DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. Ii, pp. 431-434, 1987. 0145-305X87 $3.00 + .00 Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.

Workshop I. 51

IMMUNE RESPONSES OF NON-MAMMALIAN VERTEBRATES

SIXTH INTERNATIONAL CONGRESS OF IMMUNOLOGY

July 6-11, 1986

Toronto, Canada

The Workshop on "Immune Responses of Non-Mammalian Vertebrates" was opened with a short introduction by Dr. Agust[n G. Zapata from Complutense University of Madrid (Spain) who rewieved some of the main subjects repre- sented in the Workshop. On this occasion, I emphasized four general aspects which seem to govern the evolution of the vertebrate immune system: i) Anatomical/histological specialization of the lymphoid organs; 2) Diversi- fication and specialization of lymphoid populations with expression of highly specific surface markers; 3) Increased diversity of antibodies with higher numbers of immunoglobulin classes and subclasses, and 4) Higher and better control of immune reactivity with regulatory genes enclosed in a major histocompatibility complex. The discussion, therefore, was basically conducted by Dr. R. E1Ridi (Cairo University, Egypt) according to the themes for discussion previously grouped in six categories including: I) Functional heterogeneity of immunocompetent cells in lower vertebrates. Characterization, biochemical and functional properties; II) Molecules involved in the immune responses of lower vertebrates (growth factors, histocompatibility antigens, etc...); III) Avian immunology; IV) Natural immunity. Complement; V) Temperature and seasonal influences on lower vertebrate immune responses; and VI) Mucosal immunity.

In the first group, Dr. Raison (Clin. Immunology Research Center, University of Sidney, NSW, Australia) presented evidence of the existence in the hagfish of leukocyte populations analogous to mammalian lymphocytes and macrophages. He classified hagfish leukocytes on the basis of laser scatter profiles into lymphocyte-like (small WBC) and monocyte/granulocyte- like (large WBC) populations. Both populations showed remarkable phenotypic differences using a panel of monoclonal antibodies. Hagfish Ig was restrict- ively expressed on small WBC population and functional assessment of purified hagfish small and large leukocytes identified small WBC as re- sponders and large WBC as stimulators in MLR. Functional different subpopu- lations of channel catfish lymphocytes akin to T and B cells of higher vertebrates were demonstrated by Dr. Clem's group (Dept. of Microbiology, University of Mississippi, Medical Center, Jackson, MS, USA) using monoclonal antibodies reactive with surface antigenic determinants and in vitro assays of cellular function. According their results the surface immuno- globulin present on channel catfish B cells, but not on T cells consisted of ~ -like and L chains. Moreover, each of the antigens demonstrable on channel catfish T cells was different one from another and none was readily identified as any of the surface molecules known to be associated with mammalian T cells.

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Two posters, included in this first group, were devoted to reptilian immune reactions. Drs. Saad and E1 Deeb (not present in the Workshop) (Dept. of Zoology, Cairo University, Egypt) analyzed the ontogeny of ConA responsiveness and mixed leukocyte reactivity in the lizard, Chalcides ocellatus. The response of thymocytes to ConA was first detected at stages 36-37, increasing gradually to reach a maximum at stage 40 and then declined from stage 41 to birth, yielding low responses in newborn lizards. Embryonic thymocytes in two-way MLR, using several combination sets, responded significantly at all stages studied. However, embryonic thymocytes tested in one-way MLR showed low responsive indices with parental combinations in contrast to relatively high responses in non-parental sets. Dr. Pitchappan (Dept. of Immunology, School of Biological Sciences, Madurai Kamaraj University, Madurai, India) presented results on the structure and functions of thymocytes and splenocytes of thymic lineage in Calotes versicolor employing heterologous anti-lizard thymocyte sera (ATS). A pure population of lymphocytes obtained by adherence column and bouyant density methods capable of mediating antigen specific CMI as measured by migration inhibition technique was susceptible to ATS. However, sIg bearing cells or antigen specific plaque forming cells were not affected by ATS treatment'. Studies on the thymus revealed the presence of cells mediating antigen specific migration inhibition. Furthermore, quantitative absorption experiments revealed that the membrane markers detected by ATS were distributed differen- tially in different lymphoid organs and subsets of splenocytes. Thus, the splenic T-cells carried this marker 15 times more than the thymocytes suggesting that it might be a differentiation antigen.

Posters grouped under the theme "Molecules involved in the immune responses of lower vertebrates" included studies on invertebrate "cytokine"~ growth factors in Xenopus and histocompatibility antigens in reptiles. Robert A. Prendergast (Dept. of Ophthalmology, John Hopkins University, Baltimore, Maryland) reported characterization of a 39 kd protein (SSF) isolated from coelomocytes of the sea star Asterias forbesi with biological properties which resembled those of vertebrate lymphokines and monokines. They included macrophage migratory inhibition capacity, induction of skin lessions similar to those of delayed hypersensitivity and activation of macrophages for tumor cell cytostasis. Discussion was focused on the methods used to purify and characterize the factor and the possible phylo- genetical significance of a primitive cytokine of invertebrate origin. Mitogen- and serum-free-supernatants (SNs) from cultures of PHA-stimulated Xenopus splenocytes were biochemically characterized and biologically assayed by David Watkins and Nicholas Cohen (University of Rochester, Medical Center, Rochester, New York). These SNs induced proliferation of splenic and thymic T lymphoblasts and supported the continued growth of alloreactive T-cell lines but had no effect on "resting" lymphocytes. Precipitation with saturated ammonium sulfate and passage over a Sephacryl- 200 sizing column revealed the stimulatory activity in the 20-40 kd fraction. Functional and biochemical characteristics of these molecules of Xenopus suggest a growth factor significance although no cross reactivity between human recombinant IL-2 and Xenopus splenic lymphoblasts was observed in the blast cell assay. On the other hand, these same authors reported costimulation by splenocytes of Xenopus laevis by phorbol 12-myristate-13- acetate (PMA) and LPS, suggesting activity for a possible T-cell derived B-cell stimulation factor. Dr. E1 Ridi presented evidence for the first time concerned with the structural markers of the putative snake (Psammophis sibilans) MHC. Class I-like molecules were immunoprecipitated from P. sibilans peripheral blood mononuclear cells (PBM) with alloantisera directed against MHC-linked antigens. The heavy chains, depending on the allele

