Veterinary Immunology and Immunopathology, 26 ( 1990 ) 81-92
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Elsevier Science Publishers B.V., Amsterdam
Characterization of monoclonal antibodies to channel catfish, Ictalurus punctatus, leucocytes* A.J. Ainsworth, C. Dexiang and T. Greenway Department of Basic and Applied Sciences, College Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, U.S.A. (Accepted 2 January 1990) ABSTRACT Ainsworth, A.J., Dexiang, C. and Greenway, T., 1990. Characterization of monoclonal antibodies to channel catfish, Ictalurus punctatus, leucocytes. Vet. Irnmunol. Immunopathol., 26:81-92. Four monoclonal antibodies (MAbs) were evaluated for specific reactions to various catfish peripheral blood leucocytes and anterior kidney cells. The MAb 9El served as a standard and positive control for all test reactions because of its defined reactivity with channel catfish immunoglobulin and immunoglobulin bearing cells. Of the four MAbs, two have been characterized as being specific for a non-immunoglobulin marker on lymphocytes, thus marking T lymphocytes and two were specific for catfish neutrophils. Morphological, flow cytometric and functional analysis of the reactive cells verified these findings.
INTRODUCTION
The preparation of monoclonal antibodies (MAbs) against leucocyte surface antigens has greatly facilitated immunological research in numerous species. For instance, MAbs are tools being used for research on granulocyte and lymphocyte differentiation and functional analysis (Pescovitz et al., 1984; Mackay et al., 1985; Howard et al., 1988; Kunita et al., 1988). Due to the importance of domestic animals in medical research, efforts to produce MAbs recognizing cell markers has been underway for several years; however, minimal work has been done in minor species such as fish. Presently and increasing in the future, fish will serve as the preferable animal model for specific investigations, e.g. toxicological (Weeks and Wariner, 1986 ). Because of the primitive nature of fish and their position on the phylogenetic scale these animals will be overlooked unless detailed physiological characterization is performed. There are basic facts about catfish physiology still to be proven; however, our knowledge of the catfish immune system is improving (Finco-Kent and Thune, 1987; Sizemore et al., 1984). MAbs have been characterized to catfish immunoglobulin (Lobb and Clem, * Supported by Mississippi Agriculture and Forestry Experiment Station grant number MIS6806. Contribution No. J-7245 from the Mississippi Agriculture and Forestry Experiment Station. 0165-2427/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
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1982; Lobb et al., 1984) and catfish T lymphocytes (Miller et al., 1987; Ellsaesser et al., 1988). Bly et al. (1988) has reported on the production of a MAb to catfish neutrophils. Whether the MAb characterized to catfish T lymphocytes by Miller et al. (1987) is reactive to a specific subpopulation or what antigen the MAb reported by Bly et al. ( 1988 ) is reactive to is uncertain. Due to the importance of neutrophils in non-specific immune responses and the need for MAbs identifying T lymphocyte subpopulations, the present study was undertaken to produce MAbs to channel catfish leucocytes that would react with these cell types. MATERIALS AND METHODS
Animals Channel catfish, Ictalurus punctatus Rafinesque, blood leucocytes used for production of MAbs were isolated from fish maintained in 1201 flow-through tanks held at 22_+ 1 °C and were fed 5% of body weight on alternating days. All other methods utilized channel catfish cells isolated from fish maintained in 1/ 10th acre earthen ponds on the Leveck Animal Center, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University. Pond held catfish were fed commercial growth ration daily. R B F / D n mice used in the production of MAbs were held in the Laboratory Animal Care Facilities, College of Veterinary Medicine, MSU. Mice were given food and water ad libitum.
Leucocyte isolation Channel catfish peripheral blood leucocyte (PBL) populations used for inoculation of mice and characterization of MAbs were isolated from heparinized catfish blood using a modification of the procedure of Waterstrat et al. ( 1988 ). Briefly, washed and diluted blood cells were layered onto four layer gradients consisting of 1.060, 1.065, 1.070 and 1.080 layers of Percoll (Pharmacia Fine Chemicals, Piscataway, N J). The cells at the 1.065-1.070 interface (lymphocytes) and at the 1.070-1.080 interface (primarily neutophils) were used for inoculations of mice and in screening assays. Single cell suspensions of anterior kidney were isolated in an identical fashion as above and the cells at the 1.065-1.070 interface were removed and used in assays.
Monoclonal antibody (MAb) production R B F / D n mice (Jackson Laboratories, Bar Harbour, ME) were primed with an intraperitoneal injection of 0.5 ml ( 1 × 108 cells) of either isolated catfish lymphocytes, neutrophils or immunoglobulin (100/tg/mouse). Second and third inoculations were given 4 and 6 weeks after the priming inoculation, respectively. Inoculations of cells or immunoglobulin were administered intraperitoneally for the second and intravascularly for the third inoculation.
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Three days after the third injection, the spleen was removed and a single cell suspension prepared in RPMI basal medium (Gibco, Grand Island, NY). The splenocytes were mixed with the FOX-NY myeloma cell line (Hyclone Laboratories, Logan, UT), at a 10:1 ratio (Taggart and Samloff, 1983) and allowed to fuse in the presence of 50% polyethylene glycol-4000 (Gefter et al., 1977 ). To select for the myeloma-lymphocyte hybrids, AAT medium (final concentration 7.5X 10-5M adenine, 8.0× 10-7M aminopterin, 1.6X 10-SM thymidine) (Taggart and Samloff, 1983) made in RPMI supplemented with 15% fetal bovine serum, 1% L-glutamine and 100 U penicillin-streptomycin/ml was added to the cells as they were dispensed into the wells of microtitration plates at a volume of 0.2 ml/well (4 × 105 spleen cells ). Hybridomas were assayed for specific antibody production to channel catfish PBL by modification of a described enzyme-linked immunosorbent assay using rabbit anti-mouse immunoglobulin G heavy chain specific conjugate (Engvall et al., 1971; Cobbold and Waldman, 1981; Epstein and Lunney, 1985). The hybridomas producing antibody to the catfish PBL were then cloned on soft agar and propagated in vitro in tissue culture flasks containing RPMI with 15% fetal bovine serum, 1% L-glutamine, and 100 U ofpenicillinstreptomycin/ml. Cells were overgrown at 37°C until dead. Tissue culture fluid containing antibody was harvested from each clone and stored at 48°C. Flow cytometry Analysis by fluorescent activated cell sorting (FACS) was done using a FACStar (Becton-Dickinson, Mountain View, CA) standardized with glutaraldehyde fixed chicken red blood cells and fluorescence compensated using Calibrite beads (Becton-Dickinson, Mountain View, CA). All cells for analysis were washed and resuspended at 4 × 107 cells/ml in catfish-RPMI (CFRPMI, 9 parts RPMI 1640 and l part distilled water) (Faulman et al., 1983 ) containing 0.5 M sodium azide, pH 7.8 (Ellsaesser et al., 1985). Fifty/A of cells and 50 al of MAb ( 1 : 2) were incubated for 30 min on ice. Cold CFRPMI (0.5 ml) with 0.5 M sodium azide was added to the cells, centrifuged for l0 min at 4 0 0 × g and the supernatant aspirated. The pellet was resuspended in 100 #1 of rabbit anti-mouse IgG-FITC conjugate, incubated 30 min on ice, and washed with 500/11 of cold catfish-RPMI. Upon removing the supernatant, the pellet was resuspended in 700 al of cold catfish-RPMI. Initially, cells were analyzed for forward light scatter (FSC) versus 90 ° light scatter (SSC) to set the appropriate gain settings. Upon establishing the gain settings, l 0 000 events per sample were collected and analyzed for fluorescence at 515-545 nm and SSC. Appropriate negative (rabbit anti-mouse IgGFITC plus catfish cells ) and positive (9E l plus conjugate plus cells) controls were run. The MAb, 9E l, has been characterized and reacts with catfish immunoglobulin (Lobb and Clem, 1982) and therefore, it identifies catfish B cells.
