Early ontogeny of monocytes and macrophages in the pig

Prenatal development of cord blood monocytes and tissue macrophages was studied in pig foetuses by immunophenotyping and functional assays. The function of peripheral blood monocytes was compared in germ-free and conventional piglets. First macrophages were identified by electron microscopy in foetal liver on the 25th day of gestation. Monoclonal antibodies against porcine CD45 and SWC3 antigens were used for flow cytometric identification of myelomonocytic cells in cell suspensions prepared from the yolk sac, foetal liver, spleen and cord blood. Leukocytes expressing the common myelomonocytic antigen SWC3 were found in all organs studied since the earliest stages of development. Opsonized zymosan ingestion assay was used to determine the phagocytic capacity of foetal mononuclear phagocytes isolated from cord blood, liver and spleen. In the foetal liver, avid phagocytosis of apoptic cells had been found to occur before cells were able to ingest zymosan in vitro. The first cells capable of ingesting zymosan particles were found on the 40th day of gestation in umbilical blood and 17 days later in foetal spleen and liver. Their relative proportion increased with age. Cord blood monocytes and peripheral blood monocytes in germ-free piglets had low oxidatory burst activity as shown by iodonitrophenyl tetrazolium reduction assay. A remarkable increase of oxidatory burst activity was observed in conventional piglets, probably due to activation of immune mechanisms by the microflora colonizing gastrointestinal tract.     

Neutrophils and B-cells in Atlantic cod (Gadus morhua L.)

Proportions of leucocytes from head kidney, blood and spleen were identified as B-cells and neutrophils using a polyclonal antibody to cod IgM and a monoclonal antibody which previously has been shown to bind specifically to salmon and trout neutrophils. The cell specific binding of the antibodies was supported by double immunostaining. The morphology of isolated leucocytes was examined on Diff Quick stained slide preparations, and myeloperoxidase positive neutrophils were identified by diaminobenzidine staining. The antibodies clearly identified distinct cell populations. Using flow cytometry, high proportions of neutrophils were observed in peripheral blood leucocytes and high proportions of B-cells were found in head kidney leucocytes when compared to proportions of these cells in Atlantic salmon (Salmo salar L.). The spleen contained the highest proportion of B-cells. Cytoplasmic staining of immunoglobulin positive cells in slide preparations indicated that plasma cells were present, but not strikingly abundant, in head kidney, spleen and peripheral blood. Staining for myeloperoxidase identified, in accordance with the flow cytometry results, a large number of neutrophils, especially in peripheral blood leucocytes. The neutrophil nucleus was not clearly segmented, but appeared more irregular than rounded. The findings of high proportions of neutrophils in peripheral blood suggest that these cells of the innate immune system might have a central role in defence and protection against infections in cod.     

Phagocytosis and Respiratory Burst Activity in Lumpsucker (Cyclopterus lumpus L.) Leucocytes Analysed by Flow Cytometry

In the present study, we have isolated leucocytes from peripheral blood, head kidney and spleen from lumpsucker (Cyclopterus lumpus L.), and performed functional studies like phagocytosis and respiratory burst, as well as morphological and cytochemical analyses. Different leucocytes were identified, such as lymphocytes, monocytes/macrophages and polymorphonuclear cells with bean shaped or bilobed nuclei. In addition, cells with similar morphology as described for dendritic cells in trout were abundant among the isolated leucocytes. Flow cytometry was successfully used for measuring phagocytosis and respiratory burst activity. The phagocytic capacity and ability were very high, and cells with different morphology in all three leucocyte preparations phagocytised beads rapidly. Due to lack of available cell markers, the identity of the phagocytic cells could not be determined. The potent non-specific phagocytosis was in accordance with a high number of cells positive for myeloperoxidase, an enzyme involved in oxygen-dependent killing mechanism present in phagocytic cells. Further, high respiratory burst activity was present in the leucocytes samples, verifying a potent oxygen- dependent degradation. At present, the specific antibody immune response could not be measured, as immunoglobulin or B-cells have not yet been isolated. Therefore, analyses of the specific immune response in this fish species await further clarification. The present study presents the first analyses of lumpsucker immunity and also the first within the order Scopaeniformes.     

