j.0022-1112.2005.00668.x

8/2/2019 j.0022-1112.2005.00668.x

1/15

American paddlefish leukocytes demonstratemammalian-like cytochemical staining characteristics in

lymphoid tissues

L. P E T R I E -H A N S O N * A ND A. E . P E T E R M A N

Department of Basic Sciences, College of Veterinary Medicine, Mississippi StateUniversity, P.O. Box 6100, Mississippi State, MS 39762, U.S.A.

(Received 27 April 2004, Accepted 13 December 2004)

American paddlefish Polyodon spathula leukocytes demonstrated cytoplasmic staining patternsvery similar to mammalian leukocytes when stained with acid phosphatase, a -naphthyl butyrateesterase and b-glucuronidase. American paddlefish monocytes, lymphocytes and granulocytesstained positive for acid phosphatase. Monocytes stained positive for a -naphthyl butyrateesterase. Lymphocytes that stained positive for a -naphthyl butyrate esterase were designatedtype A. Lymphocytes that stained positive with antibodies to the L chain of white sturgeonAcipenser transmontanus immunoglobulin (Ig) were designated type B. Type A and type Blymphocytes stained positive for b-glucuronidase. All leukocytes observed were negative forSudan Black B. Monocytes, lymphocytes and granulocytes were present in the renal haemato-poietic tissue, spleen, thymus, pericardial myeloid tissue, lamina propria of the spiral valve, andin meningeal myeloid tissue located dorsal to the brain, at the base of the brain and around the

notochord. Peyers patches were present in the gut. Morphological characteristics of leukocytesstained with Wrights and haematoxylin and eosin and appeared very similar to those of otherfish species. # 2005 The Fisheries Society of the British Isles

Key words: chondrosteans; immunocytochemistry; leukocytes.

INTRODUCTION

Two species of paddlefish exist, the American paddlefish Polyodon spathula(Walbaum) and the Chinese paddlefish Psephurus gladius (Martens). These

fishes are modern representatives of the phylogenetic branch that was close tothe divergence of ray-finned and lobe-finned fishes that led to the divergence of tetrapods. Although paddlefish immune processes have not been researched,paddlefish immunoglobulin was characterized by early immunological methods(Pollara et al ., 1968), and paddlefish have demonstrated antibody responses(Acton et al ., 1971).

*Author to whom correspondence should be addressed. Tel.: 1 662 325 1291; fax: 1 662 325 1031;email: [email protected]

Present address: University of South Mississippi Gulf Coast Research Laboratory, 710 E. Beach Blvd,Ocean Springs, MS 39564 U.S.A.

Journal of Fish Biology (2005) 66, 11011115doi:10.1111/j.1095-8649.2005.00668.x,availableonlineathttp://www.blackwell-synergy.com

1101# 2005 The FisheriesSociety of theBritish Isles

8/2/2019 j.0022-1112.2005.00668.x

2/15

Enzyme histochemical stains are important for identifying cell types in tissuesand are critical for identifying immunological cell types involved in develop-mental and pathological processes. Leukocyte staining characteristics deter-mined by enzyme cytochemistry and used in conjunction with standardhistological stains allow more accurate assessments of the tissue distributionof leukocytes. Acid phosphatase (AP) is a phosphoric monoester hydrolase thatacts on ester bonds. In mammals, AP is present in lymphocytes, plasma cells,monocytes, histiocytes and all stages of granulocyte development (Catovsky &Enno, 1977). a -naphthyl butyrate esterase (NBE) occurs in lymphocytes withsurface receptors for IgM; T lymphocytes stain positive, while B lymphocytesare negative (Bevan et al ., 1980). Non-specific esterases in monocytes andmacrophages are also demonstrated by NBE. b-glucuronidase (BG) is a glyco-side hydrolase that occurs in mature thymocytes, circulating T lymphocytes anda subpopulation of B lymphocytes (Machin et al ., 1980). Acid phosphatase,NBE and BG also identify lymphocyte developmental stages. Histochemicalstaining characteristics of teleost leukocytes have been described in Atlanticsalmon Salmo salar L. (Ellis, 1977), carp Cyprinus carpio L. (Secombes et al .,1983), rainbow trout Oncorynchus mykiss (Walbaum) (Razquin et al ., 1990;Kaattari, 1992; Sanchez et al ., 1995), European flounder Platichthys flesus (L.)(Pulsford et al ., 1994) and channel catfish Ictalurus punctatus (Rafinesque)(Petrie-Hanson & Ainsworth, 2000).

