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AbstractRecent evidence show that the classical distinction between innate and acquired immunity can no longer be employed to thoroughly characterize immune defenses. As an example, both insects and vertebrates possess non-canonical defense activities that differ from those identified so far; namely, insects display immune memory and mammals show innate-like lymphocytes. At the base of these observations, it can be speculated that multicellular eukaryotes share immune cells deriving from ancestral granular and non-granular immunocytes as well as sets of soluble effectors and signaling immune-related molecules. On one hand, evolutionary pressure has selected genes coding for group-specific defense molecules; on the other, some cell types and gene products can be presently considered as being enriched with group-specific adaptations deriving from most ancient protostomes and deuterostomes. The immune defenses of more recent extant animal groups could then be seen as overlying layers constituted by both ancient cells retaining broad features for non-self defenses and more specialized cells and immunomodulatory molecules for group-specific capabilities.

It appears evident that teleost fish are at present the vertebrate group in which, excluding mammals, most information on the immune system is available. However, despite the great impetus on the discovery of genes homologous to mammalian immunomodulatory molecules, the knowledge on biological activities exerted by cytokines is meager. This review reports the present knowledge on the biological activities of cytokine-like and cytokines in invertebrates and coldblooded vertebrates.

In this paper the cloning of interleukin-1β (IL-1β) from the fish Dicentrarchus labrax(sea bass) is described. Using degenerate primers designed from known IL-1β sequences, a cDNA fragment was amplified by PCR and elongated by 3′ and 5′ RACE to give the full-length coding sequence for sea bass IL-1β. The cDNA is 1292bp, lacks a putative ICE cut site, and codes for a deduced peptide of 29·4kDa with a pI of 5·1. Sequence analysis showed highest amino acid similarity with rainbow trout (62%), Xenopus(46%), and carp (45·5%) IL-1β sequences. Expression studies show that sea bass IL-1β can be upregulated by bacterial lipopolysaccharide both in vitroand in vivoin leucocytes from blood, head-kidney, spleen, gills and liver, whereas the IL-1β transcript was not detectable in thymus and gut-associated lymphoid tissue. Northern blot analysis with head-kidney leucocyte RNA showed a main LPS-upregulated band at 1·3kb, and two minor bands at 0·9 and 3·0kb, respectively. Phylogenetic comparisons with IL-1β from other vertebrates is presented.

The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fishDicentrarchus labrax(sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The purification was performed by immuno-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a single step, the percentage of DLT15-purified cells was 88±10% for gut-associated lymphoid tissue and 79±18% for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligonucleotides corresponding to the peptide sequence MYWY and VYFCA of the trout TcRβ chain, a 203bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the trout TcRVβ3. By 3′-RACE the cDNA was elongated to obtain the TcR constant region, with high similarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes.

The monoclonal antibody DLE7 was obtained against 44- to 50-kDa polypeptides solubilized from the vitelline envelope of the Mediterranean sea bass Dicentrarchus labrax.In Western blot analysis of chorion lysates it recognized cross-reactive bands at 44 kDa, 48 kDa, and 110 kDa. Previous affinity blotting with concanavalin-A showed that most of solubilized bands were glycosylated. Enzymatic deglycosylation of chorion proteins followed by Western blot analysis with DLE7 showed that the 48-kDa and 110-kDa antigens were differentially affected by endoglycosidase-F treatment. When DLE7 was employed in immunofluorescence analysis, isolated chorions and ovarian cryosections stained intensely. Positivity was also observed in liver cryosections of spawning females but not in liver of males and nonspawning females. To study the origin and delivery of chorion proteins, DLE7 was used in Western blot analysis of liver homogenates and blood serum of spawning females. Cross-reacting bands were detected in liver (90 kDa) and serum (180 kDa, 50 kDa). DLE7 was also used for the first time to set up an indirect ELISA assay to detect egg antigens in the blood of egg-producing females, raising the possibility of using DLE7 as a female-specific marker of spawning for sea bass.