The use of innate immune responses as biomarkers in a programme of integrated biological effects monitoring on flounder (Platichthys flesus) from the southern North Sea

Immunological biomarkers that reflect the effects of exposure to environmental contaminants in coastal marine habitats were sought in European flounder (Platichthys flesus) from five locations in the German Bight with different anthropogenic impacts. During a 2-year period of sampling, innate immune responses were monitored from a total of 331 individual flounder of a body length of 18 to 25 cm. From the fish, plasma lysozyme, phagocytosis and respiratory burst activity of head kidney leucocytes were analysed and implemented as part of an integrated biological effects monitoring programme. As the measurements of the parameters applied here varied within wide ranges at some locations, spatial differences could not always be established, but some general trends could be drawn: plasma lysozyme activity was decreased in flounder contaminated with DDT adducts and some PCBs, while cellular functions such as phagocytosis and respiratory burst were stimulated by some chlorinated hydrocarbons. Correlation analysis also revealed connections not only between the parameters applied here and some contaminants but also with some biochemical parameters used as biomarkers in pollution monitoring: in flounder with decreased integrity of hepatocyte lysosomal membranes, immune functions also were impaired, and plasma lysozyme as well as phagocytosis activity of head kidney cells were impaired when the activity of cytochrome P450 1A was induced. The data presented here indicate that innate immune responses may be useful parameters to monitor cellular functions in a battery of biomarkers of different levels of biological organisation. Communicated by H. v. Westernhagen, A. Diamant     

Characterization of carp thrombocytes with specific monoclonal antibodies

Monoclonal antibodies (WCL6) specific for carp Cyprinus carpio thrombocytes were produced by immunizing mice with membrane lysates of IgM-negative peripheral blood leucocytes (PBL) and selected on the negative reaction with B cells. WCL6 was reactive with a membrane molecule of approximately 90 kDa and to a lesser extent with molecules of approximately 95 and 110 kDa. In general, between 30 and 40% of PBL were WCL6⁺ and appeared to be round to spindle-shaped cells. Immunohistochemical analysis of cryo-sections showed much higher numbers of WCL6⁺ cells in the spleen than in the pronephros, intestine and thymus. Flow cytometric analysis of cell suspensions isolated from these organs only revealed WCL6⁺ cells (4–10%) in the spleen. Electron microscopy of immunogold-stained WCL6⁺ PBL showed round but also some spindle-shaped cells with canalicular and granular structures, and a more irregular and electron-dense nucleus than found in lymphocytes. WCL6⁺ cells with electrondense pyknotic nuclei (without a clear nuclear envelope) were found also and their frequency increased with the length of the isolation and staining procedure used. In the spleen, several differentiation steps of WCL6⁺ cells were found and hence the spleen seems to be the thrombopoietic organ in carp. Thrombocytes from blood could be activated with collagen; the collagen-activated cells showed a higher side (90°) scatter by flow cytometric analysis and finally considerable cell death.     

Flow cytometry assays of respiratory burst in Atlantic salmon (Salmo salar L.) and in Atlantic cod (Gadus morhua L.) leucocytes

The oxidation of dihydrorhodamine 123 (DHR) to the fluorescent rhodamine 123 (RHO) was detected using flow cytometry. This assay for detection of respiratory burst activity was established in peripheral blood leucocytes (PBL) and head kidney leucocytes (HKL) of Atlantic salmon and Atlantic cod. The leucocytes were stimulated by phorbol 12-myristate 13-acetate (PMA). For cod cells 10 times lower concentration of PMA had to be used compared to salmon cells, as higher concentrations were toxic and resulted in considerable cell death. The cells found to be RHO-positive were monocytes/macrophages and neutrophils based on the scatter dot plots, but for salmon also some small cells were found to have high fluorescence intensity both in the flow cytometry analyses and by fluorescence microscopy of cytospin preparations. The nature of these cells is not known. For cod leucocytes, such cells were not obvious. The instrument settings are a bit more demanding for cod, as cod cells die more easily compared to salmon cells. In both assays the limit between negative and positive cells has to be carefully considered. The presented flow cytometry protocols for measurements of respiratory burst in salmon and cod leucocytes can be applied in various studies where respiratory burst functions are involved, such as to verify if it is activated or suppressed in connection with infections and immunostimulation.     