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Additive studies Additive studies were done in a similar manner as those described for characterization of MAb to catfish immunoglobulin (Lobb and Clem, 1982 ) and provided an initial indication as to similarities of MAbs. Additivity experiments were done on the four uncharacterized MAbs. Additive studies were interpreted by looking at the changes in the intensity of fluorescence and percent of positive cells when only one MAb was used compared to when two or more were used in the same assay. Generally, the results were interpreted as follows. If there was an increase in fluorescent intensity and no increase in the percent of positive cells from the single MAb assay to the additivity assay then the same cells were being identified but a different marker was being detected on the cell. However, when comparing the results of single and additive assays, if the fluorescent intensity remains approximately the same, but the percent of positive cells increases then a different population of cells or a more inclusive cell population was being identified by the MAb question. For instance, PBL were reacted with the MAb being characterized as noted above: however, after this step 9El or another uncharacterized MAb was reacted with the cells for 30 min on ice followed by rabbit anti-mouse FITC conjugate. The percent of total cells marked by a MAb alone or in conjunction with another MAb was determined and compared to reaction of the MAb alone with the cells. The reaction sequence of the uncharacterized monoclonal antibodies was reversed to ensure that steric hinderance and affinity was not influencing FACS results.
Histochemical staining Cytocentrifuge slides were prepared following cell isolation, phagocytic assays or upon FACS sorting of the fluorescent positive cells. Slides were stained with either Wright a n d / o r Sudan black B (Sigma Chemical Company, St. Lousi, MO) (Ellsaesser and Clem, 1986). Positive fluorescent cells were sorted into glass test tubes containing 0.1 ml pooled normal catfish serum by using fluorescent activated cell sorting. Typically, 1 × 10 6 positive cells were collected. The collected cells were pelleted at 4 0 0 × g for 10 min and resuspended in Hank's balanced salt solution, calcium-magnesium free (HBSSCMF), containing 15% catfish serum. Slide preparations were made in the following manner. Slides were washed with 0.5 ml HBSS-CMF for 3 min at 1500 rpm in a cytocentrifuge (CYTO-Tek, Miles Scientific, Elkhart, IN). Three-tenths ml of cell suspension containing a minimum of 5 X 105 cells was added to the slide cassette and centrifuged onto the slide at 500 rpm for 2 min. Slides were air dried and stained with either modified Wright or Sudan black B before viewing by light microscopy. The Sudan black B stain was carried out in the following manner. After fixing the cells over formaldehyde vapor for 10 min, the slides were incubated in Sudan black B stain for 2.5 to 3.0 h. The cells were decolorized for 10 rain in propylene glycol, rinsed in
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distilled water, 90% ethanol and a final rinse in distilled water. The slides were counter stained with Gill No. 3 (Sigma Chemical Co, St. Louis, MO ).
Phagocytic assay Peripheral blood cells isolated as described above were washed and resuspended in HBSS-CMF at a concentration of 5 X 10 6 cells/ml. Five-tenths ml of the cells were incubated with 0.5 ml ofAeromonas hydrophila ( 1 X 108 bacteria/ml, a final bacteria to cell ratio of 20:1 ) and 0.2 ml of normal catfish serum for 30 min at 27°C (Sakai, 1984; Ainsworth et al., 1990). One-tenth ml of the cell-bacteria mixture was used to make cytocentrifuge preparations. The cytocentrifuge slides were stained and examined for percent phagogocytosis and phagocytic index. The percent phagocytosis was determined by calculating the neutrophils out of 100 that contained bacteria. The phagocytic index was determined by counting 100 neutrophils (phagocytic and nonphagocytic ) and dividing the total number of bacteria engulfed by the neutrophils by 100 to yield an average number of bacteria per cell.
Lymphocyte panning and culture assay Channel catfish PBL were separated into marker ( + ) and marker ( - ) populations by indirect panning (Buttke et al., 1983; Sizemore et al., 1984). Polystyrene Petri dishes were coated with 10 ml of a rabbit anti-mouse IgG (Pel-Freeze, Rogers, AR) (0.2 m g / m l in phosphate buffer saline, PBS, pH 7.4) for 36 h at 4 oC. The antibody solution was aspirated, the dishes washed three times with sterile PBS, and CF-RPMI (9 parts RPMI 1640, 1 part distilled water with 10 m M HEPES, 1% L-glutamine, 100 U pencillin and 100 /tg streptomycin ) containing 2% bovine calf serum and 0.1 mg normal rabbit s e r u m / m l was added to the dishes for a 40 min incubation at 22°C. Isolated catfish leukocytes (2 X 108/ml) were mixed with either C2-3A or C2-4A tissue culture fluid at a final dilution of 1 : 3 for 30 min at 4 ° C with occasional mixing. The cells were washed twice in CF-RPMI and diluted to 6 x 107/ml in CF-RPMI and 10 ml of the cell suspension added to the rabbit anti-mouse IgG coated dishes. U p o n incubating the dishes at 4°C for 60-90 min with gently swirling every 15 min, the nonadherent cells were aspirated and retained and the fluid from one wash saved. The dishes were washed twice more and the fluid discarded. The adherent cells were scraped into CF-RPMI, both cell populations were washed twice and used in lymphocyte stimulation cultures. Accessory cell coated plates were prepared by first allowing 0.5 ~g of fibronectin (Sigma Chemical Company, St. Louis, MO) in 0.05 of water to evaporate in each well. Subsequently, 1 × 10 6 PBL in CF-RPMI containing 10% h u m a n plasma and 5% catfish serum were added to each well and incubated for 3 h in a 27°C humid 5% CO2 incubator. The wells were thoroughly washed and the plates used in stimulation assays (Clem et al., 1985 ). Channel catfish PBL, nonadherent, and adherent cells were cultured using the method de-
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scribed by Faulmann et al. ( 1983 ) in accessory cell coated 96-well tissue culture plates. Mitogen concentrations used were as follows; concanavalin A (Con A) 0.25 m g / m l (ICN Biomedicals, Lisle, IL) and lipopolysaccharide (LPS) 1.0 m g / m l (Difco, Baxter S/P, Harahan, LA) dispensed at 10 #l/well. Cultures were incubated in a 27°C, humid, 5% CO2 incubator for 66 h. Eighteen h before harvesting, the cultures were pulsed with 10/~l/well of 3H thymidine ( New England Nuclear, Boston, MA) at 0.5 #Ci/ml. Cultures were harvested onto glass fiber filters, dried and counted in the liquid scintillation counter. Results are expressed as counts per minute (cpm) and as the ratio of Con A to LPS.
