During the course of studies involving the in vitro manipulation of channel catfish peripheral blood leukocytes, spontaneous proliferation was observed with unexpectedly high frequency. Propagation ...of these spontaneously proliferating cells has resulted in the development of long-term (> 11 mo.) cell lines which stain positively for nonspecific esterase and peroxidase, are phagocytic for latex beads, and morphologically resemble mammalian monocytes or macrophages. These long-term cell lines also exhibit two important additional functional features. First, induction with lipopolysaccharide results in the secretion of relatively high levels of catfish high and low molecular weight species of interleukin-1 active on channel catfish and mouse T cells, respectively. Second, these cell lines are efficient antigen-presenting cells to autologous peripheral blood leukocytes for antigen specific in vitro proliferative and antibody responses. This antigen-presenting function is blocked by inhibitors known to prevent antigen processing and presentation by mammalian monocytes. Allogeneic mixtures of cell line (used as antigen-presenting cells) and responding peripheral blood leukocytes, however, resulted in strong mixed leukocyte reaction but not in specific antibody responses. The availability of such cell lines should facilitate further studies on accessory cell functions in fish immune responses.
Objective
Lymphotoxin β (LTβ), a cytokine produced by T cells and B cells, plays a central role in the normal development of lymph nodes and is critical in the formation of ectopic germinal center ...reactions in rheumatoid synovitis. Because resident fibroblast‐like synoviocytes (FLS) express receptors for LTβ, we examined the consequences of FLS activation by LTβ.
Methods
FLS from patients with rheumatoid arthritis were isolated and examined for the expression of LTβ receptor. FLS were incubated with LTα1β2 and assayed for the production of cytokines and chemokines and the up‐regulation of adhesion molecules.
Results
Exposure of FLS to recombinant LTα1β2 resulted in the production of multiple inflammatory cytokines and metalloproteinases, implicating FLS as amplifiers of the inflammatory process in the inflamed joint. Additionally, LTα1β2 was found to up‐regulate the expression of cell adhesion molecules, rendering FLS to efficient adhesion substrates for T cells. LTα1β2 also induced production of the chemokines CCL2 and CCL5, which elicited transmigration activity of T cells. Upon stimulation with LTα1β2, FLS did not acquire characteristics of follicular dendritic cells.
Conclusion
These data document that FLS are involved in multiple stages of the inflammatory process, including the recruitment and retention of lymphocytes in the synovial microenvironment. We propose that the heterotypic interaction between LTβ‐producing lymphocytes and responding FLS contributes to the establishment of complex lymphoid microstructures, and that this may be one element that defines susceptibility of the synovial membrane to lymphoid organogenesis.
Recent analyses of the upstream regulatory regions of the class I major histocompatibility complex genes in higher primates provided a generalized structural basis for the differential expression of ...A- and B-locus gene products in response to specific physiological stimulus. Among the regulatory sequences that differ between the loci is the interferon-responsive element (IRE). While the B-IRE is conserved, the A-IREs have species-specific sequence variation. We previously demonstrated that the B-IRE was an interferon (IFN)-inducible enhancer, whereas none of the A-IREs were functional. In the present study, we examined the biochemical basis for the enhancer activity of the conserved B-IRE and found that this may be attributed to a novel γ-IFN-inducible factor. This factor accumulated in nuclei of cells within minutes of exposure to γ-IFN. Its appearance was independent of de novo protein synthesis. However, it was not detected in nuclei of cells treated with herbimycin A, suggesting that its appearance depends on a protein kinase activation pathway. Supershift assays indicated that it was distinct from STAT1α, IFN regulatory factor-1, and p48, transcription factors known to bind IRE-like sequences found in regulatory regions of many non-major histocompatibility complex γ-IFN-responsive genes. Competition assays show that this novel factor bound B-IRE with relatively high affinity, about 100-fold more than that for the A-IRE sequence. This factor was also present in STAT1α and p48 somatic mutants that also exhibited B-IRE enhancer activity in reporter gene bioassays in a manner similar to those seen with wild type cells. These observations indicate the existence of a novel γ-IFN-dependent transcriptional activation pathway that correlates with the differential enhancer activity of the HLA-B IRE.
