Craig J. Lobb

Secretory immunity induced in catfish, Ictalurus punctatus, following bath immunization

Individual adult channel catfish were immunized by immersion in an antigen bath containing dinitrophenylated-horse serum albumin. Anti-DNP hemagglutination titers of serum and cutaneous mucus were determined following both primary and secondary bath immunization. The results showed that five of the six fish had a cutaneous mucosal anti-DNP titer following the bath immunizations. In contrast, only one of the six catfish was shown to have any demonstrable change in its serum anti-DNP titer following the bath immunizations. The mucous anti-DNP hemagglutinin was shown to be antibody (Ab). The affinity-purified mucous Ab was found to have the same complex tetrameric architecture as well as the same molecular weight heavy and light chains as serum anti-DNP Ab. Histological studies showed that the catfish epidermis was richly vascularized. Within the epidermis there were numerous lymphocytes which were predominantly associated with the basal layer. These studies indicate that the secretory immune system of catfish can be stimulated by external antigens. Secondly, these studies show that bath immunization can differentially effect the relative antibody response of the catfish secretory and systemic immune systems.

Fish Lymphocytes Differ in the Expression of Surface Immunoglobulin

Catfish peripheral blood and splenic lymphocytes were assayed for surface immunoglobulin using fifteen different mouse hybridoma antibodies to catfish immunoglobulin (Ig). These studied showed that this battery of monoclonal antibodies did not detect significant amounts of Ig on all lymphocytes. Unlike polyclonal antisera which demonstrated nearly 100% surface Ig+ cells, the monoclonal antibodies detected approximately 40% surface Ig+ cells. Furthermore, the percentage of Ig+ cells reactive with two of these monoclonals, tentatively shown to react with two different types of catfish light chains, was found to be nearly additive when the two antibodies were mixed. Thus it seems that fish lymphocytes, like their mammalian counterparts, have two different populations of lymphocytes; one which contains abundant surface Ig and one which does not. Whether these two types of cells represent the fish equivalents of B and T cells remains to be determined.

Temperature-mediated processes in teleost immunity: Differential effects of invitro and invivo temperatures on mitogenic responses of channel catfish lymphocytes☆

The in vitro mitogenic responses of channel catfish peripheral blood leucocytes to ConA and LPS were differentially affected by both in vitro and in vivo temperatures. The magnitude of the response to LPS was relatively independent of both in vitro culture temperature and in vivo acclimation temperature. The magnitude of the response to ConA was suppressed at lower in vitro temperatures although this suppression could be reduced by lower in vivo acclimation temperatures. In vitro temperature-shift experiments indicated that channel catfish PBL could respond to ConA at a lower in vitro temperature if first stimulated with ConA at a higher in vitro temperature. The converse, however was not true in that channel catfish PBL did not respond at a higher in vitro temperature after an initial stimulation with ConA at a lower in vitro temperature. This latter failure to respond could not be attributed to the induction of a suppressor cell (or factor) by exposure to ConA at a lower temperature. These studies, when coupled with other available data on channel catfish PBL subpopulations, are interpreted as supporting the hypothesis that low temperature immunosuppression in fish may result from preferential inhibitory effects on T cells rather than B cells.

A G Protein-coupled Receptor from Zebrafish Is Activated by Human Parathyroid Hormone and Not by Human or Teleost Parathyroid Hormone-related Peptide IMPLICATIONS FOR THE EVOLUTIONARY CONSERVATION OF CALCIUM-REGULATING PEPTIDE HORMONES

Genomic and cDNA clones encoding portions of a putative catfish parathyroid hormone (PTH) 2 receptor (PTH2R) led to the isolation of a cDNA encoding a full-length zebrafish PTH2R (zPTH2R). The zPTH2R shared 63 and 60% amino acid sequence identity with human and rat PTH2Rs, respectively, 47–52% identity with mammalian and frog PTH/PTHrP receptors (PTH1R), and less than 37% with other members of this family of G protein-coupled receptors. COS-7 cells expressing zPTH2R(43), a 5′ splice variant that lacked 17 amino acids in the amino-terminal extracellular domain, showed cAMP accumulation when challenged with [Tyr34]hPTH(1–34)-amide (hPTH) (EC50, 1.64 ± 0.95 nm) and [Ile5,Trp23,Tyr36]hPTHrP-(1–36)-amide ([Ile5, Trp23]hPTHrP) (EC50, 46.8 ± 12.1 nm) but not when stimulated with [Tyr36]hPTHrP-(1–36)-amide (hPTHrP), [Trp23,Tyr36]hPTHrP-(1–36)-amide ([Trp23]hPTHrP), or [Ala29,Glu30,Ala34,Glu35,Tyr36]fugufish PTHrP-(1–36)amide (fuguPTHrP). FuguPTHrP also failed to activate the human PTH2R but had similar efficiency and efficacy as hPTH and hPTHrP when tested with cells expressing the human PTH1R. Agonist-dependent activation of zPTH2R was less efficient than that of zPTH2R(43), and both receptor variants showed no cAMP accumulation when stimulated with either secretin, growth hormone-releasing hormone, or calcitonin. The zPTH2R thus has ligand specificity similar to that of the human homolog, which raises the possibility that a PTH-like molecule exists in zebrafish, species which lack parathyroid glands.