Statistical analysis Statistical analysis of the lymphocyte stimulation data was done using the SAS statistical computer software (SAS, 1985 ). RESULTS
Characteristics of MAb reacting with PBL and anterior kidney Results reported in Table 1, 2 and 3 are from different catfish and can only be compared on a qualitative basis. The MAbs (C2-3a, C2-4a, C3-1, and 51A) were reacted to enriched populations of catfish PBL and isolated anterior kidney cells. The data in Table 1 are given as mean percent positive reaction; the MAbs C2-3a and C2-4a reacted with PBL populations ranged from 11.0 to 35.1% and with anterior kidney preparations from 10.2 to 56.0%. The MAbs C3-1 and 51A reacted with all cell populations but in varying percentages depending on the number of neutrophils present. The mean percent positive reaction of MAbs to catfish leucocytes ranged from 5.0 to 14.0% and to isoTABLE 1 Reactivity of MAb with leucocytes as determined by FACS Cells ~
PBL Anterior
Mean percent positive reaction Positive control 2
C2-3a 3
C2-4a
C3-1
51A
Sudan positive
37.3 18.3
20.1 28.8
25.3 41.6
11.2 47.0
10.6 49.2
12.3 56.0
tPBL=peripheral blood leucocytes and were a combination of the cells from the interface of a 1.070 and 1.080 Percoll gradient from individual fish. Anterior kidney cells from individual fish were collected from Histopaque gradients. -'The positive control was the reaction of 9El (known positive for catfish immunoglobulin thus it reacts with B lymphocytes) with the cells being tested. 3The number of catfish tested varied from 5 to 7 per MAb.
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lated anterior kidney cells ranged from 12.7 to 57.6% (Table 1 ). Another measurement obtained by using the FACS data was the correlation of the SSC and fluorescence of the positive cells. The SSC of cells reacting with the MAbs was as follows: 9El, 2.5; C2-3a, 2.23; C2-4a, 2.15; C3-1, 14.02; C2-4a, 14.22. The cells reactive with C3-1 and 51A had a greater SSC indicating more granularity.
Additive reactions of the MAb The additivity studies provided proof that C2-3a, C2-4a, C3-1 and 5 I A were marking cells other than those bearing surface immunoglobulin. Additivity with various combinations of MAbs resulted in essentially the sum of the mean percent positive reaction of each individual MAb in question (Table 2 ) except in cases where the MAb were reacting with the same population of cells resulting in no change in the percent positive. The MAbs noted with the prefix C2 reacted with a different cell type than did the MAb noted as C31 or 51A. Additivity reactions within the C2 group of MAb confirmed that these MAbs were reacting with the same cells. Both MAbs varied in their reaction to T lymphocytes depending on the fish. Likewise, C3-1 and 51A MAbs reacted with the same group of cells. However, C2-3a and C2-4a reacted with a different cell population than those cells marked with C3-1 and 51A. Histochemical staining Wright stained cytocentrifuge preparations of FACS sorted cells positive for the C2-3a and C2-4a had morphological characteristics consistent with lymphocytes. To determine if the cells in FACS analysis were positive for both TABLE2 Additive results o f c h o s e n M A b I as d e t e r m i n e d by FACS analysis First M A b reacted to PBL 2
% Positive 3
9El C2-3a C2-4a C3-I 51A
11.7 28.9 41.1 6.9 10.2
Percent positive u p o n reacting the second M A b C2-3a
C2-4a
C3-1
51A
56.4 ND 4 35.6 34.5 34.9
65.5 26.6 ND 52.4 50.1
14.6 34.5 53.2 ND 10.3
20.2 34.9 51.1 10.7 ND
t M o n o c l o n a l a n t i b o d i e s were r u n in every possible additive c o m b i n a t i o n . T h e Table is representative o f the results obtained. 2PBL = peripheral blood leukocytes isolated as described previously. 3Results are expressed as the percent positive fluorescence. 4ND = not d e t e r m i n e d .
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TABLE 3 A representative comparison of MAb reactivity in FACS versus Sudan black B staining of PBL Fish ~
5D 5D 4C 4C
MAb
C2-3a C2-4a C3-1 5 IA
% positive before FACS sorting
% positive after FACS sorting
FACS
Sudan
FACS
Sudan
37.0 38.1 11.5 15.4
29.02 29.0 22.5 22.5
74.0 87.5 85.0 81.2
3.0 4.5 84.0 82.0
~Catfish were stressed 18 h prior to bleeding for PBL and this is why percent positive by FACS for each marker is higher than in Table 1. :Sudan black B staining was performed on cytocemrifuge slide preparations of 3-5.0 X 10 s FACS sorted fluorescent positive cells.
fluorescence and Sudan black B staining, cytocentrifuge preparations of presorted and FACS sorted fluorescent positive cells were made (Table 3). In the case of C3-1 and 51A there was a direct correlation in the percent fluorescent positive sorted cells and positive Sudan black B staining. However, this was not the case for fluorescent positive C2-3a and C2-4a cells where an increase in FACS sorted purity resulted in a decrease in positive Sudan black B staining.
Assays offimctional ability The decision as to which functional assay a MAb reactive cell type would be included was based on Sudan black B histochemical staining after FACS sorting of fluorescent positive cells. Sorted cells staining predominantly positive for Sudan black B were analyzed for function in phagocytic assays and excluded from lymphocyte panning and culture assays and visa versa for Sudan black B negative cells. Assays for phagocytic ability of the cells staining positive for Sudan black B were carried out on C3-1 and 51A positive cells and were similar. Function based on C3-1 fluorescent positive FACS sorted cells resulted in a 87.8% phagocytosis and a phagocytic index of 17.1. Results of the indirect panning procedure and lymphocyte culture indicated that C23a and C2-4a were reacting with T lymphocytes (Table 4). The cpm, although not extremely high in the adherent cell cultures, were always significantly higher for the Con A stimulated cultures when compared to the cultures stimulated with LPS. Accessory cell presence in cultures also provided the necessary factors to maximize the blastogenesis of T lymphocytes. Another measurement used to assess whether the adherent cells were T lymphocytes was the comparison of Con A to LPS ratios for cultures. The PBL and nonadherent cultures for both mAbs had an average Con A: LPS ratio of 0.54
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TABLE 4 Blastogenic activity of C2-3a and C2-4a adherent positive cells after being panned t MAb
Culture
ConA /
LPS
Neg
Con A: LPS ratio
C2-3a
PBL Adherent Nonadherent PBL Adherent Nonadherent
50363 1073* 441 l 9152 1370* 8775
8284* 289 8220* 22126" 1180 14952"
1008 139 1648 2937 210 2864
0.614 3.71 0.59 0.41 1.16 0.59
C2-4a
t Perpheral blood leukocytes (PBL) were reacted with a 1:3 dilution of tissue culture fluid containing either C2-3A or C2-4A for 30 min at 4°C, washed, and incubated for 60 min on plates coated with rabbit anti-mouse Ig. Nonadherent cells were aspirated from plates and adherent cells scraped off the plates. Isolated PBL were dispensed ( 1 × 106 cells/well ) into flbronectin coated 96-well tissue culture plates and incubated for 3 h in a 5% CO2, 27°C humid incubator. Wells were washed with CF-RPMI leaving fibronectin adherent accessory cells. Different fish were used for each MAb. -'Cultures were incubated in the presence of Con A, LPS, or only media (negative) for the duration of the experiment. 3Results are expressed as the least square mean of counts per minute (cpm) of cultures and analyzed for significant differences ( P < 0 . 0 0 1 ) between Con A and LPS within a given treatment. CPM being significantly higher in mitogen stimulated cultures, i.e. Con A and LPS, within a treatment are denoted with an *. The standard of error of the least square means was less than 6% in all cases. 4The Con A: LPS ratio was calculated by dividing the cpm of the Con A cultured by the cpm of the LPS culture.
whereas the adherent cultures had a ratio of 3.71 and 1.16 for C2-3a and C24a, respectively.
