L. William Clem

Phylogeny of lymphocyte heterogeneity: II. Differential effects of temperature on fish T-like and B-like cells

Abstract Lymphocytes from the bluegill, a freshwater fish, were observed to undergo in vitro mitogenic responses to a variety of “classical” mitogens. Using cell fractionation approaches based upon surface markers and in vitro mitogenesis, bluegill lymphocytes could be divided into two populations. One population responded to PHA and Con A but not to LPS, contained surface antigens in common with bluegill brain, and did not form spontaneous rosettes with rabbit erythrocytes. The other population responded to LPS but not to PHA or Con A, did not appear to contain surface antigens in common with brain, and did form rosettes with rabbit erythrocytes. The former population responded to mitogenic stimulation very well at 32 °C, whereas the latter population responded better at 22 than at 32 °C. The pattern of mitogenic responses and brain antigen distribution coupled with the observation that mixed lymphocyte responses were obtained at 32 but not at 22 °C makes it likely that the 32 °C responsive population represents the fish equivalent of T cells. The other population may be B cells. These data suggest that the immunosuppressive effects of low temperatures on cold-blooded animals may be effects on the generation of functional T cells and not on B cells.

Phylogeny of immunoglobulin structure and function-X. Humoral immunoglobulins of the sheepshead, Archosargus probatocephalus☆

Abstract The sheepshead has two readily isolatable humoral immunoglobulins (Igs), a ∼16S tetrameric form(s) and a ∼6S monomeric form. The 16S tetrameric population, while seeming to contain but one antigenic species, was observed to be composed of two subpopulations when analyzed for mass heterogeneity in the presence of denaturing solvents. The first subpopulation was a disulfide linked form of ∼700, 000 daltons likely composed of 8 heavy (H) and 8 light (L) chains. The second tetrameric subpopulation also appeared to contain 8 H and 8 L chains but was assembled as two noncovalent subunits of predominantly disulfide linked dimers, each with 4 H and 4 L chains and having a molecular mass of ∼350, 000 daltons. The molecular weights of the H and L chains from these tetrameric species were ∼70,000 and ∼25, 000 respectively. In vitro experiments indicated the two tetrameric subpopulations were not interconvertible. Alkaline-urea gel electrophoretic analysis of the pooled tetrameric subpopulations indicated that J chain was present in amounts that suggested only one of the subpopulations actually contained J chain. The 6S Ig (∼140, 000 daltons) was composed of two subunits (∼70, 000 daltons) linked to one another noncovalently. The subunits each contained 1 H (∼45,000 daltons) and 1 L (∼25, 000 daltons) chain. Tje 6S Ig was antigenically deficient to the 16S Igs, and it is suggested that this deficiency may be due to a ∼25, 000 dalton segment present in the H chain of the 16S molecules and missing from the 6S molecules.

Phylogeny of Immunoglobulin Structure and Function. XI. Secretory Immunoglobulins in the Cutaneous Mucus of the Sheepshead, Archosargus Probatocephalus

Abstract Sheepshead cutaneous mucus was shown to contain immunoglobulin (Ig) which was antigenically identical to the serum high molecular weight (HMW) Ig. Ion exchange and gel filtration chromatography were used to fractionate the mucous Ig into two distinct molecular weight populations. One population had a molecular weight of ∼700,000 daltons, dissociated in denaturing solvents similar to the serum HMW Ig, and was composed of heavy (H) and light (L) chains of ∼70,000 and ∼25,000 daltons respectively. The second molecular weight population chromagraphed as a dimeric Ig population of ∼350,000 daltons. A portion of these dimeric Ig proteins were bonded noncovalently and dissociated on sodium dodecyl sulfate polyacrylamide gels into monomeric forms. The other portion was observed to be covalently bonded and associated covalently with a ∼95,000 dalton protein which might be the teleost equivalent to the secretory piece of mammals. Both of the dimeric Ig populations had H and L chains of ∼70,000 and ∼25,000 daltons respectively. Additional work is required to assess the role possible secretory piece and the function of the secretory immune system in teleosts.

Recognition of viruses and xenogeneic proteins by the blue crab, Callinecyes sapidus. I. Clearance and organ concentration

Abstract The blue crab, Callinectes sapidus , was able to clear radio-labeled viruses and xenogeneic proteins from the circulation in a manner indicating recognition of self from non-self. Xenogeneic proteins and poliovirus particles were concentrated in the central axis of the gills, whereas bacteriophages appeared to be concentrated predominately in the hepato-pancreas. These results clearly indicate that the blue crab does not simply degrade and externalize foreign substances, but is capable of a relatively sophisticated recognition phenomenon in the normal or “non-immunized” state.

Phylogeny of immunoglobulin structure and function—XII. Secretory immunoglobulins in the bile of the marine teleost Archosargus probatocephalus

Abstract Sheepshead bile was shown to contain immunoglobulin which was antigenically identical to the high molecular weight tetrameric immunoglobulin found in sheepshead serum. Although the bile immunoglobulin appeared dimeric (mol. wt ~ 320,000 daltons) in physiological buffers, molecular weight determinations in the detergent sodium dodecyl sulfate indicated this dimeric protein dissociated into monomeric units (~ 160,000 daltons) each composed of two heavy and two light chains. Furthermore, the molecular weight (~ 55,000 daltons) of the heavy chains from the bile immunoglobulin was intermediate between those of the H chains from the serum high molecular weight and low molecular weight immunoglobulins (~ 70,000 and ~45,000 daltons, respectively). These findings have revealed a previously unappreciated heterogeneity in fish immunoglobulins and suggest that fish, like higher animals, may have the ability to synthesize immunoglobulins that are destined to function in secretions.