At the initiation of this study, histochemical characterization of leukocytesfrom the Polyodontidae had not been performed. To address this void, isolatedperipheral blood leukocytes, tissue imprints and the leukocyte component of primary and secondary lymphoid tissues of P. spathula were characterized.Selected mammalian leukocytic enzyme substrate stains and monoclonal anti-bodies against the light chain of white sturgeon Acipenser transmontanusRichardson immunoglobulin (Adkison et al ., 1996) were utilized and the find-ings compared to mammalian leukocyte staining patterns.

MATERIALS AND METHODS

FISH PRODUCTION

Female American paddlefish were artificially spawned at Kentucky State University,Frankfort, KY, U.S.A. Lutenizing hormone releasing hormone was intra-muscularlyadministered at a rate of 100 mg kg

1 of body mass. The priming dose was 10 % of thetotal dose followed by a resolving dose of 90 % of the total dose 12 h later. When theybegan to release eggs consistently, the females were immobilized and the eggs removed.American paddlefish milt was added to water (1:200) and immediately combined with theeggs. Fullers earth was added to remove the adhesive properties of the eggs and the eggswere stirred with a turkey feather for 20 min. The fertilized eggs were placed in shippingbags, inflated with oxygen and transported to Mississippi State University College of Veterinary Medicines Aquatic Facility. The eggs were incubated in 15 C well water in165 l McDonald jars (30 to 40 % exchange per min) set in 1135 5 l circular fibreglasstanks filled to a depth of 35 cm. After 5 to 6 days of incubation, 1 mm mesh hapas wereplaced below the outflow of the McDonald jars to collect the fry after hatching. After theyolk-sac was absorbed, larvae were released from the hapas into the tanks and fedsalmon starter (Rangen Feeds, ID, U.S.A.) at a rate of 30 % of their body mass perday ( M day

1 ) for the first week, 20 % M day1 for the next 2 weeks and 15 % M day

1

1102 L . P E T RI E -H A NS O N A N D A . E . P E T ER M AN

# 2005 TheFisheries Society of theBritish Isles, Journal of FishBiology 2005, 66, 11011115

8/2/2019 j.0022-1112.2005.00668.x

3/15

until 4 weeks post hatch (wph). Belt feeders continuously provided feed. When theAmerican paddlefish were 4 wph, their diet was supplemented with freeze-dried krilland algae. To administer these feeds, the water flow was turned off, 227g of krill and227 g of algae were added to each tank daily. After 4 h, water flow was resumed. The fishwere maintained in this manner until used for experiments.

P E RI P HE R A L B L OO D L E UK O CY T ES

Blood was collected from 18 month post-hatch (mph) American paddlefish that weresedated with 50 mgl

1 tricaine methane sulphonate (MS-222) by caudal venipuncture into4 ml heparinized tubes. Aliquots of whole blood were layered on 1077, 1083 and 1119histopaque gradients (Sigma Chemical Company). The gradients were centrifuged at 700 gfor 30 min. Each layer was collected, placed in a 15 ml centrifuge tube and labeled A (1077),B (1083) and C (1119), respectively. The volume of each fraction was brought to 5 ml withphosphate buffered saline (PBS) (0 01M, pH 7 0) and mixed. The cells were centrifuged at250 g for 10 min. The supernate was discarded and the cells were resuspended in 10 ml PBS.The cells were transferred onto Poly-Prep microscope slides (Sigma Chemical Company) by

cytocentrifugation using a Cyto-Tek Centrifuge (Miles, Inc., Elkhart, IN, U.S.A.). One andone-half ml of cell suspension were added to each of six cyto-tubes and centrifuged for 5 minat 470 g. For each stain, 100 cells were observed and the per cent of cells staining positive wasnoted. Positive staining was described either as a concentrated focus or blushing (when thecolour appeared diffusely scattered throughout the cytoplasm). Whole blood smears wereprepared to determine differential leukocyte cell counts. Five differential leukocyte counts of whole blood smears were performed and averaged for each stain type.