Cloning and characterisation of a cDNA encoding Japanese flounder Paralichthys olivaceus IgD

A cDNA containing the gene for Japanese flounder IgD consisted of 3240 bp encoding 998 amino acid residues. The amino acid sequence of the constant region of Japanese flounder IgD shares 38-80% identity with the sequences of previously reported teleost IgDs. The structure of the constant region of Japanese flounder IgD, which contains the micro1, delta1, delta2, delta3, delta4, delta5, delta6, delta7, and TM regions, is similar to the structures of the constant regions of the IgDs of channel catfish and Atlantic salmon. Southern blot hybridisation showed that the Japanese flounder IgD gene exists as a single locus. The Japanese flounder IgD gene was mainly detected in peripheral blood leucocytes (PBLs) and small amounts were detected in the spleen, head and trunk kidney, although IgM mRNA was detected in similar amounts in PBLs, the head kidney, and spleen. The copy number of IgM mRNA in Japanese flounder PBL was 56-fold higher than that of IgD.     

Molecular characterization and gene expression of a CXC chemokine gene from Japanese flounder Paralichthys olivaceus

Chemokines are small, secreted cytokine peptides that have the ability to recruit a wide range of immune cells to sites of infection and disease. A novel CXC chemokine was obtained from Japanese flounder Paralichthys olivaceus. This chemokine cDNA contains an open reading frame of 333 nucleotides encoding 111 amino acid residues containing four conserved cysteine residues. The gene is composed of four exons and three introns as are those of mammalian and fish CXC chemokines. Results of homology and phylogenetic analysis revealed that the Japanese flounder CXC chemokine is closest to CXCL13 subgroup. The gene was expressed in immune-related organs, including head kidney, trunk kidney, spleen and peripheral blood leukocytes (PBLs). Japanese flounder CXC chemokine gene expression was observed at 3 and 6h after induction by LPS, but not at 3 and 6h after induction by poly I:C. These results suggest that the Japanese flounder CXC chemokine is probably associated with inflammatory as well as homeostatic functions.     

Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.)

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans.     

Morphological and flow cytometric characterization of leukocytes from the notothenioid teleosts Dissostichus eleginoides, Notothenia coriiceps, and Trematomus hansoni

To date, the adaptive immune response of the notothenioid fishes of the Sub- and High-Antarctic has received little attention. Here we characterize leukocyte preparations derived from head (pronephric) kidney, spleen, and intestine of three notothenioid species, Dissostichus eleginoides, Notothenia coriiceps, and Trematomus hansoni. Cells were collected from head kidney, spleen, and intestine, and were fixed in paraformaldehyde and analyzed by flow cytometry and optical microscopy. The three species displayed typical leukocyte populations composed of lymphocytes, granulocytes, monocytes/macrophages and thrombocytes with a peculiar organ distribution and peculiar flow cytometric patterns. This work represents the first characterization of cells involved in immune reactions in the investigated species.     

Molecular cloning, expression, and functional analysis of caspase-10 from Japanese flounder Paralichthys olivaceus

We isolated and sequenced caspase-10 cDNA and gene from Japanese flounder, Paralichthys olivaceus. The Japanese flounder (JF)-caspase-10 cDNA consisted of 2282 bp and encoded 495 amino acid residues. The characteristic death effector domains (DEDs) of caspases were observed in JF-caspase-10 as well as the three aspartic acid residues (D-186, -382 and -392), which are potential cleavage sites for the large and small subunit structures. The amino acid residue (His-325) and pentapeptide (QACQG), which are involved in catalytic activity, were absolutely conserved in Japanese flounder-caspase-10. JF-caspase-10 gene has a length of 6.6 kb and consists of 11 exons and 10 introns similar to that of human. The strong expression of JF-caspase-10 mRNA was detected in the gills, peripheral blood leukocytes, spleen and posterior kidney, while the weak expression was observed in the head kidney, heart, intestine, skin and stomach. The over-expression analysis of JF-caspase-10 in Japanese flounder cell line HINAE was shown to induce apoptosis 24h post-transfection using TUNEL assay.     

