17), as judged by Western blotting. Peptide mapping provided additional proof that p17 is a fragment which comprises the entire FK506 binding domain I of chicken hsp56, terminating with an Arg-Lys which might represent a processing site. Binding of radiolabeled dihydro FK506 to p17 is saturable with a calculated KD of 42 nM. Since size exclusion chromatography of drug-p17 complexes indicates that the active species is a homodimer with a mass of 30-40 kDa, the stoichiometry calculated for the drug-protein complex is approximately 1:1. Furthermore, unlike FKBP-12, chicken p17 bound to FK506 does not bind to calcineurin-calmodulin complexes. This work demonstrates the excision of a domain from an hsp56 protein that is active in binding FK506 and functionally distinct from FKBP-12, a protein of similar size and structure.

Presence of Lesions without Virus Replication in the Thymus of Chickens Exposed to Infectious Bursal Disease Virus

Specific-pathogen-free (SPF) chickens were exposed to the IM and VA isolates of virulent infectious bursal disease virus (IBDV). Both viruses induced rapidly progressing lymphoid cell depletion in the bursa. The bursal lesions persisted through the observation period of 16 days. The virus-exposed birds also had histologic lesions in the thymus. Thymic lesions peaked at 3-4 days postinoculation (PI) and then subsided. Immunofluorescence (IF) and antigen-capture enzyme-linked immunosorbent assay (ELISA) detected abundant viral antigen in the bursa, but not in the thymus, of chickens during the first week after infection with IM-IBDV or VA-IBDV. This result indicated that the presence of histologic lesions in the thymus was not associated with active infection and replication of the virus in thymic cells. Inoculation of homogenates of bursal and thymic tissues from virus-exposed chickens into embryonated chicken eggs revealed the presence of infectious virus from both tissues. We speculated that the virus recovered from thymus may have been contributed by virus-infected cells that were circulating through the thymus at the time when this tissue was homogenized.

Depletion of CD4 + and CD8 + T Lymphocyte Subpopulations by CIA-1, a Chicken Infectious Anemia Virus

The suppressive effect of chicken infectious anemia virus (CIAV) on T-lymphocyte subpopulation was evaluated in vivo by flow cytometry and dual immunostaining on frozen sections. Between 14 and 21 days postinoculation (PI), the percentage of CD4-, CD8-, and CT1-positive (CD4+, CD8+, and CT1+) cells was significantly lower in chickens infected at 1 day of age with CIA-1 strain of CIAV than in controls. The mean percentage of CD4+ cells in the thymus was only 43%, whereas in the controls it was 77%. The mean percentages of CD8+ cells in the thymus in infected and control chickens was 54% and 90%, respectively, and of CT1+ cells, 44% and 92%, respectively. At 28 days PI, the percentage of CD4+, CD8+, and CT1+ cells was similar in infected and control chickens. Also at 14 and 21 days PI, immunoperoxidase staining demonstrated fewer CD4+, CD8+, and CT1+ cells in the thymus of infected chickens than in controls. In frozen sections of thymus stained with CIA-1 antibodies and CD4, CD8, or CT1, few of the cells positive for CIAV antigen seen in the outer zone of the cortex carried CD4, CD8, or CT1 molecules. These results suggest that CIA-1 infection either destroys cells expressing CD4, CD8, or CT1 molecules on their surface or interferes with the expression of these molecules.

Protection against Apoptosis in Chicken Bursa Cells by Phorbol Ester in Vitro

Like the thymus, the bursa of Fabricius is a site of massive lymphopoiesis accompanied by cell death in vivo. In the present study we have, therefore, examined whether chicken bursa and thymus cells exhibit apoptosis. Bursa and thymus cells from SC chickens, 4-10 weeks of age, were incubated for 8-24 hr with various reagents. Genomic DNA was isolated, electrophoresed in 3% Nusieve agarose gels, and examined for patterns of DNA fragmentation. A laddering of DNA in multiples of 200 base pairs, indicative of apoptosis, was observed particularly with bursa and, to a much smallest extent, with thymus or spleen cells. These patterns of DNA fragmentation from bursa cells could be prevented by adding phorbol myristate acetate (PMA) but not by its inactive analog 4α-PMA during culture. Ionomycin is not required for this effect and, alone, appears to be slightly toxic for bursa cells, although it does not inhibit the effect of PMA. PMA did not affect the degree of DNA fragmentation in spleen or thymus cells. The addition of the protein kinase C (PKC) inhibitor staurosporine abolished both the preventive effect of PMA on apoptosis and its protective effect on bursa cells, as assayed by [ 3H]thymidine incorporation 24-48 hr after the initiation of cultures. PKC inhibitors also prevented the proliferation-inducing effect of PMA + ionomycin on spleen cells. It is concluded that the activation of protein kinase C and perhaps other kinases protects against apoptosis in cultured bursa cells.