Temperature-Mediated Processes in Teleost Immunity: Homeoviscous Adaptation in Teleost Lymphocytes

Abstract In vitro studies with pinfish (Lagodon rhomboides) lymphocytes demonstrated that the temperature to which a fish is acclimated in vivo affects the temperature dependence of lymphocyte responses to mitogenic stimulation with conconavalin A (ConA) in vitro, i.e., at an in vitro temperature of 20°, the lymphocytes of 17° acclimated fish responded to ConA better than the lymphocytes of 27° acclimated fish. Fluidity measurements of plasma membranes from lymphocytes isolated from both cold- and warm-acclimated pinfish were made using fluorescence polarization spectrophotometry and the fluorescent membrane probe, 1,6-diphenyl-1,3,5-hexatriene. While the membranes of cold-acclimated pinfish were more fluid than those of warm-acclimated fish when both were measured at the same temperature, the fluidities were equal when measured at the respective acclimation temperatures. These results support the hypothesis that the process of homeoviscous adaptation of lymphocyte plasma membranes may be an important aspect of teleost immunity.

Phylogeny of lymphocyte heterogeneity. IV. Evidence for T-like and B-like cells in reptiles.

Abstract Peripheral blood lymphocytes from the Florida alligator were separated into at least two subpopulations based upon surface markers and mitogen responsiveness. Fractionation on glasswool columns yielded two subpopulations, a nonadherent one responsive in vitro to PHA and Con A but not to LPS, and an adherent one responsive to LPS (and to PHA). Depletion of cells bearing surface immunoglobulin determinants (by complement-mediated cytotoxicity and by anti-immunoglobulin affinity columns) ablated the responsiveness to LPS (and PWM) but not to PHA. LPS, but not PHA, induced a subpopulation of alligator lymphocytes to differentiate into immunoglobulin producing cells. These data, and other related studies, indicate the Florida alligator likely has T and B cells akin to those of mammals and birds.

Phylogeny of lymphocyte heterogeneity. I. Membrane immunoglobulins of teleost lymphocytes

Immunofluorescence studies of bream lymphoid tissues revealed that our 90% of the lymphocytes from blood, anterior kidney, spleen and thymus exhibited membrane immunoglobulin determinants. Furthermore, a majority of such cells were observed to undergo patching and capping when the membrane proteins were complexed with antisera to fish serum immunoglobulins. Lactoperoxidase catalysed radioiodination, detergent lysis and immunoprecipitation with appropriate antisera were employed to study the properties of this membrane immunoglobulin. Quantification, by inhibition of immunoprecipitation with serum immunoglobulin, indicated the average amount of immunoglobulin determinants for bream lymphocytes from either blood, anterior kidney, spleen or thymus to be about half that present on mouse B cells. Physiochemical characterization of labeled membrane immunoglobulin from bream lymphocytes suggested that only one class of immunoglobulin heavy chain was present and that about one-half of this material resembled the monomeric (2H-2L chain) IgM-like proteins present in bream serum. A major unanswered question raised by this study was whether or not certain of the bream membrane immunoglobulin determinants were associated with molecules that did not resemble classical immunoglobulins.

Phylogeny of lymphocyte heterogeneity. III. Mitogenic responses of reptilian lymphocytes

Abstract Peripheral blood lymphocytes from Florida alligators were isolated and observed to undergo mitogenesis when cultured in vitro with Con A, PHA, LPS, PWM and PPD. These cells were also observed to participate in two-way mixed lymphocyte reactions. Optimal culture conditions included using mammalian medium (MEM) supplemented with adult alligator serum and additional sodium chloride and incubation at 32°C. In contrast to previous studies with lymphocytes from another ectotherm, the bluegill, a bony fish, alligator lymphocytes exhibited no differential effects of incubation temperatures when different mitogens were employed.

Phylogeny of immunoglobulin structure and function—VIII: Intermolecular heterogeneity of shark 19S IgM antibodies to pneumococcal polysaccharide☆

Abstract Significant IgM antibody responses to the pneumococcal capsular polysaccharide were demonstrated in nurse sharks immunized with pneumococcal vaccines. The antibodies isolated from immune sera by affinity chromatography were exclusively of the 19S variety; no 7S antibodies were isolated from any of six animals studied for periods up to twelve months. Equilibrium dialysis studies of the isolated antibodies demonstrated several important points: (1) the IgM antibodies contained ten combining sites per 19S molecule, (2) in all cases, the isolated antibodies showed marked heterogeneity of combining sites, (3) the antibodies were all of low average affinity, and (4) there was no increase in the average affinity of the antibodies isolated from any single animal for periods of up to twelve months after the start of immunization. In order to determine if a single IgM molecule contains ten equivalent combining sites, the antibodies isolated from several animals were fractionated by liquid isoelectric focusing. Equilibrium dialysis experiments using focused fractions showed the presence of ten functionally identical combining sites per IgM molecule. As a proof of the structural homogeneity of focused fractions, antibodies were separated into H and L chains, § recombined and tested for the regeneration of the original combining site by equilibrium dialysis. The results indicated that recombinants of focused antibody fractions contained binding sites identical to those of the intact antibody, whereas heterogeneous (unfocused) recombinants and isolated H and L chains failed to show any binding activity. The conclusion from this study is that the heterogeneity of ligand binding exhibited by nurse shark 19S antibodies to the capsular polysaccharide of the Type III pneumococcus can be attributed to intermolecular heterogeneity.