Analysis of Channel Catfish Peripheral Blood Leucocytes by Bright-Field Microscopy and Flow Cytometry

Abstract Peripheral blood leucocytes of channel catfish Ictalurus punctatus were characterized by Wrights stain, differential cytochemical stains, and immunofluorescence; lymphocytes, thrombocytes, neutrophils, and monocytes were identified. The morphologically similar neutrophils and monocytes were distinguished with a Sudan black B stain for neutrophils and an assay of nonspecific esterase activity for monocytes. Monocytes also phagocytized polystyrene beads. Leucocytes were physically separated by cytofluorography based upon their forward and 90° light-scattering properties. One population of sorted cells contained almost exclusively lymphocytes, a second contained predominantly thrombocytes, and the third contained both neutrophils and monocytes. Multiparameter cytofluorography with mouse monoclonal antibodies to channel catfish immunoglobulin revealed that a vast majority of leucocytes exhibiting surface immunofluorescence were lymphocytes, though only about 40% of the lymphocytes contained demonstr...

An effective culture system for studying in vitro mitogenic responses of channel catfish lymphocytes

Abstract Lymphocytes of channel catfish Ictalurus punctatus can be stimulated by lipopolysaccharide (LPS) and concanavalin A (ConA) to synthesize deoxyribonucleic acid (DNA) in vitro. The importance of the proper serum supplement and tonicity of the culture medium was established empirically. Responses to LPS were temperature-independent whereas the responses to ConA were suppressed at lower temperatures. Although peripheral blood and splenic lymphocytes exhibited similar responses to both mitogens, anterior kidney cells did not respond, probably due to the high background levels of DNA synthesis in this highly hematopoietic organ. Received September 27, 1982 Accepted June 12, 1983

Distinctive subpopulations of catfish serum antibody and immunoglobulin

High molecular weight antibodies and immunoglobulin of the channel catfish were found to be composed of heterogeneous mixtures of covalent subpopulations. Although these proteins appeared tetrameric in physiological buffers (mol. wt approximately 700,000), molecular weight estimates in the presence of the detergent sodium dodecyl sulfate indicated that they dissociated into eight distinct subpopulations. Each dissociative subpopulation was found to be stable under the conditions of electrophoretic analysis, and furthermore each was apparently composed of equimolar H and L chains. Based upon relative mobilities it seems probable that the catfish antibody was composed of various combinations of covalent linkages between covalently-linked H-L chain pairs, i.e. 8, 7, 6, 5, 4, 3, 2 and 1. Further studies suggest the presence of size heterogeneity in catfish light chains. Whether or not this distinction represents isotypic L chain differences is not yet known but these findings coupled with the complex architecture of the physiological tetramer suggest an unusual and previously unappreciated level of complexity present in fish antibody.

Covalent structure and affinity of channel catfish anti-dinitrophenyl antibodies☆

Serum antibody (Ab) of channel catfish, Ictalurus punctatus, is predominantly a tetrameric, approximately 700,000-dalton protein. This tetrameric Ab dissociates into 8 distinctive subpopulations, each composed of equimolar heavy and light chains, when in the presence of denaturing solvents. The present studies utilizing purified anti-DNP Abs obtained from catfish monitored for either short (1-2 months) or long (1-2 years) periods post-primary immunization with dinitrophenylated horse serum albumin showed no demonstrable differences in the ratio of the covalent Ab subpopulations. Although significant humoral Ab levels were sustained during the 1-2 year monitoring period, there were no significant changes in the binding affinities or heterogeneity of the induced Ab.

Immunoglobulin in the eggs of the channel catfish (Ictalurus punctatus)

Egg yolk proteins obtained from 2-3-day-old fertilized channel catfish eggs were analyzed to determine if immunoglobulin (Ig) was present. Using both monoclonal and polyclonal antibodies to catfish Ig, egg Ig was isolated and structurally characterized. The egg Ig when analyzed in SDS-gels under nonreducing conditions dissociated into eight distinct subpopulations with relative mobilities identical to that previously described for the serum Ig of the channel catfish. In addition, the component heavy (H) and light (L) chains of the egg Ig had similar relative mobilities as the H and L chains identified in serum Ig. Immunocytological studies using both polyclonal and monoclonal anti-catfish Ig showed that Ig was dispersed throughout the yolk of the egg. In addition these analyses indicated that Ig was localized within the external membranes (eggshell) of the egg.

The Nucleotide Targets of Somatic Mutation and the Role of Selection in Immunoglobulin Heavy Chains of a Teleost Fish

Sequence analysis of H chain cDNA derived from the spleen of an individual catfish has shown that somatic mutation occurs within both the VH- and JH-encoded regions. Somatic mutation preferentially targets G and C nucleotides with approximately balanced frequencies, resulting in the predominant accumulation of G-to-A and C-to-T substitutions that parallel the activation-induced cytidine deaminase nucleotide exchanges known in mammals. The overall mutation rate of A nucleotides is not significantly different from that expected by sequence-insensitive mutations, and a significant bias exists against mutations occurring in T. Targeting of mutations is dependent upon the sequence of neighboring nucleotides, allowing statistically significant hotspot motifs to be identified. Dinucleotide, trinucleotide, and RGYW analyses showed that mutational targets in catfish are restricted when compared with the spectrum of targets known in mammals. The preferential targets for G and C mutation are the central GC positions in both AGCT and AGCA. The WA motif, recognized as a mammalian hotspot for A mutations, was not a significant target for catfish mutations. The only significant target for A mutations was the terminal position in AGCA. Lastly, comparisons of mutations located in framework region and CDR codons coupled with multinomial distribution studies found no substantial evidence in either independent or clonally related VDJ rearrangements to indicate that somatic mutation coevolved with mechanisms that select B cells based upon nonsynonymous mutations within CDR-encoded regions. These results suggest that the principal role of somatic mutation early in phylogeny was to diversify the repertoire by targeting hotspot motifs preferentially located within CDR-encoded regions.