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examined showed a molecular weight of 46-53 kd, and the possible equivalent

to ~ 2-microglobulin 12kd. Alloantisera against adherent cells separated from PBM and splenocytes inhibited MLR but not ConA-mediated stimulation, lysed in complement-dependent cytotoxicity assays i00 and 5.0% of B and

thymus lymphocytes respectively, and immunoprecipitated from adherent cells of P. sibilans molecules composed of two different chains with molecular weight of 32 and 25-28 kd respectively.

Despite absence of various authors, subjects related to avian immunology provided new information on the role of the bursa of Fabricius as a peri- pheral lymphoid organ and the chemotactic properties of chicken mononuclear

cells. Paul Niewenhuis (Dept. of Histology, University of Groningen, The Netherlands) reported experimental manipulation of embryonic bursa

of Fabricius (BF) which causes profound alterations in the development

of the B cell system in young chickens, suggesting the bursa as the major channel for the interaction between intestinal (flora) antigens and the

developing immune system in the early post-hatching period. Thus, ligation of the bursal duct in ovo (BDL) on the 19th day of gestation prevented the development of antigen specific PFCs in the spleen, decreased the levels of "natural" antibodies, the B development, germinal centre formation and changed the number of B cells and the distribution of Ig-isotypes in bursa follicles. Discussion was consequently focused on the role of BF as a central and/or peripheral lymphoid organ in birds. Golemboski and Dietert (Dept. of Poultry and Avian Sciences, Cornell University, Ithaca) analyzed the in vitro chemotaxis of blood chicken mononuclear leukocytes to the peptide N-Met-Leu-Phe in blind-well chambers with poly- carbonate filter. Their results demonstrated that the N-formyl group is required for chemotaxis. The effects of in vivo response to an inflamma- tory stimulus on in vitro blood mononuclear leukocyte chemotaxis was examined 6 hours after peritoneal irritation by injection of Sephadex. Under these conditions chicken mononuclear leukocytes exhibited no in vitro chemotaxis to the chemoattractant suggesting that the in vivo response to an inflammatory stimulus may deplete the blood monocyte subpopulations capable of responding in vitro to chemoattractant peptides.

Dr. Donald D. Ourth (Dept. of Biology, Memphis State University,

Memphis) analyzed new aspects of the activation of the alternative complement pathway (ACP) in the channel catfish, Ictalurus punctatus. He found very little bactericidal activity against those pathogens that lacked sialic acid. A relative lack of sialic acid or no sialic acid correlated with

a strong bactericidal response by the catfish ACP. Consequently, neuraminid- ase treatment of the bacterial fish pathogens greatly increased the bacter- icidal response against them.

Once more, Dr. Clem's group presented two posters showing the effects of low temperature on fish lymphocyte reactivity. Flow cytometry was used to monitor the kinetics of capping induced by mouse monoclonal anti-

bodies specific for channel catfish B cell membrane immunoglobulin (mAb IHI2) and for common antigenic determinants present on channel catfish T and B cell membranes (mAb fIG3). The kinetic of capping induced by mAb IHI2 was dependent both on the length of time of in vivo temperature acclimation and on the in vivo assay temperatures. Energies of activation required for mIg capping were between 32.6-23.9 Kcal/mol. and decreased with lower temperature acclimation. Studies on capping with the mAb IIG3 demonstrated that only after acclimation to lower temperatures were differ- ences in the kinetics of capping apparent between the two cell types. On the other hand, low (nonpermissive) in vitro temperatures inhibited

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the in vitro responses of channel catfish T cells, but not B cells to both mitogenic (ConA) and antigenic (TD antigens) stimulation. In order to identify the nature of the low temperature sensitive step, channel catfish T cells were subjected to various treatments with a calcium ionophore (A23187), phorbol ester and/or mitogens. The results indicated that low temperature suppression of fish T cell responses is not due to an inherent inability of these cells to synthesize DNA at low temperature.

Dr. Kehayov's poster (Inst. of Zoology, L Russki str., Sofia, Bulgaria) (not present in the Workshop) revealed information on seasonal fluctuations of the humoral immune response in the tortoise, Testudo sraeca iberica, emphasizing more intensive primary and secondary anti-BSA and anti-LPS responses in summer than in winter.

Finally, a morphological study by S. Hart et al. (not present in the Workshop) (Dept. of Biological Sciences, Plymouth Polytechnic, England) on the presence of lymphoid tissue throughout the intestinal tract and the female genitourinary tract of the dogfish, Scyliorhinus canicula was included in the theme of discussion on mucosal immunity. The authors reported high concentration of lymphoid cells in the gut, few intraepithelial leukocytes and a significant number of plasma cells in the connective tissue of the genitourinary tract. Furthermore, IgM-like was detected in the bile, intestinal mucus and genitourinary secretions.

Dr. Agust~n G. Zapata

This report was basically elaborated from the author's summaries