LYMPHOID TISSUES

Histological samples of the renal haematopoietic tissue (RHT), thymus, spleen, meningealmyeloid tissue (MMT) and pericardial myeloid tissue (CMT) were obtained from 18 mphAmerican paddlefish. Samples were either fixed in phosphate buffered 10 % formalin,paraffin embedded, sectioned at 36 mm and stained with haematoxylin and eosin (HE) orplaced in cryomolds with optimal cutting temperature (OCT) freezing medium (10 24%polyvinyl alcohol and 4 25% polyethylene glycol), snap frozen in liquid nitrogen and held at 70 C until used for cytochemistry and immunohistochemistry. Frozen blocks were seriallysectioned at 48 mm thicknesses using a cryostat Tissue Tek II (Lab Tek Products, Divisionof Miles Laboratories, Inc., Naperville, IL, U.S.A.). Sections were air dried and alternateserial sections cut from the same block were fixed and stained with different enzymesubstrates, monoclonal antibodies, Sudan Black B (SBB), HE or Wrights stain (WR).

STAINING

Enzyme staining procedures were done using commercially available kits with mod-ifications of fixation and incubation times and temperatures to optimize staining of fishtissues (Petrie-Hanson & Ainsworth, 2000). Acid phosphatase, NBE and BG stainingwere performed using a Lymphocyte Enzyme Kit (Sigma Diagnostics). Air-dried sectionswere fixed in a citrate-acetone-formaldehyde (CAF) solution (25 ml 18 mM citric acid,9 mM sodium citrate, 12 mM sodium chloride, 65 ml acetone and 8 ml 37 % formalde-hyde, adjusted to pH 3 6) for 30 s and rinsed in running deionized water for 4560 s.Sections evaluated for AP were incubated in the dark for 45 min at 30 C in a 4 g l

1

naphthol AS-BI phosphoric acid/methanol in 2 5 M acetate buffer solution with70mgl

1 Fast Garnet GBC (C 14 H 15 N 3 ) base in 0 4MHCl, at pH 5 2. Sectionsevaluated for NBE were incubated in the dark for 30 min at 30 C in a 2 4 g l

1 a -naphthyl butyrate esterase/methanol pH 7 7, 0067M phosphate buffer solution with40gl

1 pararosaniline in 2 M HCl. Sections evaluated for BG were incubated in the darkfor 60min at 30 C in a 2 5 gl1 naphthol AS-BI b-D-glucuronic acid/methanol in 2 5 Macetate buffer solution pH 5 2 with 40 g l

1 pararosaniline in 2 M HCl. The sections wererinsed in tap water for 2 min and then counterstained.

PA D D L E F IS H L E U K O C Y TE S 1103

# 2005 The FisheriesSociety of theBritish Isles, Journal of Fish Biology 2005, 66, 11011115

8/2/2019 j.0022-1112.2005.00668.x

4/15

Sections evaluated for SBB were fixed in a 26 C glutaraldehyde solution (3 % glutar-aldehyde, 25 % acetone in borate buffer pH 7 6) for 1min at 32 C and then rinsed indeionized water. The sections were stained in SBB staining reagent (0 18% w/v SudanBlack B in 69 % ethanol) for 2 5 min and rinsed in 70 % ethanol until dye was not visiblyleaching from the sections. After a distilled water rinse, they were counterstained.

