No evidence for the presence of tissue factor in high-purity preparations of immunologically isolated eosinophils.

BACKGROUND: To date, there is no unequivocal opinion on whether human eosinophils express tissue factor (TF). Therefore, we studied the expression of TF protein and activity in resting or stimulated immunologically purified human eosinophils. METHODS AND RESULTS: By use of immunologic isolation, we achieved over 96% purity of eosinophil preparations, and contamination by CD14-positive cells was below 0.3%. Flow cytometric [fluorescence-activated cell sorting (FACS)] analysis of eosinophils revealed no surface expression of TF antigen in resting or stimulated eosinophils. Immunoblotting of eosinophil lysates did not show any TF protein under resting or stimulated conditions. The lysates of resting or stimulated eosinophils contained no detectable levels of TF procoagulant activity. In contrast, monocytes, stimulated in plasma or medium, possessed readily detectable TF levels on the cell surface and in cell lysates as detected by FACS and immunoblotting. This was active TF antigen, as confirmed by TF activity assay (19.2 +/- 4.2 and 28.6 +/- 3.1 mU per 10(6) cells, stimulated in medium or plasma, respectively). We found no detectable TF mRNA levels in resting or stimulated eosinophils by real-time polymerase chain reaction (PCR), whereas in monocytes TF mRNA levels were significantly increased after stimulation. CONCLUSIONS: Our data indicate that there is no evidence for TF expression in high-purity preparations of immunologically isolated eosinophils.

Immunoglobulin genes and antibody responses in the spotted wolffish (Anarhichas minor Olafsen).

The spotted wolffish Anarhichas minor Olafsen is a promising new species in aquaculture in the cold waters of northern Norway. In this paper, some basic immunological studies of this marine species are reported. Of comparative interest are the cDNA sequences of the immunoglobulin transcript and the antibody responses to model antigens. Of more practical importance are the humoral immune responses and antibody specificities to potentially pathogenic bacteria. Full length cDNA clones encoding the immunoglobulin heavy and light chains in the spotted wolffish were sequenced demonstrating variable degrees of similarity to other teleost fish species. Also in the spotted wolffish the CH4 domain was deleted in the transmembrane form of the immunoglobulin heavy chain (IgH) as a receptor on B cells, with the transmembrane exon spliced directly to the CH3 domain. The antibody responses to various antigens like hapten-carrier molecules, protein antigens and bacterial pathogens were relatively high, but with some interesting exceptions. Anti-hapten responses to NIP and FITC were high while anti-DNS responses were low, but more surprisingly, there was hardly any B-cell response to the carrier molecule LPH. On the other hand, protein antigens like CGG and BSA were highly immunogenic in the spotted wolffish as were the bacterial antigens Vibrio anguillarum, V. salmonicida and Aeromonas salmonicida.

Diversity of the immunoglobulin heavy chain in the Atlantic salmon (Salmo salar L.) is contributed by genes from two parallel IgH isoloci.

Immunoglobulin heavy chain (IgH) variable (V) region cDNAs from the Atlantic salmon, Salmo salar L., have been isolated and analysed with respect to diversity and transcription of the two parallel IgH isoloci in this species. A total of nine V(H) families were defined according to the 80% identity criterion, of which seven were highly related (>80% identity) to the V(H) families defined in rainbow trout and arctic charr. The variability of the CDR1 and 2 was low, although mutational hot-spot consensus sequences were accumulated in these regions. The CDR3 showed largest variability, expressing at least eight different groups of D motifs diversified by fusion of the D motifs, possible N and P nucleotide insertions and exonuclease activity. Presumably functional transcripts expressing D motifs in all three reading frames were identified for two of the motifs. The cDNAs were mapped to either of the two parallel loci, and sequence analysis revealed that the repertoire of V(H) segments was contributed by transcription of genes from both of the IgH isoloci. Transcription of genes from both isoloci generated no obvious effects on variability in the CDR3 of the Atlantic salmon IgH chains, although one additional J(H)-segment with altered N-terminal was generated by the process of duplication and divergence. Thus, the issue of biological significance of the two IgH isoloci remains unclear.