A pore-forming protein,perforin, from a non-mammalian organism,Japanese flounder,<Emphasis Type="Italic"> Paralichthys olivaceus</Emphasis>

A perforin cDNA of Japanese flounder, Paralichthys olivaceus, was cloned from a cDNA library of kidney stimulated with ConA/PMA. The full-length cDNA is 2,157 bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3 kb. The amino acid sequence of the Japanese flounder perforin is 36% identical to that of rat perforin and 37% identical to amino acid sequences of mouse and human perforin. The Japanese flounder perforin also showed low homology to human and mouse complement components (C6, C7, C8 and C9), ranging from 19% to 24%. However, the membrane attack complex/perforin domain is conserved. A phylogenetic analysis placed the Japanese flounder perforin in the same cluster with other known mammalian perforins. RT-PCR analysis revealed that the perforin gene was expressed in the peripheral blood leukocytes, head kidney, trunk kidney, spleen, heart, gill and intestine of healthy fish. Recombinant perforin produced in insect cells using the baculovirus expression system showed calcium-dependent hemolytic activity.     

Effect of temperature on immune response of Japanese flounder (Paralichthys olivaceus) to inactivated lymphocystis disease virus (LCDV)

Using flow cytometric analysis, the dynamics of surface immunoglobulin positive (sIg+) cells in lymphoid organs of Japanese flounder (Paralichthys olivaceus) reared at 9, 15, 21 and 26 °C, was investigated following intraperitoneal injection with inactivated lymphocystis disease virus (LCDV). The results showed that the percentages of sIg+ cells were suppressed in peripheral blood leucocytes (PBL), spleen leucocytes (SL) and head kidney leucocytes (HKL) from 9 °C to 15 °C immunized groups, and arrived at their peaks (9 °C: 26.12% in PBL, 18.84% in SL, 17.53% in HKL; 15 °C: 38.82% in PBL, 25.38% in SL, 23.95% in HKL) at 9th and 7th week after immunization, respectively. While the proportions of sIg+ cells in PBL, SL and HKL increased most prominent in the 21 °C group and reached the peaks (54.16% in PBL, 30.32% in SL, 30.23% in HKL) at 5th week. The responses of sIg+ cells from 26 °C group were similar to that from 21 °C group and reached the peaks (35.3% in PBL, 26.24% in SL, 21.83% in HKL) at 5th week. Simultaneously, the kinetics of the specific antibody titer against LCDV in sera was determined. It was shown that the antibody response in the 21 °C group was most prominent and reached the peak earliest. These results indicated inactivated LCDV elicited the most powerful immune response when Japanese flounder maintained at the optimal temperature (21 °C) and obtained the most effective immunization, while the response were suppressed at 9 °C, 15 °C or 26 °C.     

Technique and staining optimization leucoconcentration

In cytometric clinical application, it is important to obtain cell suspensions rapidly with as little cytological alteration as possible. A procedure has been achieved to prepare cell suspensions for flow cytometric analysis. The leucoconcentration technique, first described by Herbeuval for cytologic analysis, has been modified to be applied in cytometry. This technique involves Saponin lysis of red cells of peripheral blood or bone marrow samples that have been previously fixed with picric acid alcohol solution. Cells in suspension are not shifted and tinctorial affinity is not modified. Then cells have been stained with Mithramycin. Each parameter defined by Crissman has been analyzed to define the best staining conditions. The availability of Leucoconcentration with Mithramycin-DNA-staining permits determination of cell cycle with a fine resolution.     