Slides evaluated for HE were fixed in a glutaraldehyde solution for 1 min at 6 C,rinsed in deionized water and stained using the same procedure used for formalin fixed,paraffin embedded sections. Slides were stained in 6 g l

1 haematoxylin in 0 6 g l1

sodium iodide and 52 8 g l1 aluminum sulphate then rinsed with deionized water until

the water ran clear. Ammonium hydroxide (1 5 ml per 500 ml water) was added for 1 minto raise the pH and set the haematoxylin. The slides were rinsed with deionized water andimmersed in 95 % alcohol for 1 min. The slides were stained with eosin-Y for 30 s,immersed in 95 % alcohol followed by immersion in 100 % alcohol and a final immersionin xylene. For WR, air-dried sections were fixed in methanol for 1 min. They were dippedin Wrights stain (0 3% w/v buffered methanol, pH 6 9) for 15 s. The sections were rinsedin deionized water for 30 s and allowed to air dry.

Acid phosphatase, NBE and BG were counterstained with 15 % methylene blue or01% malachite green and mounted in an aqueous based mounting medium. Negativecontrols omitted the corresponding substrate. Sudan Black B and HE stained sectionswere counterstained with 6 g l

1 haematoxylin in 0 6 g l1 sodium iodide and 52 8 g l

1

aluminium sulphate. Sudan Black B, HE and WR were mounted in Permount (SigmaChemical Company). Slides were viewed on an Olympus BHA microscope and photo-graphed using an Olympus C35-A camera.

IMMUNOHISTOCHEMISTRY

The DAKO fast red substrate system (Carpinteria, CA, U.S.A.) was utilized for immu-nohistochemistry. Two monoclonal antibodies (mAb) were obtained from the Universityof California, Davis, CA, U.S.A. These two antibodies, designated II-26 and II-32,recognize the light-chain of white sturgeon immunoglobulin (Ig) and also recognizeAmerican paddlefish Ig (Adkison et al ., 1996). Cryosections and cytospins were fixed inacetone for 10 s, then incubated in 0 05 M Tris-HCl buffer (8 g NaCl, 0 2g KCl and 3gTris base- hydroxy methyl amino methane in 800 ml deionized water with pH adjusted to80 with 1 M HCl and the volume adjusted to 1 l) or tris buffered saline, pH 7 276 for10 min. The buffer was also used as the rinse solution between steps.

Slides were placed in immunostaining chambers in a Shandon 1 Sequenza slide rack. Fourdrops of a 1 : 20 dilution of both mAbs and Tris-HCl buffer were added to each slide for anincubation time of 1 h. After four rinses, four drops of biotinylated porcine anti-rabbit, anti-goat and anti-mouse immunoglobulin in PBS containing carrier protein and 15 mM sodiumazide was added and incubated on the slides for 10 min. After four rinses, four drops of streptavidin conjugated to alkaline phosphatase in PBS, containing carrier protein and15 mM sodium azide was added and incubated on the slides for 10 min. The chromagen

used was fast red. One tablet containing naphthol phosphate and fast red was dissolved in2 ml 0 1 M Tris-HCL buffer, pH 8 2. Levamisole (0 1 M) was added to the chromagen toblock endogenous alkaline phosphatase activity. The chromagen was placed on the slidesand allowed to incubate for 20 min. The sections were rinsed with deionized water, air driedand mounted in an aqueous based mounting medium. Control slides consisted of slides inwhich the primary only, or secondary only, or primary and secondary were omitted. Theserepresented non-specific binding, endogenous enzyme activity and biotin background.

RESULTS

P E RI P HE R AL B L OO D L E UK O CY T ES

Cells stained very similarly to those described in other fish species (Table I).Fraction A was predominately lymphocytes. Monocytes and thrombocytes were



8/2/2019 j.0022-1112.2005.00668.x

5/15

also present. All fraction A cells were positive for AP and demonstrated 14 mmcytoplasmic foci. Forty-five per cent were positive for NBE, with 19 % demonstrat-ing either 12 mm cytoplasmic foci and 26 % demonstrating cytoplasmic blushing.Forty-nine per cent were positive for BG; of these, 10 % demonstrated 23 mmcytoplasmic foci and 90 % demonstrated cytoplasmic blushing. When stained forIg, 90 % were positive and demonstrated cytoplasmic staining. Fraction A cells werenot positive for SBB. Lymphocyte nuclei stained dark bluish-purple and thecytoplasm stained clear blue when stained with WR and HE. Cell diameters rangedfrom 5 to 9 mm. Small lymphocytes had a higher nuclear to cytoplasmic ratio anddemonstrated a small rim of cytoplasm around the nucleus. Larger lymphocytesdemonstrated more of a lighter coloured cytoplasm visible around the nucleus.