Studies were conducted to address the role(s) of antigen (Ag) processing/presentation in channel catfish immune responses. Vigorous and specific secondary in vitro proliferative and antibody (Ab) ...responses were obtained to keyhole limpet and Limulus polyphemus hemocyanins with peripheral blood leukocytes (PBL) from catfish previously primed in vivo with Ag. In addition, such antigen-specific in vitro proliferative and Ab responses were efficiently elicited by antigen-pulsed and subsequently paraformaldehyde-fixed autologous PBL used as putative antigen-presenting cells (APC) but not by APC fixed prior to Ag pulsing. Treatment of these putative APC with lysosomotropic agents, protease inhibitors, or the ionophore monensin prior to or during pulsing with Ag significantly inhibited both in vitro responses. Furthermore, the use of radiolabeled protein indicated that both untreated and inhibitor-treated PBL but not erythrocytes take up Ag; however, only untreated PBL were able to degrade Ag. Immune restriction was indicated by the use of allogeneic PBL as APC in that only strong MLRs were generated with no detectable antibodies produced in vitro. Finally, the employment of isolated leukocyte subpopulations demonstrated that both catfish B (sIg+) lymphocytes and monocytes were efficient Ag presentors.
Using the structurally defined protein antigen cytochrome C, studies were conducted in an attempt to delineate the fine specificities of channel catfish immune repertoires. We have previously ...reported that species variants of cytochrome C were cross-stimulatory to peripheral blood leukocytes (PBL) from catfish immunized with the pigeon variant. Molecular database analyses revealed the existence of overlapping epitopes that appear to define the specificity of the immune response to a "family" of closely related antigens. To further explore these observations, studies were conducted to determine the contribution of peptide 81-104 to the immunogenicity of cytochrome C. Current data showed that peptide 81-104 and intact cytochrome C were stimulatory to PBL from fish previously immunized with the native molecule. In contrast, PBL from fish previously primed with the peptide 81-104 responded only to the immunizing peptide as well as to some, but not all, variants of the peptide 81-104. The differences in the stimulatory capacities of the peptide variants appeared to correlate with amino acid substitutions at various positions of the peptide and changes in their predicted secondary structures.
This work was undertaken to investigate whether or not antigen processing and presentation are important in channel catfish in vitro secondary immune responses elicited with structurally defined ...proteins, namely, pigeon heart cytochrome C (pCytC), hen egg lysozyme, and horse myoglobin. The use of in vitro antigen-pulsed and fixed B cells or monocytes as antigen presenting cells (APC) resulted in autologous peripheral blood leukocytes (PBL) responding with vigorous proliferation and antibody production in vitro. In addition, several long-term catfish monocyte lines have been found to function as efficient APC with autologous but not allogeneic responders. Subsequent separation of the responding PBL into sIg- (T-cell-enriched) and B (sIg+) cells subsets showed that both underwent proliferative responses to antigen-pulsed and fixed APC. Moreover, allogeneic cells used as APC were found to induce only strong mixed leukocyte reactions without specific in vitro antibody production. Initial attempts at identifying the immunogenic region(s) of the protein antigens for catfish indicated there are two such regions for pCytC, namely, peptides 66-80 and 81-104.
Studies were conducted to address further the role(s) of antigen processing and presentation in the induction of immune responses in a phylogenetically lower vertebrate, specifically a teleost, the ...channel catfish. In particular, studies were aimed at determining the subcellular compartments involved in antigen degradation by channel catfish antigen-presenting cells (APC) as well as ascertaining the reexpression of immunogenic peptides on the surfaces of APC. The results showed that exogenous protein antigens were actively endocytosed by APC as detected by flow cytometry. Use of radiolabeled antigen and subcellular fractionation protocols also showed that antigen localized in endosomes/lysosomes. Furthermore, there was an apparent redistribution of antigen between these organelles and the plasma membrane during the course of antigen pulsing. Functional assays for the induction of in vitro antigen-specific proliferation of immune catfish peripheral blood leukocytes (PBL) showed that membrane preparations from antigen-pulsed autologous APC were highly stimulatory. The magnitude of responses elicited with such membrane preparations was very similar to that of PBL cultures stimulated with native antigen-pulsed and fixed intact APC or prefixed intact APC incubated with a peptide fragment of the nominal antigen. Current data further corroborate our previous findings that steps akin to antigen processing and presentation are clearly important in the induction of immune responses in lower vertebrates like fish, in a manner similar to that seen in mammalian systems. Consequently, it would appear that many immune functions among the diverse taxa of vertebrates are remarkably conserved.
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