Interleukin-1 (IL-1) is a family of polypeptides that is thought to play an important role in the regulation of the systemic response to invasive stimuli such as infection, trauma or antigenic challenge, exerting both immunopotentiating and inflammatory actions on a wide variety of cellular targets. Binding studies with radiolabeled IL-Is showed that structurally distinct receptors are present on different cell types, suggesting that the different biological activities might be the consequence of IL-1 binding to distinct receptor subtypes.The highly detrimental activities of IL-1 represent a major drawback for its therapeutic application. Studies with IL-1 fragments have shown that the nonapeptide corresponding to the 163–171 sequence of human IL-1β is able to mimic the immunopotentiating activities of IL-1 while being devoid of its pro-inflammatory potentials, thus it might find clinical application as an immunostimulating agent. Since IL-1 is involved in some pathological conditions, strategies for anti-IL-1 therapy should also be developed. In this regard a recently characterized naturally occurring IL-1 receptor antagonist might play an important role and its structural relationships with IL-1β are hereafter discussed.

Although rapid progress has been made in knowledge of the teleost immune system, specific antibodies recognizing cellular populations are few and, among lymphocytes, they are restricted to the B cell lineage. In this work, we describe the preparation and characterisation of a monoclonal antibody directed against thymocytes of the mediterranean sea bass (Dicentrarchus labrax) (1 year group). The antibody was obtained by immunising mice with paraformaldehyde-fixed thymocytes, and by screening hybridoma culture media by indirect immunofluorescence and FACS analysis using both fixed and living cells. The hybridoma cell line which after limiting dilution cloning gave the best IIF staining of living thymocytes, and the sharpest immunostaining of Bouin fixed sections was selected and named DLT15. DLT15 stained 74·9% of thymocytes, 2·4% of cells in the whole blood, 0·8% in the spleen, and 2·6% in the head-kidney in immunofluorescence and FACS analysis. Immunohistochemical analysis revealed DLT15-immunoreactive cells inside the thymus, the spleen, and the head-kidney. In these two latter organs immunoreactive cells were enriched in regions where erythropoiesis was less abundant, and were present either as isolated cells or as small clusters of two to four cells.

Defence properties of hemoctyes were investigated using the anti-hemocyte monoclonal antibody BrH1 obtained by immunizing mice with 2% paraformaldehyde-fixed hemoctyes of the stick insect Bacillus rossius. In Western blot analysis, the antibody recognized a 180 kDa antigen in hemocyte cell lysates, whereas fat body lysates and cell-free hemolymph were negative. In immunofluorescence analysis of cultured or freshly collected hemoctyes, BrH1 stained intracellular antigen(s) in detergent-treated cells. Transverse cryosections of adult stick insects probed by immunofluorescence with BrH1 showed in situthe scattered distribution of hemoctyes inside the haemocoel. The antigen(s) recognized by BrH1 appears to be involved in cell defence hemocyte-mediated mechanisms, as evidenced by the fact that cryosections of insects challenged in vivowith yeast cells, bacteria, or polystyrene latex particles and probed with BrH1 showed an accumulation of antigen surrounding the injected stimuli. © 1997 Elsevier Science Ltd. All rights reserved

Sea bass immunoglobulins were single-step purified from the whole serum by affinity chromatography on protein A-sepharose. The purified immuno-globulins had an apparent molecular weight of 78kDa (heavy chain) and 27kDa (light chain) in SDS-PAGE. Immunoglobulins were used as immunogens in mice, and after fusion, the hybridomas were screened by indirect immunofluorescence on living cells, western blotting, and immunohistochemical staining of Bouin fixed tissues. Among the positive hybridomas obtained, three clones were selected according to their ability to recognise either the immunoglobulin light chain (DLIg3) or the heavy chain (DLIg13 and DLIg14). DLIg3 stained in FACS analysis 1% of cells in the whole blood and 19·7% of PBL, 1·1% in the thymus, 22·2% in the spleen, 12·2% in the head-kidney, and 1·5% in the midgut. DLIg13 and DLIg14 were unable to stain living cells by immunofluorescence and FACS, whereas recognised fixed cells following ABC-immunoperoxidase staining of spleen, head-kidney and midgut. DLIg3 was used to set up a sensitive ELISA assay to detect and quantify purified and serum immunoglobulins. Using purified immunoglobulins the assay sensitivity resulted 1·2ng ml−1, whereas the serum immunoglobulin content resulted 3·92±2·16mg ml−1with an assay detection limit of 3·9ng ml−1, corresponding to a serum dilution of 9·7×105times.