Fraction B was predominately large monocytes. Granulocytes were alsopresent. When stained with AP, 100 % of fraction B cells demonstrated cyto-plasmic foci. Ninety-seven per cent demonstrated cytoplasmic blushing whenstained for NBE. Fraction B cells were not positive for BG, Ig or SBB. Theshape of the nucleus varied from round to unevenly lobulated and the cyto-plasm appeared a pale blue-grey when stained with WR and HE.

Fraction C was predominately granulocytes. Granulocytes ranged in sizefrom 8 to 10 mm, were positive for AP, demonstrated 1 mm cytoplasmic foci

and cytoplasmic blushing and were not positive for NBE, BG, Ig or SBB.Nuclei appeared round or bi-lobed, a few were multi-lobed. The cytoplasmstained neutrally and appeared pale with WR stain. These cells frequentlyruptured in smears, and the released granules stained pale blue. A second typeof granulocyte was observed in which the cytoplasmic granules were morenumerous and larger. Nuclei were compressed and represented a smaller portionof the cell. When stained with WR and HE, these granulocytes demonstratedprominent red-orange cytoplasmic granules.

Blood smears stained with AP demonstrated 51 % of positive cells weremonocytes, 43 % were lymphocytes and 6 % were granulocytes. In the blood

smears stained with NBE, 53 % of positive cells were monocytes and 47 % werelymphocytes. In the blood smears stained with BG, 91 % of positive cellsdemonstrated cytoplasmic blushing and 9 % demonstrated foci. In the blood

TABLE I. Staining properties of American paddlefish peripheral blood leukocytes

Stain type

Cell type AP NBE BG Ig SBB WR

Monocyte blue cytoplasmType A lymphocyte blue cytoplasmType B lymphocyte blue cytoplasmGranulocyte pale cytoplasm

light blue granulesEGC orange/red granules

AP, acid phosphatase; NBE, a -naphthyl butyrate esterase; BG, b-glucuronidase; Ig, monoclonalantibody specific for white sturgeon and American paddlefish immunoglobulin; SBB, Sudan BlackB; WR, Wrights stain; EGC, eosinophilic granular cell. , seen; , not seen.



8/2/2019 j.0022-1112.2005.00668.x

6/15

smears evaluated for Ig, 86 % of all cells were positive and exhibited a focus.None of the peripheral blood cells stained positive for SBB. When stained withWR, 47 % of leukocytes present in the whole blood smears were identified asmonocytes, 46 % lymphocytes and 7 % granulocytes.

L E UK O CY T E D I ST R IB U TI O N I N T I SS U ES

The anterior, middle and posterior kidney regions were similar in cellularorganization. Immediately adjacent to the notochord, the tissue was predomi-nately haematopoietic and interrenal tissue. Acid phosphatase, NBE, BG and Igpositive cells were observed mixed throughout the haematopoietic tissue. Allpositive cells demonstrated foci and cytoplasmic blushing. No SBB positive cellswere observed. Tissue monocytes, lymphocytes, erythrocytes and eosinophilicgranular cells (EGCs), at varying stages of development, were present in thehaematopoietic tissue. As the tissue expanded laterally and dorsally, nephronswere mixed with haematopoietic tissue. Aggregations of renal corpuscles andtubules were located adjacent to aggregations of haematopoietic tissue.

The thymus was narrow and discrete, lying on the upper inside edge of the oper-culum. Two regions, a less dense medullaand denser cortex were apparent. The thymuscontained AP, NBE, BG and Ig positive cells. Acid phosphatase and NBE positive cellsdemonstrated foci and cytoplasmic blushing, while BG and Ig positive cells demon-strated cytoplasmic blushing. Whorls of epithelial cells and whorls of eosinophilic,degenerated epithelial cells or early Hassalls corpuscles were seen.