DISCUSSION
Monoclonal antibodies are known to be useful tools for investigations into cell differentiation and as markers for cell distribution studies. We have produced a panel of monoclonal antibodies which should be useful for those types of studies. Based on the histochemical staining, FSC/SSC FACS analysis and the functional assays, two of the MAbs were found to react with channel catfish non-immunoglobulin bearing lymphocytes (T lymphocytes) and two to neutrophils. Recent work in another laboratory has produced a MAb which reacts with channel catfish T lymphocytes and the cells of the thymus (Ellsaesser et al., 1988) and have a preliminary report on a MAb to channel catfish neutrophilic granulocytes (Bly et al., 1988 ). The FACS software (Becton-Dickinson, Mountainview, CA) assigns an arbitrary number to the location of cells
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based on their SSC or granularity. Cells in the granulocyte series are known to have a greater SSC than lymphoid cells and can therefore be tentatively identified using this measurement. The cells reactive with C2-3a and C2-4a had very little SSC and were considered to be lymphoid-like cells whereas the MAbs C3-1 and 51A identified cells having a greater SSC consistent with that of neutrophils. Panning and lymphocyte stimulation assays run on C2-3a and C2-4a reactive cells indicated that T lymphocytes were the adherent cell population. For example, mitogen induced blastogenesis of C2-3a adherent cells resulted in a cpm of 1073 for Con A and 289 cpm for LPS, whereas the nonadherent cells gave the opposite result with a cpm of4411 for Con A and 8220 for LPS. Although the cpm for C2-4a adherent cells did not contrast as well between Con A and LPS, there was a significant difference in the two cpm ( P < 0.001 ). The Con A:LPS ratio in the C2-4a experiment was 1.16 for adherent cultures as compared to 0.59 for the nonadherent Con A:LPS. The smaller Con A: LPS ratio of the C2-4a adherent cell population suggest that a subpopulation of cells might be adherent. The low cpm obtained in the adherent cell cultures was due to the low number of accessory cells that bound to the fibronectin coated microtiter culture wells. Sizemore et al. ( 1984 ) found that optimum blastogenic responses in Con A responding cells were obtained when 4 × 103 accessory cells per well were used and Clem et al. (1985) has shown the ability of channel catfish monocytes to act as accessory cell in Con A stimulated immune responses. Of concern was the finding that a T lymphocyte response was detectable in the nonadherent cell population. This phenomenon can be explained as a technical problem with the panning assay related to the absolute number of cells that can be bound to the rabbit antimouse coated Petri dishes and the affinity to which the cells are bound to the plates. Also, one must consider that a subpopulation of T lymphocytes was adhering to the plates leaving the remaining T lymphocytes to be aspirated out of the plates with the nonadherent cells. There was a direct correlation between C3-1 and 51A FACS sorted fluorescent positive cells and the Sudan black B stain; therefore these cell populations were analyzed in the phagocytic assays only. When FACS sorted fluorescent positive peripheral blood neutrophils were incubated with Aeromonas hydrophila in the presence of 16.6% normal catfish serum, the percent phagocytosis was 87.8% with a phagocytic index of 17.1. Phagocytosis was evident by marked morphological changes in the neutrophils consisting of vacuoles containing bacteria within cells and a frequent finding of the nucleus being flattened and pushed to the periphery of a cell heavily laden with bacteria. Additivity studies using the newly produced catfish lymphocyte MAbs and previously characterized 9E 1 (Lobb et al., 1982 ) provided further evidence for the conclusion that a T-lymphocyte was being marked because the percent of fluorescent positive cells increased when C2-3a and C2-4a were added to the assay with 9E 1. When assaying all the various combinations of C2-3a and
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C2-4a with each other we obtained mixed results. In additivity experiments if C2-3a was reacted with the PBL then the percent positive cells was lower than if C2-4a was reacted first. These results are quite interesting considering that various stressing events caused the results to be reversed. Further studies will be carried out to determine if subpopulation reactions are occurring. The results of additive experiments of the uncharacterized MAb also provided evidence that C2-3a and C2-4a were reacting with a different cell population than C3-1 and 51A. Since accepted distribution values for various peripheral blood cells of the channel catfish are only now being established, one must be cautious when stating that a particular number of range of a cell type is normal (Ellsaesser and Clem, 1986). Using the MAbs discussed in the present work, we have been able to demonstrate dramatic shifts in surface immunoglobulin bearing cells (B-lymphocytes), non-immunoglobulin bearing cells (T-lymphocytes) and neutrophils in channel catfish under various stress situations (manuscript in preparation ). The MAbs produced in the present study will not only be useful in basic research but will have practical value, for example, in monitoring changes in cell numbers during physiological or environmental stress. ACKNOWLEDGEMENTS
I wish to thank G. Capley for assistance in producing the monoclonal antibodies, J. Brazil for assistance in the characterization studies and B. Boyd for assistance in the characterization studies and for performing flow cytometry analysis. I also wish to thank Dr. S. Pruett for his valuable comments concerning this manuscript.
REFERENCES Ainsworth, A.J. and Dexiang, C., 1990. Differences in the phagocytosis of four bacteria by channel catfish neutrophils: opsonic effect and killing ability. Dev. Comp. Immunol., in press. Bly, J.E., Miller, N.W. and Clem, L.W., 1988. Identification of channel catfish neutrophils using monoclonal antibodies. The kinetics of stress-induced neutrophilia (Abstract). South Central Branch of the American Society of Microbiology and Midsouth Biochemists Annual Meeting. Baton Rouge, LA, p 48. Buttke, T.M., Mallett, G.S. and Cuchen, M.A., 1983. Positive selection of mouse B and T lymphocytes and analysis of isolated populations by flow cytometry. J. Immunol. Methods, 58: 193-199. Clem, L.W., Sizemore, R.C., Ellsaesser, C.F. and Miller, N.W., 1985. Monocytes as accessory cells in fish immune responses. Dev. Comp. Immunol., 9: 803-809. Cobbold, S.P. and Waldmann, H., 1981. A rapid solid-phase enzyme-linked binding assay for screening monoclonal antibodies to cell surface antigens. J. Immunol. Methods, 44: 125133.