Expression of immunoglobulin heavy chain transcripts (VH-families, IgM, and IgD) in head kidney and spleen of the Atlantic cod (Gadus morhua L.).

Expression of the immunoglobulin (Ig) heavy chain transcripts in spleen and head kidney of the Atlantic cod (Gadus morhua L.) was investigated using in situ hybridization (ISH) and northern blotting. Specific detection of plasma cells was done with a probe for secretory IgM transcripts (mu 4). The plasma cells were often clustered close to blood vessels. Cells expressing surface IgM and IgD transcripts were detected using ISH with tyramide signal amplification (TSA). The positive cells were more abundant than plasma cells, had a lymphocyte-like morphology, and were evenly distributed throughout the tissues. This suggests that cod IgD mainly is expressed as a B-cell receptor akin to IgD in mammals. The VH-III family dominated the repertoire within the plasma cells, in agreement with data from cDNA cloning. Immunization with hapten-carrier antigen did not induce a systemic antibody response, and neither was any change in the clustering or distribution pattern of plasma cells within the tissues seen. A few clusters of plasma cells expressed only the rare VH-I and VH-II families, suggesting an ongoing clonal expansion and differentiation in these regions independently of immunization.

Variable region diversity of the Atlantic cod (Gadus morhua L.) immunoglobulin heavy chain.

This study was undertaken to determine whether a lack of VH domain diversity could explain, in part, the failure of Atlantic cod to respond to immunization with the production of specific antibodies. The variability of cod VH regions was studied in 113 cDNA and 2 genomic clones. A fourth VH family and a second putative JH element were identified. The expressed VH repertoire showed a clear bias in the pattern of VH family utilization, with about 80% of the clones belonging to the VH-III family. Furthermore, the VH-III family was complex and could be subdivided into several subfamilies, while little variation was seen within the other families. The VH family bias gives a somewhat reduced variability of the VH gene region of cod, but not lower than that of the rabbit IgM repertoire. The H chain CDR3 region of cod was longer than that of trout, frog and mouse, and also highly variable in sequence, probably reflecting a relative importance of this region in cod. On the other hand, the CDR3 length variability was restricted, and this may reduce the diversity of the cod VH region.

Immunoglobulin D (IgD) of Atlantic cod has a unique structure.

A new immunoglobulin heavy-chain gene with some homology to mammalian IgD was recently cloned from the channel catfish and Atlantic salmon, two species of teleost fish. We have cloned and sequenced a new H-chain gene from Atlantic cod (Gadus morhua L.) which has clear similarities to these genes, but which also differs in several ways. The similarities of catfish, salmon, and cod delta to the mammalian delta genes are sequence homology, location immediately downstream of IgM (mu), and expression by alternative splicing rather than class switching. A unique feature of catfish, salmon, and cod delta is the chimeric nature of the gene product, as the mu1 exon is spliced to the delta1 exon. Several unique features of cod IgD were found: (1) a deletion of the delta3, delta4, delta5, and delta6 domains described in catfish and salmon IgD, (2) a tandem duplication of a part of the delta locus including the delta1 and delta2 domains, (3) the presence of a truncated delta7 domain downstream of the deltaTM exons, and (4) the separation of the duplicated domains by a short exon (deltay) which has homology to a conserved part of the transmembrane exon 1 (TM1) of some H-chain isotypes. This unique organization of the delta locus of cod probably developed after the evolutionary split from the catfish and salmon branches.

Humoral immune parameters in Atlantic cod (Gadus morhua L.) I. The effects of environmental temperature.