The splenic parenchyma consisted of red and white pulp organized intoerythrocytic and leukocytic compartments, respectively. Acid phosphatase posi-tive cells concentrated around arterioles and were scattered throughout theparenchyma. The NBE positive cells that demonstrated cytoplasmic focioccurred in zones adjacent to arterioles, while larger cells demonstrating cyto-plasmic blushing occurred scattered throughout the parenchyma. The BG posi-tive cell population occurred immediately adjacent to the arterioles,demonstrating peri-arteriole lymphoid sheathing (PALS), and also occurredalong some of the larger veins. Eosinophilic granular cells were also seen.

The MMT was dissected from the dorsal surface of the brain (inside thecranium), external, but adjacent to the base of the cranium and from aroundthe notochord. All MMT stained positive for AP, NBE, BG (Fig.1) and Ig. The

parenchyma of the section from around the cranium consisted of densely packedsmall stem cells. The positive cells followed trabeculae, with AP and NBE stainingthe most cells, followed by BG. The tissue isolated from around the notochordconsisted of densely packed lymphocytes adjacent to adipose tissue.

Small aggregations of CMT were associated with the dorsal apex of the heart.At specific sites, several vessels continued from the epicardium into a mass of thelymphopoietic tissue. This tissue was positive for AP, NBE, BG (Fig. 2) and Ig.

Acid phosphatase, BG and Ig positive cells were seen diffusely scatteredthroughout the submucosa and lamina propria of the gut. Peyers patches werepresent and stained positive for AP (Fig. 3), NBE, BG and Ig. These aggregations

consisted of lymphocytes, and were surrounded by connective tissue. Large cellscontaining densely staining eosinophilic granules occurred throughout themucosa (Fig. 4). The size of these cells ranged from 14 14 mm to 10 35 mm.



8/2/2019 j.0022-1112.2005.00668.x

7/15

(a)

(b)

(c)

F IG . 1. American paddlefish meningeal myeloid tissue demonstrates (a) acid phosphatase positive cells (b)a -naphthyl butyrate esterase positive cells and (c) b -glucuronidase positive cells ( *). Bar 200 mm.



8/2/2019 j.0022-1112.2005.00668.x

8/15

CMT

M

CM

**

F IG . 2. American paddlefish pericardial myeloid tissue (CMT) demonstrates b -glucuronidase positivecells (*), melanin aggregates (M) and negative cardiac muscle (CM). Bar 200 mm.

GALT

int villi

F IG . 3. Peyers patches (GALT) tissue in the American paddlefish spiral valve demonstrates acid phos-phatase positive cells ( *). Intestinal villi (int villi) are seen on the luminal surface. Bar 200 mm.



8/2/2019 j.0022-1112.2005.00668.x

9/15

DISCUSSION

P E RI P HE R A L B L OO D L E UK O CY T ES

Acid phosphatase staining patterns observed in American paddlefish lympho-cytes were similar to those described in mammals and fishes. In mammals, AP ispresent in all stages of lymphocyte development, and these cells demonstrate

cytoplasmic blushing or foci (Catovsky & Enno, 1977; Bevan et al ., 1980).Younger lymphocytes demonstrate foci while older cells demonstrate a diffusestaining pattern. In channel catfish, AP positive T lymphocytes demonstrate acytoplasmic focus, while B lymphocytes demonstrate a focus or cytoplasmicblushing (Petrie-Hanson & Ainsworth, 2000).

In the American paddlefish, type A lymphocytes were positive for NBE anddemonstrated staining patterns very similar to mammalian T cells. Human Bcells and immature T cells are NBE negative, while mature T cells demonstrateup to four cytoplasmic foci (Bevan et al ., 1980). In the channel catfish, T cellsdemonstrate NBE positive cytoplasmic foci (Petrie-Hanson & Ainsworth, 2000).

Some American paddlefish lymphocytes were positive for BG. b-glucuronidaseoccurs in mammalian mature thymocytes, circulating T lymphocytes and a sub-population of immature B lymphocytes (Catovsky & Enno, 1977). Channel

25 m

F IG . 4. Large cells containing eosinophilic granules ( ) in the gut lamina propria and epithelium of theAmerican paddlefish, haematoxylin and eosin.