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Ellsaesser, C.F. and Clem, L.W., 1986. Haematological and immunological changes in channel catfish stressed by handling and transport. J. Fish Biol., 28:511-521. Ellsaesser, C.F., Bly, J.E. and Clem, L.W., 1988. Phylogeny of lymphocyte heterogeneity: The thymus of the channel catfish. Dev. Comp. lmmunol., 12: 787-799. Ellsaesser, C.F., Miller. N.W., Cuchens, M.A. and Lobb, C.J., 1985. Analysis of channel catfish peripheral blood leucosytes by bright-field microscopy and flow cytometry. Trans. Am. Fish. Soc., 114: 279-285. Engvall, E. and Perlman. P., 1971. Enzyme-linked immunosorbent assay of lgG Immunochemistry, 8: 871-874. Epstein, S.L. and Lunney, J.K., 1985. A cell surface ELISA in the mouse using only poly-llysine as a cell fixative. J. Immunol. Methods, 76: 63-72. Faulman, E., Cuchens, M.A., Lobb, C.J., Miller, N.W. and Clem, L.W., 1983. An effective culture system for studying in vitro mitogenic responses of channel catfish lymphocytes. Trans. Am. Fish. Soc., 112: 673-679. Finco-Kent, D. and Thune, R.L., 1987. Phagocytosis by catfish neutrophils. J. Fish Biol., 31 ( supplement A ): 41-49. Gefter, M.L., Marguiles, P.H. and Schraff, M.D., 1977. A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Sore. Cell Gen., 3:231-236. Howard, C.J. Parsons, K.R., Jones, B.V. Sopp, P. and Pocock, D.H., 1988. Two monoclonal antibodies (CC17, CC29 ) recognizing an antigen (Bo5) on bovine T lymphocytes, analogous to human CD5. Vet. Immunol. Immunopathol., 19:127-139. Kunita, S., Koyama, H. and Saito, H., 1988. Preparation and characterization of monoclonal antibodies against bovine lymphocyte surface antigens. Vet. Immunol. Immunopathol.. 18: 201-212. l o b b , C.J. and Clem, L.W., 1982. Fish lymphocytes differ in the expression of surface immunoglobulin. Dev. Comp. Immunol., 6: 473-479. Lobb, C.J., Olson, M.O.J. and Clem, L.W., 1984. Immunoglobulin light chain classes in a teleost fish. J. |mmunol., 132: 1917-1923. Mackay, C.R., Maddox, J.F., Gogolin-Ewens, K.J. and Brandon, M.R., 1985. Characterization of two sheep lymphocyte differentiation antigens, SBU-T1 and SBU-T6. Immunology, 55: 729-737. Miller, N.W., Bly, J.E., VanGinkel, F., Ellsaesser, C.F. and Clem, L.W., 1987. Phylogeny of lymphocyte heterogeneity: Identification and separation of functionally distinct subpopulations of channel catfish lymphocytes with monoclonal antibodies. Dev. Comp. lmmunol., 1 I: 739-747. Pescovitz, M.D., Lunney, J.K. and Sachs, D.H., 1984. Preparation and characterization of monoclonal antibodies reactive with porcine PBL. J. lmmunol., 133: 368-375. Sakai, D.K., 1984. Opsonization by fish antibody and complement in the immune phagocytosis by peritoneal exudate cells isolated from salmonid fishes. J. Fish Dis., 7: 29-38. SAS, 1985. SAS User's Guide: Statistics (Version 5 edition). Car3', North Carolina, CA, pp. 956. Sizemore, R.C., Miller, N.W., Cuchens, M.A. Lobb, C.J. and Clem, L.W.. 1984. Phylogeny of lymphocyte heterogeneity: the cellular requirements for in vitro mitogenic responses of channel catfish leukocytes. J. Immunol.. 133: 2920-2924. Taggart, R.T. and Samloff, I.M., 1983. Stable antibody-producing murine hybridomas. Science. 219: 1228-1230. Waterstrat, P.R., Ainsworth, A.J. and Capley, G., 1988. Use of discontinuous Percoll gradient technique for the separation of channel catfish peripheral blood leukocytes. J. Fish Dis., 11 (4): 289-294. Weeks, B.A. and Wariner, J.E., 1986. Functional evaluation of macrophages in fish form a polluted estuary. Vet. Immunol. lmmunopathol., 12:313-320.
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Characterization of monoclonal antibodies to channel catfish, Ictalurus punctatus, leucocytes* A.J. Ainsworth, C. Dexiang and T. Greenway Department of Basic and Applied Sciences, College Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, U.S.A. (Accepted 2 January 1990) ABSTRACT Ainsworth, A.J., Dexiang, C. and Greenway, T., 1990. Characterization of monoclonal antibodies to channel catfish, Ictalurus punctatus, leucocytes. Vet. Irnmunol. Immunopathol., 26:81-92. Four monoclonal antibodies (MAbs) were evaluated for specific reactions to various catfish peripheral blood leucocytes and anterior kidney cells. The MAb 9El served as a standard and positive control for all test reactions because of its defined reactivity with channel catfish immunoglobulin and immunoglobulin bearing cells. Of the four MAbs, two have been characterized as being specific for a non-immunoglobulin marker on lymphocytes, thus marking T lymphocytes and two were specific for catfish neutrophils. Morphological, flow cytometric and functional analysis of the reactive cells verified these findings.
INTRODUCTION
The preparation of monoclonal antibodies (MAbs) against leucocyte surface antigens has greatly facilitated immunological research in numerous species. For instance, MAbs are tools being used for research on granulocyte and lymphocyte differentiation and functional analysis (Pescovitz et al., 1984; Mackay et al., 1985; Howard et al., 1988; Kunita et al., 1988). Due to the importance of domestic animals in medical research, efforts to produce MAbs recognizing cell markers has been underway for several years; however, minimal work has been done in minor species such as fish. Presently and increasing in the future, fish will serve as the preferable animal model for specific investigations, e.g. toxicological (Weeks and Wariner, 1986 ). Because of the primitive nature of fish and their position on the phylogenetic scale these animals will be overlooked unless detailed physiological characterization is performed. There are basic facts about catfish physiology still to be proven; however, our knowledge of the catfish immune system is improving (Finco-Kent and Thune, 1987; Sizemore et al., 1984). MAbs have been characterized to catfish immunoglobulin (Lobb and Clem, * Supported by Mississippi Agriculture and Forestry Experiment Station grant number MIS6806. Contribution No. J-7245 from the Mississippi Agriculture and Forestry Experiment Station. 0165-2427/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
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1982; Lobb et al., 1984) and catfish T lymphocytes (Miller et al., 1987; Ellsaesser et al., 1988). Bly et al. (1988) has reported on the production of a MAb to catfish neutrophils. Whether the MAb characterized to catfish T lymphocytes by Miller et al. (1987) is reactive to a specific subpopulation or what antigen the MAb reported by Bly et al. ( 1988 ) is reactive to is uncertain. Due to the importance of neutrophils in non-specific immune responses and the need for MAbs identifying T lymphocyte subpopulations, the present study was undertaken to produce MAbs to channel catfish leucocytes that would react with these cell types. MATERIALS AND METHODS
Animals Channel catfish, Ictalurus punctatus Rafinesque, blood leucocytes used for production of MAbs were isolated from fish maintained in 1201 flow-through tanks held at 22_+ 1 °C and were fed 5% of body weight on alternating days. All other methods utilized channel catfish cells isolated from fish maintained in 1/ 10th acre earthen ponds on the Leveck Animal Center, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University. Pond held catfish were fed commercial growth ration daily. R B F / D n mice used in the production of MAbs were held in the Laboratory Animal Care Facilities, College of Veterinary Medicine, MSU. Mice were given food and water ad libitum.
Leucocyte isolation Channel catfish peripheral blood leucocyte (PBL) populations used for inoculation of mice and characterization of MAbs were isolated from heparinized catfish blood using a modification of the procedure of Waterstrat et al. ( 1988 ). Briefly, washed and diluted blood cells were layered onto four layer gradients consisting of 1.060, 1.065, 1.070 and 1.080 layers of Percoll (Pharmacia Fine Chemicals, Piscataway, N J). The cells at the 1.065-1.070 interface (lymphocytes) and at the 1.070-1.080 interface (primarily neutophils) were used for inoculations of mice and in screening assays. Single cell suspensions of anterior kidney were isolated in an identical fashion as above and the cells at the 1.065-1.070 interface were removed and used in assays.
Monoclonal antibody (MAb) production R B F / D n mice (Jackson Laboratories, Bar Harbour, ME) were primed with an intraperitoneal injection of 0.5 ml ( 1 × 108 cells) of either isolated catfish lymphocytes, neutrophils or immunoglobulin (100/tg/mouse). Second and third inoculations were given 4 and 6 weeks after the priming inoculation, respectively. Inoculations of cells or immunoglobulin were administered intraperitoneally for the second and intravascularly for the third inoculation.