Characterization of monoclonal antibodies against heavy and light chains of flounder (Paralichthys olivaceus) immunoglobulin

Flounder (Paralichthys olivaceus) Immunoglobulins (Igs) were purified from the serum of mouse IgG-immunized flounder by using affinity chromatography. Under denaturing conditions in SDS-PAGE, the flounder Igs appeared to be composed of 2 heavy (H) chains (72 and 77 kDa) and two light (L) chains (26 and 28 kDa). Monoclonal antibodies (MAbs) were produced by the fusion of myeloma cells (SP2/0) with Balb/c mouse spleen cells that were previously sensitized against affinity-purified flounder Igs. In a Western blot analysis, the produced MAbs, FIM511, FIM519, and FIM562 recognized both the 72 and 77 kDa H chains, 26 kDa, and 28 kDa L chain, respectively. Mouse antiserum against flounder Igs reacted more strongly with the L chain of 28 kDa than with 26 kDa, suggesting that the 28 kDa molecule is more immunogenic than the 26 kDa L chain molecule. In a FACS analysis, the ratios of the Ig+ cell population in the flounder head kidney and spleen cells were 49% and 24%, respectively. Unexpectedly, however, the ratios of the Ig+ B-like cell population in the flounder were not significantly augmented, even after the immunization of an immunogenic antigen. This suggests that the humoral immune response in fish could be considerably different from that in mammals. The produced MAbs in this study would be useful in characterizing flounder Ig+ B-like cells and in developing flounder Ig detecting an immunoassay system.     

Orange-spotted grouper (Epinephelus coioides) toll-like receptor 22: molecular characterization, expression pattern and pertinent signaling pathways

The toll-like receptors (TLRs) are an important gene family in host innate immunologic surveillance. The TLR22 gene is an essential member of the TLRs that is only found in aquatic animals and has been detected in some bony fish. Here, a TLR22 homolog, EcTLR22, was characterized in the orange-spotted grouper (Epinephelus coioides) via homology cloning. The 3321 bp full-length cDNA sequence of EcTLR22 was obtained, which included an open reading frame of 2880 bp encoding a putative peptide of 960 amino acids containing three highly typical domains with the characteristics of TLR family members. The deduced amino acid sequence of EcTLR22 showed a relatively high similarity to flounder TLR22. Phylogenetic analysis showed that the orange-spotted grouper TLR22 sequence was clustered with those of Perciforme, such as flounder and croaker. Real-time quantitative PCR analysis revealed broad expression of EcTLR22, with relatively high expression detected in the head kidney, trunk kidney, spleen, peripheral blood leukocytes (PBLs) and heart of orange-spotted grouper. After injection with Vibrio alginolyticus, there was significant up-regulation of the expression of EcTLR22 in the spleen. In evaluating unstimulated/stimulated head kidney leukocytes and spleen leukocytes, a significant increase in EcTLR22 mRNA expression was detected, which implied a sensitive immune response. Furthermore, four important molecules for signal transduction, MyD88, TRIF, TNF-α and IRF3, were chosen to analyze the role of the EcTLR22 signaling pathway in anti-pathogen responses. Upon LPS or Poly I:C challenge, expression of the four genes was induced, with an increasing tendency detected in head kidney leukocytes, suggesting that the four genes might work with EcTLR22 in host defense against pathogenic microbes.     

Production, characterisation and applicability of monoclonal antibodies to immunoglobulin of Japanese flounder (Paralichthys olivaceus)

Immunoglobulin (Ig) of Japanese flounder (Paralichthys olivaceus) was purified by a combination of salting-out and DEAE Sepharose Column chromatography. The purified immunoglobulin had an apparent molecular weight of 74 kDa (heavy chain) and 24 kDa (light chain) in SDS-PAGE. Eighteen hybridomas secreting monoclonal antibodies (MAbs) against Japanese flounder Ig were obtained by immunisation of Balb/C mice with purified Ig preparations, which were selected on the basis of the double indirect enzyme-linked immunosorbent assay (D-ELISA). Two of them designated as 2D8 and 2H1 were cloned by limiting dilution and characterised with western blotting, indirect immunofluorescence assay test (IIFAT) and fluorescence-activated cell sorter (FACS) analysis. Under reducing conditions in western blotting, both MAb 2D8 and MAb 2H1 were specific for the heavy chain of Japanese flounder Ig. MAb 2D8 was used to identify surface Ig-positive lymphocytes in the peripheral blood, spleen and pronephros of healthy Japanese flounder by flow cytometry. FACS analysis revealed that 40.48% of lymphocytes in the peripheral blood, 17.32% in the spleen and 9.67% in the pronephros were reactive to 2D8.     