The effects of environmental temperature on certain humoral immune parameters in Atlantic cod (Gadus morhua L.) were studied. Serum samples were collected from captive cod, of wild origin, kept at different temperatures for 12 months. It was found that immunoglobulin and natural antibody levels increased with increasing temperature whereas the total serum protein concentration, anti-protease activity, iron concentration, unsaturated and total iron binding capacity decreased with increasing temperature. Haemolytic activity and percentage iron saturation also tended to decrease with increasing temperature although this was not statistically significant.

Humoral immune parameters in Atlantic cod (Gadus morhua L.) II. The effects of size and gender under different environmental conditions.

The effects of size and gender on several humoral immune parameters in cod were examined under different environmental conditions. Serum samples were collected from wild cod of different sizes. Two samplings were undertaken: In the spring in relatively cold waters off the north west coast of Iceland and in the fall in relatively warm waters off the west coast of Iceland. Most of the parameters increased with increasing cod size, except the haemolytic activity which decreased. Higher serum protein levels were seen in cod sampled in the fall than in the spring. In cod sampled in the spring there was an apparent difference between specimens < 75 cm in length and the larger specimens with respect to haemolytic activity and iron concentration. None of the parameters were influenced by the gender of the cod.

Ontogeny of lymphoid organs and immunoglobulin producing cells in Atlantic cod (Gadus morhua L.).

The ontogeny of lymphoid organs and the development of cells expressing immunoglobulin heavy chain (IgH) mRNA as well as cells containing immunoglobulin (IgM) were studied in Atlantic cod (Gadus morhua L.), a marine teleost. Head kidney and spleen appeared as the first lymphoid organs, present at the time of hatching, whereas thymus was observed in 9 mm larvae. Fully developed lymphoid organs were not achieved until after metamorphosis. Cells expressing IgH mRNA were detected in paraffin sections of larvae and juveniles by in situ hybridization. Positive cells were not detected in fish smaller than 33 mm (58 days after hatching). IgH mRNA expression coincided with the first appearance of immunoglobulin-positive cells as revealed by immunohistochemistry in the same animals.

Immunoregulation in fish II: intermolecular-induced suppression of antibody responses studied by haptenated antigens in atlantic salmon (Salmo salar L).

Here we report evidence for T cell dependent intermolecular-induced suppression of antibody responses in fish, using a panel of T cell dependent (TD) and T cell independent (TI) hapten-carrier antigens. Atlantic salmon were immunized intraperitoneally either with protein antigens: Limulus polyphemus hemocyanin (LPH), chicken gammaglobulin (CGG), A. salmonicida surface A-layer protein (ALPAsal) or lipopolysaccharide (LPS) antigens isolated from A. salmonicida and Escherichia coli. The various antigens were given as a mixture of the native and haptenated (4-hydroxy-3-iodo-5-nitrophenyl-acetic acid, NIP; 2,4,6-trinitrophenyl-acetic acid, TNP; fluorescein-5-iso-thiocyanate, FITC) forms. The salmon immune system responded to the antigen mixtures by eliciting high anti-hapten titers whereas the antibody titers against protein determinants were low (suppress 65-95%) as determined by ELISA. The suppression was induced between haptens (NIP and FITC) and between heterologous antigens (NIP-CGG and LPH) indicating that the mechanisms involved were non-specific. Moreover, suppression was induced by TD antigens only, indicating that the mechanism was T cell dependent. Injection of antigen mixtures containing variable amounts of the competing antigens showed that the kinetics of suppression was dose-ratio and dose dependent. In a time-course study it was found that the suppressed anti-LPH response was unchanged until native LPH was injected almost 2 years after the primary immunization, showing that permanent tolerance had not been induced. Sequential immunization showed that the antibody response was most sensitive to suppression during the initial 10 days after immunization. Moreover, the carrier antigen was also able to induce suppression of hapten epitopes, but only if the anti-carrier response was allowed to develop for 14 days before the hapten-carrier antigen was injected. This shows that AIS in fish is elicited as a result of the immune response to the dominant antigen, and can be induced against either antigens if the temporal order of administration is reversed. A possible model for AIS as a normal immunoregulatory process in fish is proposed and discussed.