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Three days after the third injection, the spleen was removed and a single cell suspension prepared in RPMI basal medium (Gibco, Grand Island, NY). The splenocytes were mixed with the FOX-NY myeloma cell line (Hyclone Laboratories, Logan, UT), at a 10:1 ratio (Taggart and Samloff, 1983) and allowed to fuse in the presence of 50% polyethylene glycol-4000 (Gefter et al., 1977 ). To select for the myeloma-lymphocyte hybrids, AAT medium (final concentration 7.5X 10-5M adenine, 8.0× 10-7M aminopterin, 1.6X 10-SM thymidine) (Taggart and Samloff, 1983) made in RPMI supplemented with 15% fetal bovine serum, 1% L-glutamine and 100 U penicillin-streptomycin/ml was added to the cells as they were dispensed into the wells of microtitration plates at a volume of 0.2 ml/well (4 × 105 spleen cells ). Hybridomas were assayed for specific antibody production to channel catfish PBL by modification of a described enzyme-linked immunosorbent assay using rabbit anti-mouse immunoglobulin G heavy chain specific conjugate (Engvall et al., 1971; Cobbold and Waldman, 1981; Epstein and Lunney, 1985). The hybridomas producing antibody to the catfish PBL were then cloned on soft agar and propagated in vitro in tissue culture flasks containing RPMI with 15% fetal bovine serum, 1% L-glutamine, and 100 U ofpenicillinstreptomycin/ml. Cells were overgrown at 37°C until dead. Tissue culture fluid containing antibody was harvested from each clone and stored at 48°C. Flow cytometry Analysis by fluorescent activated cell sorting (FACS) was done using a FACStar (Becton-Dickinson, Mountain View, CA) standardized with glutaraldehyde fixed chicken red blood cells and fluorescence compensated using Calibrite beads (Becton-Dickinson, Mountain View, CA). All cells for analysis were washed and resuspended at 4 × 107 cells/ml in catfish-RPMI (CFRPMI, 9 parts RPMI 1640 and l part distilled water) (Faulman et al., 1983 ) containing 0.5 M sodium azide, pH 7.8 (Ellsaesser et al., 1985). Fifty/A of cells and 50 al of MAb ( 1 : 2) were incubated for 30 min on ice. Cold CFRPMI (0.5 ml) with 0.5 M sodium azide was added to the cells, centrifuged for l0 min at 4 0 0 × g and the supernatant aspirated. The pellet was resuspended in 100 #1 of rabbit anti-mouse IgG-FITC conjugate, incubated 30 min on ice, and washed with 500/11 of cold catfish-RPMI. Upon removing the supernatant, the pellet was resuspended in 700 al of cold catfish-RPMI. Initially, cells were analyzed for forward light scatter (FSC) versus 90 ° light scatter (SSC) to set the appropriate gain settings. Upon establishing the gain settings, l 0 000 events per sample were collected and analyzed for fluorescence at 515-545 nm and SSC. Appropriate negative (rabbit anti-mouse IgGFITC plus catfish cells ) and positive (9E l plus conjugate plus cells) controls were run. The MAb, 9E l, has been characterized and reacts with catfish immunoglobulin (Lobb and Clem, 1982) and therefore, it identifies catfish B cells.
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Additive studies Additive studies were done in a similar manner as those described for characterization of MAb to catfish immunoglobulin (Lobb and Clem, 1982 ) and provided an initial indication as to similarities of MAbs. Additivity experiments were done on the four uncharacterized MAbs. Additive studies were interpreted by looking at the changes in the intensity of fluorescence and percent of positive cells when only one MAb was used compared to when two or more were used in the same assay. Generally, the results were interpreted as follows. If there was an increase in fluorescent intensity and no increase in the percent of positive cells from the single MAb assay to the additivity assay then the same cells were being identified but a different marker was being detected on the cell. However, when comparing the results of single and additive assays, if the fluorescent intensity remains approximately the same, but the percent of positive cells increases then a different population of cells or a more inclusive cell population was being identified by the MAb question. For instance, PBL were reacted with the MAb being characterized as noted above: however, after this step 9El or another uncharacterized MAb was reacted with the cells for 30 min on ice followed by rabbit anti-mouse FITC conjugate. The percent of total cells marked by a MAb alone or in conjunction with another MAb was determined and compared to reaction of the MAb alone with the cells. The reaction sequence of the uncharacterized monoclonal antibodies was reversed to ensure that steric hinderance and affinity was not influencing FACS results.
Histochemical staining Cytocentrifuge slides were prepared following cell isolation, phagocytic assays or upon FACS sorting of the fluorescent positive cells. Slides were stained with either Wright a n d / o r Sudan black B (Sigma Chemical Company, St. Lousi, MO) (Ellsaesser and Clem, 1986). Positive fluorescent cells were sorted into glass test tubes containing 0.1 ml pooled normal catfish serum by using fluorescent activated cell sorting. Typically, 1 × 10 6 positive cells were collected. The collected cells were pelleted at 4 0 0 × g for 10 min and resuspended in Hank's balanced salt solution, calcium-magnesium free (HBSSCMF), containing 15% catfish serum. Slide preparations were made in the following manner. Slides were washed with 0.5 ml HBSS-CMF for 3 min at 1500 rpm in a cytocentrifuge (CYTO-Tek, Miles Scientific, Elkhart, IN). Three-tenths ml of cell suspension containing a minimum of 5 X 105 cells was added to the slide cassette and centrifuged onto the slide at 500 rpm for 2 min. Slides were air dried and stained with either modified Wright or Sudan black B before viewing by light microscopy. The Sudan black B stain was carried out in the following manner. After fixing the cells over formaldehyde vapor for 10 min, the slides were incubated in Sudan black B stain for 2.5 to 3.0 h. The cells were decolorized for 10 rain in propylene glycol, rinsed in
MAbs TO ICTALURUS PUNCT,4TUS LEUCOCYTES
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distilled water, 90% ethanol and a final rinse in distilled water. The slides were counter stained with Gill No. 3 (Sigma Chemical Co, St. Louis, MO ).
Phagocytic assay Peripheral blood cells isolated as described above were washed and resuspended in HBSS-CMF at a concentration of 5 X 10 6 cells/ml. Five-tenths ml of the cells were incubated with 0.5 ml ofAeromonas hydrophila ( 1 X 108 bacteria/ml, a final bacteria to cell ratio of 20:1 ) and 0.2 ml of normal catfish serum for 30 min at 27°C (Sakai, 1984; Ainsworth et al., 1990). One-tenth ml of the cell-bacteria mixture was used to make cytocentrifuge preparations. The cytocentrifuge slides were stained and examined for percent phagogocytosis and phagocytic index. The percent phagocytosis was determined by calculating the neutrophils out of 100 that contained bacteria. The phagocytic index was determined by counting 100 neutrophils (phagocytic and nonphagocytic ) and dividing the total number of bacteria engulfed by the neutrophils by 100 to yield an average number of bacteria per cell.