The development of the lymphoid organs of flounder, Paralichthys olivaceus, from hatching to 13 months

The growth of the lymphoid organs, such as head kidney, spleen and thymus were studied in flounder, Paralichthys olivaceus Temminck & Schlegel, from hatching to 13 months of age. Except for the thymus, all organs grew as the fish grew. By 2 months of age the lymphoid organs attained their maximum relative weight. The organ weight showed a closer correlation to body weight than they did to age. The total number of leucocytes in the lymphoid organs increased with age, but the number per milligram of lymphoid organ remained constant. A micro and ultrastructural study of the lymphoid organs showed that the full development of the lymphoid organs was not achieved until the juvenile stage. The spleen and head kidney had mixed populations of "red" and "white" cells. The head kidney was more lymphoid than the spleen. The thymus involuted quickly during the first 6 months. The blood components had no obvious relationship with age or season during the period studied.     

Studies of Atlantic salmon (Salmo salar L.) blood, spleen and head kidney leucocytes using specific monoclonal antibodies, immunohistochemistry and flow cytometry

Monoclonal antibodies (mabs) raised against Atlantic salmon serum IgM (C7G7 and G2H3) and isolated peripheral blood leucocytes (PBL) (E3D9, C4B6 and D8B3) were applied in this study. Using immunoenzymehistochemistry, immunofluorescence and flow cytometry, the distribution of mab+ cells in blood, spleen and head kidney from Atlantic salmon were studied. Immunostaining on cytospin preparations and flow cytometry of isolated PBL showed that the Ig+ cells recognised by C7G7 and G2H3 were mononuclear leucocytes (MNL). The cytospin preparations showed some Ig+ cells with strong cytoplasmic staining, most likely plasma cells. The salmon blood neutrophils were the only E3D9+ cells in cytospin preparations of PBL, and E3D9 recognised about 94% of the defined neutrophil fraction in flow cytometry. The reactivities of C4B6 and D8B3 were to a large degree similar in both immunoenzymehistochemistry and flow cytometry, recognising both MNL and blood neutrophils. Immunofluorescence double staining of PBL with C4B6 and D8B3 showed double staining of all mab+ cells and D8B3 was apparently not able to block the binding of C4B6 to PBL. Immunofluorescence double staining of PBL also revealed more E3D9+ than C4B6+ neutrophils. In immunostaining on cryostat sections of spleen and head kidney, staining of cells was observed with all the mabs, the head kidney generally containing more positive cells than the spleen. Some potential applications for immunological studies using these mabs are suggested.     

Studies of Atlantic salmon (Salmo salar L.) blood, spleen and head kidney leucocytes using specific monoclonal antibodies, immunohistochemistry and flow cytometry

Monoclonal antibodies (mabs) raised against Atlantic salmon serum IgM (C7G7 and G2H3) and isolated peripheral blood leucocytes (PBL) (E3D9, C4B6 and D8B3) were applied in this study. Using immunoenzymehistochemistry, immunofluorescence and flow cytometry, the distribution of mab+ cells in blood, spleen and head kidney from Atlantic salmon were studied. Immunostaining on cytospin preparations and flow cytometry of isolated PBL showed that the Ig+ cells recognised by C7G7 and G2H3 were mononuclear leucocytes (MNL). The cytospin preparations showed some Ig+ cells with strong cytoplasmic staining, most likely plasma cells. The salmon blood neutrophils were the only E3D9+ cells in cytospin preparations of PBL, and E3D9 recognised about 94% of the defined neutrophil fraction in flow cytometry. The reactivities of C4B6 and D8B3 were to a large degree similar in both immunoenzymehistochemistry and flow cytometry, recognising both MNL and blood neutrophils. Immunofluorescence double staining of PBL with C4B6 and D8B3 showed double staining of all mab+ cells and D8B3 was apparently not able to block the binding of C4B6 to PBL. Immunofluorescence double staining of PBL also revealed more E3D9+ than C4B6+ neutrophils. In immunostaining on cryostat sections of spleen and head kidney, staining of cells was observed with all the mabs, the head kidney generally containing more positive cells than the spleen. Some potential applications for immunological studies using these mabs are suggested.