Abstract: Immunoglobulins from the Antarctic fish species Trematomus bernacchii, Trematomus hansoni, Trematomus newnesi, and Chionodraco hamatus were analysed in whole serum and after purification by affinity chromatography on protein A-sepharose. Using SDS-PAGE, the apparent masses of the heavy and light chains were, respectively, 83.5 kDa and 27.5 kDa for T. bernacchii, 83.5 kDa and 27 kDa for T. hansoni, 81 kDa and 27.5 kDa for T. newnesi, and 74.5 kDa and 30 kDa for C. hamatus. It was not possible to purify immunoglobulins from T. newnesi due to their low concentration in serum. Heterogeneity in mass of both heavy and light chains was observed in all species. By using a polyclonal antibody raised against sea bass immunoglobulins, cross-reactivity was observed with heavy and light chains of all species. With this antibody, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and results showed the relative immunoglobulin concentration in sera of the Antarctic fish species considered, using as standard sea bass immunoglobulins.

The interleukin 1 receptors (IL-1R) on the human B lymphoma RAJI and on the murine thymoma EL4-6.1 have been characterized. Equilibrium binding analysis using both 125I-labeled IL-1α and IL-1β showed that RAJI cells have a higher number of binding sites/cell for IL-1β (2400, Kd2.2 nM) than for IL-1α (316, Kd0.13 nM). On the other hand, EL4-6.1 cells have more receptors/cell for IL-1α (22 656, Kd1 nM) than for IL-1β (2988, Kd0.36 nM). Dexamethasone (DXM) induced on RAJI cells a time-dependent increase in binding sites for both IL-1β and IL-1α without affecting their binding affinities. However, while receptor-bound 125I-IL-1α was displaced with equal efficiency by both IL-1 forms, only unlabeled IL-1β could effectively displace 125I-IL-1β. Cross-linking experiments indicated that RAJI cells have a predominant IL-1R of about 68 kDa, while EL4-6.1 cells have an IL-1-binding polypeptide of 80 kDa. These results suggest that B and T cells possess structurally different IL-1R with distinct binding properties for IL-1α and IL-1β.

The interleukin 1 receptors (IL‐1R) on the human B lymphoma RAJI and on the murine thymoma EL4‐6.1 have been characterized. Equilibrium binding analysis using both 125I‐labeled IL‐1α and IL‐1β showed that RAJI cells have a higher number of binding sites/cell for IL‐1β (2400, Kd2.2 nM) than for IL‐1α (316, Kd0.13 nM). On the other hand, EL4‐6.1 cells have more receptors/cell for IL‐1α (22 656, Kd1 nM) than for IL‐1β (2988, Kd0.36 nM). Dexamethasone (DXM) induced on RAJI cells a time‐dependent increase in binding sites for both IL‐1β and IL‐1α without affecting their binding affinities. However, while receptor‐bound 125I‐IL‐1α was displaced with equal efficiency by both IL‐1 forms, only unlabeled IL‐1β could effectively displace 125I‐IL‐1β. Cross‐linking experiments indicated that RAJI cells have a predominant IL‐1R of about 68 kDa, while EL4‐6.1 cells have an IL‐1‐binding polypeptide of 80 kDa. These results suggest that B and T cells possess structurally different IL‐1R with distinct binding properties for IL‐1α and IL‐1β.