Lymphocyte panning and culture assay Channel catfish PBL were separated into marker ( + ) and marker ( - ) populations by indirect panning (Buttke et al., 1983; Sizemore et al., 1984). Polystyrene Petri dishes were coated with 10 ml of a rabbit anti-mouse IgG (Pel-Freeze, Rogers, AR) (0.2 m g / m l in phosphate buffer saline, PBS, pH 7.4) for 36 h at 4 oC. The antibody solution was aspirated, the dishes washed three times with sterile PBS, and CF-RPMI (9 parts RPMI 1640, 1 part distilled water with 10 m M HEPES, 1% L-glutamine, 100 U pencillin and 100 /tg streptomycin ) containing 2% bovine calf serum and 0.1 mg normal rabbit s e r u m / m l was added to the dishes for a 40 min incubation at 22°C. Isolated catfish leukocytes (2 X 108/ml) were mixed with either C2-3A or C2-4A tissue culture fluid at a final dilution of 1 : 3 for 30 min at 4 ° C with occasional mixing. The cells were washed twice in CF-RPMI and diluted to 6 x 107/ml in CF-RPMI and 10 ml of the cell suspension added to the rabbit anti-mouse IgG coated dishes. U p o n incubating the dishes at 4°C for 60-90 min with gently swirling every 15 min, the nonadherent cells were aspirated and retained and the fluid from one wash saved. The dishes were washed twice more and the fluid discarded. The adherent cells were scraped into CF-RPMI, both cell populations were washed twice and used in lymphocyte stimulation cultures. Accessory cell coated plates were prepared by first allowing 0.5 ~g of fibronectin (Sigma Chemical Company, St. Louis, MO) in 0.05 of water to evaporate in each well. Subsequently, 1 × 10 6 PBL in CF-RPMI containing 10% h u m a n plasma and 5% catfish serum were added to each well and incubated for 3 h in a 27°C humid 5% CO2 incubator. The wells were thoroughly washed and the plates used in stimulation assays (Clem et al., 1985 ). Channel catfish PBL, nonadherent, and adherent cells were cultured using the method de-
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scribed by Faulmann et al. ( 1983 ) in accessory cell coated 96-well tissue culture plates. Mitogen concentrations used were as follows; concanavalin A (Con A) 0.25 m g / m l (ICN Biomedicals, Lisle, IL) and lipopolysaccharide (LPS) 1.0 m g / m l (Difco, Baxter S/P, Harahan, LA) dispensed at 10 #l/well. Cultures were incubated in a 27°C, humid, 5% CO2 incubator for 66 h. Eighteen h before harvesting, the cultures were pulsed with 10/~l/well of 3H thymidine ( New England Nuclear, Boston, MA) at 0.5 #Ci/ml. Cultures were harvested onto glass fiber filters, dried and counted in the liquid scintillation counter. Results are expressed as counts per minute (cpm) and as the ratio of Con A to LPS.
Statistical analysis Statistical analysis of the lymphocyte stimulation data was done using the SAS statistical computer software (SAS, 1985 ). RESULTS
Characteristics of MAb reacting with PBL and anterior kidney Results reported in Table 1, 2 and 3 are from different catfish and can only be compared on a qualitative basis. The MAbs (C2-3a, C2-4a, C3-1, and 51A) were reacted to enriched populations of catfish PBL and isolated anterior kidney cells. The data in Table 1 are given as mean percent positive reaction; the MAbs C2-3a and C2-4a reacted with PBL populations ranged from 11.0 to 35.1% and with anterior kidney preparations from 10.2 to 56.0%. The MAbs C3-1 and 51A reacted with all cell populations but in varying percentages depending on the number of neutrophils present. The mean percent positive reaction of MAbs to catfish leucocytes ranged from 5.0 to 14.0% and to isoTABLE 1 Reactivity of MAb with leucocytes as determined by FACS Cells ~
PBL Anterior
Mean percent positive reaction Positive control 2
C2-3a 3
C2-4a
C3-1
51A
Sudan positive
37.3 18.3
20.1 28.8
25.3 41.6
11.2 47.0
10.6 49.2
12.3 56.0
tPBL=peripheral blood leucocytes and were a combination of the cells from the interface of a 1.070 and 1.080 Percoll gradient from individual fish. Anterior kidney cells from individual fish were collected from Histopaque gradients. -'The positive control was the reaction of 9El (known positive for catfish immunoglobulin thus it reacts with B lymphocytes) with the cells being tested. 3The number of catfish tested varied from 5 to 7 per MAb.
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lated anterior kidney cells ranged from 12.7 to 57.6% (Table 1 ). Another measurement obtained by using the FACS data was the correlation of the SSC and fluorescence of the positive cells. The SSC of cells reacting with the MAbs was as follows: 9El, 2.5; C2-3a, 2.23; C2-4a, 2.15; C3-1, 14.02; C2-4a, 14.22. The cells reactive with C3-1 and 51A had a greater SSC indicating more granularity.
Additive reactions of the MAb The additivity studies provided proof that C2-3a, C2-4a, C3-1 and 5 I A were marking cells other than those bearing surface immunoglobulin. Additivity with various combinations of MAbs resulted in essentially the sum of the mean percent positive reaction of each individual MAb in question (Table 2 ) except in cases where the MAb were reacting with the same population of cells resulting in no change in the percent positive. The MAbs noted with the prefix C2 reacted with a different cell type than did the MAb noted as C31 or 51A. Additivity reactions within the C2 group of MAb confirmed that these MAbs were reacting with the same cells. Both MAbs varied in their reaction to T lymphocytes depending on the fish. Likewise, C3-1 and 51A MAbs reacted with the same group of cells. However, C2-3a and C2-4a reacted with a different cell population than those cells marked with C3-1 and 51A. Histochemical staining Wright stained cytocentrifuge preparations of FACS sorted cells positive for the C2-3a and C2-4a had morphological characteristics consistent with lymphocytes. To determine if the cells in FACS analysis were positive for both TABLE2 Additive results o f c h o s e n M A b I as d e t e r m i n e d by FACS analysis First M A b reacted to PBL 2
% Positive 3
9El C2-3a C2-4a C3-I 51A
11.7 28.9 41.1 6.9 10.2
Percent positive u p o n reacting the second M A b C2-3a
C2-4a
C3-1
51A
56.4 ND 4 35.6 34.5 34.9
65.5 26.6 ND 52.4 50.1
14.6 34.5 53.2 ND 10.3
20.2 34.9 51.1 10.7 ND
t M o n o c l o n a l a n t i b o d i e s were r u n in every possible additive c o m b i n a t i o n . T h e Table is representative o f the results obtained. 2PBL = peripheral blood leukocytes isolated as described previously. 3Results are expressed as the percent positive fluorescence. 4ND = not d e t e r m i n e d .
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TABLE 3 A representative comparison of MAb reactivity in FACS versus Sudan black B staining of PBL Fish ~
5D 5D 4C 4C
MAb
C2-3a C2-4a C3-1 5 IA
% positive before FACS sorting
% positive after FACS sorting
FACS
Sudan
FACS
Sudan
37.0 38.1 11.5 15.4
29.02 29.0 22.5 22.5
74.0 87.5 85.0 81.2
3.0 4.5 84.0 82.0
~Catfish were stressed 18 h prior to bleeding for PBL and this is why percent positive by FACS for each marker is higher than in Table 1. :Sudan black B staining was performed on cytocemrifuge slide preparations of 3-5.0 X 10 s FACS sorted fluorescent positive cells.
fluorescence and Sudan black B staining, cytocentrifuge preparations of presorted and FACS sorted fluorescent positive cells were made (Table 3). In the case of C3-1 and 51A there was a direct correlation in the percent fluorescent positive sorted cells and positive Sudan black B staining. However, this was not the case for fluorescent positive C2-3a and C2-4a cells where an increase in FACS sorted purity resulted in a decrease in positive Sudan black B staining.
Assays offimctional ability The decision as to which functional assay a MAb reactive cell type would be included was based on Sudan black B histochemical staining after FACS sorting of fluorescent positive cells. Sorted cells staining predominantly positive for Sudan black B were analyzed for function in phagocytic assays and excluded from lymphocyte panning and culture assays and visa versa for Sudan black B negative cells. Assays for phagocytic ability of the cells staining positive for Sudan black B were carried out on C3-1 and 51A positive cells and were similar. Function based on C3-1 fluorescent positive FACS sorted cells resulted in a 87.8% phagocytosis and a phagocytic index of 17.1. Results of the indirect panning procedure and lymphocyte culture indicated that C23a and C2-4a were reacting with T lymphocytes (Table 4). The cpm, although not extremely high in the adherent cell cultures, were always significantly higher for the Con A stimulated cultures when compared to the cultures stimulated with LPS. Accessory cell presence in cultures also provided the necessary factors to maximize the blastogenesis of T lymphocytes. Another measurement used to assess whether the adherent cells were T lymphocytes was the comparison of Con A to LPS ratios for cultures. The PBL and nonadherent cultures for both mAbs had an average Con A: LPS ratio of 0.54
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MAbs TO ICT4LUR US PUNCTA TUS LEUCOCYTES
TABLE 4 Blastogenic activity of C2-3a and C2-4a adherent positive cells after being panned t MAb
Culture
ConA /
LPS
Neg
Con A: LPS ratio
C2-3a
PBL Adherent Nonadherent PBL Adherent Nonadherent
50363 1073* 441 l 9152 1370* 8775
8284* 289 8220* 22126" 1180 14952"
1008 139 1648 2937 210 2864
0.614 3.71 0.59 0.41 1.16 0.59
C2-4a
t Perpheral blood leukocytes (PBL) were reacted with a 1:3 dilution of tissue culture fluid containing either C2-3A or C2-4A for 30 min at 4°C, washed, and incubated for 60 min on plates coated with rabbit anti-mouse Ig. Nonadherent cells were aspirated from plates and adherent cells scraped off the plates. Isolated PBL were dispensed ( 1 × 106 cells/well ) into flbronectin coated 96-well tissue culture plates and incubated for 3 h in a 5% CO2, 27°C humid incubator. Wells were washed with CF-RPMI leaving fibronectin adherent accessory cells. Different fish were used for each MAb. -'Cultures were incubated in the presence of Con A, LPS, or only media (negative) for the duration of the experiment. 3Results are expressed as the least square mean of counts per minute (cpm) of cultures and analyzed for significant differences ( P < 0 . 0 0 1 ) between Con A and LPS within a given treatment. CPM being significantly higher in mitogen stimulated cultures, i.e. Con A and LPS, within a treatment are denoted with an *. The standard of error of the least square means was less than 6% in all cases. 4The Con A: LPS ratio was calculated by dividing the cpm of the Con A cultured by the cpm of the LPS culture.
whereas the adherent cultures had a ratio of 3.71 and 1.16 for C2-3a and C24a, respectively.
DISCUSSION
Monoclonal antibodies are known to be useful tools for investigations into cell differentiation and as markers for cell distribution studies. We have produced a panel of monoclonal antibodies which should be useful for those types of studies. Based on the histochemical staining, FSC/SSC FACS analysis and the functional assays, two of the MAbs were found to react with channel catfish non-immunoglobulin bearing lymphocytes (T lymphocytes) and two to neutrophils. Recent work in another laboratory has produced a MAb which reacts with channel catfish T lymphocytes and the cells of the thymus (Ellsaesser et al., 1988) and have a preliminary report on a MAb to channel catfish neutrophilic granulocytes (Bly et al., 1988 ). The FACS software (Becton-Dickinson, Mountainview, CA) assigns an arbitrary number to the location of cells
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based on their SSC or granularity. Cells in the granulocyte series are known to have a greater SSC than lymphoid cells and can therefore be tentatively identified using this measurement. The cells reactive with C2-3a and C2-4a had very little SSC and were considered to be lymphoid-like cells whereas the MAbs C3-1 and 51A identified cells having a greater SSC consistent with that of neutrophils. Panning and lymphocyte stimulation assays run on C2-3a and C2-4a reactive cells indicated that T lymphocytes were the adherent cell population. For example, mitogen induced blastogenesis of C2-3a adherent cells resulted in a cpm of 1073 for Con A and 289 cpm for LPS, whereas the nonadherent cells gave the opposite result with a cpm of4411 for Con A and 8220 for LPS. Although the cpm for C2-4a adherent cells did not contrast as well between Con A and LPS, there was a significant difference in the two cpm ( P < 0.001 ). The Con A:LPS ratio in the C2-4a experiment was 1.16 for adherent cultures as compared to 0.59 for the nonadherent Con A:LPS. The smaller Con A: LPS ratio of the C2-4a adherent cell population suggest that a subpopulation of cells might be adherent. The low cpm obtained in the adherent cell cultures was due to the low number of accessory cells that bound to the fibronectin coated microtiter culture wells. Sizemore et al. ( 1984 ) found that optimum blastogenic responses in Con A responding cells were obtained when 4 × 103 accessory cells per well were used and Clem et al. (1985) has shown the ability of channel catfish monocytes to act as accessory cell in Con A stimulated immune responses. Of concern was the finding that a T lymphocyte response was detectable in the nonadherent cell population. This phenomenon can be explained as a technical problem with the panning assay related to the absolute number of cells that can be bound to the rabbit antimouse coated Petri dishes and the affinity to which the cells are bound to the plates. Also, one must consider that a subpopulation of T lymphocytes was adhering to the plates leaving the remaining T lymphocytes to be aspirated out of the plates with the nonadherent cells. There was a direct correlation between C3-1 and 51A FACS sorted fluorescent positive cells and the Sudan black B stain; therefore these cell populations were analyzed in the phagocytic assays only. When FACS sorted fluorescent positive peripheral blood neutrophils were incubated with Aeromonas hydrophila in the presence of 16.6% normal catfish serum, the percent phagocytosis was 87.8% with a phagocytic index of 17.1. Phagocytosis was evident by marked morphological changes in the neutrophils consisting of vacuoles containing bacteria within cells and a frequent finding of the nucleus being flattened and pushed to the periphery of a cell heavily laden with bacteria. Additivity studies using the newly produced catfish lymphocyte MAbs and previously characterized 9E 1 (Lobb et al., 1982 ) provided further evidence for the conclusion that a T-lymphocyte was being marked because the percent of fluorescent positive cells increased when C2-3a and C2-4a were added to the assay with 9E 1. When assaying all the various combinations of C2-3a and
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C2-4a with each other we obtained mixed results. In additivity experiments if C2-3a was reacted with the PBL then the percent positive cells was lower than if C2-4a was reacted first. These results are quite interesting considering that various stressing events caused the results to be reversed. Further studies will be carried out to determine if subpopulation reactions are occurring. The results of additive experiments of the uncharacterized MAb also provided evidence that C2-3a and C2-4a were reacting with a different cell population than C3-1 and 51A. Since accepted distribution values for various peripheral blood cells of the channel catfish are only now being established, one must be cautious when stating that a particular number of range of a cell type is normal (Ellsaesser and Clem, 1986). Using the MAbs discussed in the present work, we have been able to demonstrate dramatic shifts in surface immunoglobulin bearing cells (B-lymphocytes), non-immunoglobulin bearing cells (T-lymphocytes) and neutrophils in channel catfish under various stress situations (manuscript in preparation ). The MAbs produced in the present study will not only be useful in basic research but will have practical value, for example, in monitoring changes in cell numbers during physiological or environmental stress. ACKNOWLEDGEMENTS
I wish to thank G. Capley for assistance in producing the monoclonal antibodies, J. Brazil for assistance in the characterization studies and B. Boyd for assistance in the characterization studies and for performing flow cytometry analysis. I also wish to thank Dr. S. Pruett for his valuable comments concerning this manuscript.
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