8/2/2019 j.0022-1112.2005.00668.x

10/15

catfish B and T cells demonstrate BG positive foci and cytoplasmic blushing; Igpositive cells usually coincide with the cells demonstrating the focal stainingpattern, not the blushing pattern (Petrie-Hanson & Ainsworth, 2000). Americanpaddlefish lymphocytes that demonstrated intense pink foci were assumed to betype A lymphocytes because the numbers of BG positive cells with foci wereequivalent to the numbers of NBE positive cells with foci in the peripheralblood counts. Cells exhibiting pale pink BG cytoplasmic blushing were desig-nated type B lymphocytes. The numbers of BG positive blushing cells wereequivalent to the numbers of Ig cells with foci in the peripheral blood counts.

In American paddlefish tissue sections, Ig positive cells demonstrated eithercytoplasmic blushing or positive foci. Most Ig positive cells were lymphocytes.Cells that exhibited blushing with Ig could be either mature type B cells orplasma cells. Ig positive cells that demonstrated foci could be immature type Blymphocytes, because this population occurred at the same tissue locations asthe BG blushing cells in sequential serial sections. Blushing could also resultfrom the presence of both cytoplasmic and surface Ig (mature type B lympho-cytes), while Ig foci could result from the presence of cytoplasmic Ig only(immature type B lymphocytes). A high percentage of Ig positive peripheralleukocytes could be cells expressing surface immune complexes. A putative Fcreceptor for IgM has been identified on channel catfish noncytotoxic cells(NCC) (Shen et al ., 2002). Perhaps similar receptors are present on Americanpaddlefish leukocytes, and are being visualized by immunohistochemistry in thepresent studies. American paddlefish lymphocyte morphology is similar to thatdescribed in other fish species (Zapata & Cooper, 1990; Zapata et al ., 1996 a).Lymphocyte ultrastructure is similar to comparable mammalian cells (Clawsonet al ., 1966).

Acid phosphatase was present in American paddlefish monocytes and macro-phages that demonstrated red-violet cytoplasmic blushing. In mammals, AP ispresent in all stages of development of monocytes and macrophages, and red-violet cytoplasmic blushing is the characteristic staining pattern (Catovsky &Enno, 1977; Bevan et al ., 1980). A similar staining pattern was observed inchannel catfish monocytes and macrophages (Petrie-Hanson & Ainsworth,2000). American paddlefish monocytes and macrophages demonstrated palebrown cytoplasmic blushing when stained with NBE. This pattern is verysimilar to that observed in mammalian and channel catfish macrophages and

monocytes (Catovsky & Enno, 1977; Bevan et al ., 1980; Petrie-Hanson &Ainsworth, 2000). Some channel catfish monocytes, however, also demonstratedfoci. American paddlefish monocyte and macrophage morphology is similar tothat described in other fish species (Zapata & Cooper, 1990; Zapata et al .,1996 a). Clawson et al . (1966) described the ultrastructure of these cells assimilar to comparable mammalian cells.

Many fishes have only one type of granulocyte (Ainsworth, 1992). Thepredominate granulocyte in American paddlefish is the EGC. By light micro-scopy, these cells appeared very similar to EGCs described in other fish species(Reite, 1998). Clawson et al . (1966) stated that granules in American paddlefish

eosinophils were much larger than those present in mammalian eosinophils, butother aspects of the two types of eosinophils were comparable. None of thegranulocytes observed stained positive for SBB, so they were not considered



8/2/2019 j.0022-1112.2005.00668.x

11/15

neutrophils. Chondrosteans have been reported to have eosinophils and neu-trophils (Hine, 1992). More specifically, heterophilic, acidophilic and basophilicgranulocytes are present in the shortnose sturgeon Acipenser brevirostrumLesueur (Hine & Wain, 1988). Clawson et al . (1966) described American pad-dlefish neutrophils that were very similar to mammalian neutrophils. Differen-tial counts, however, were not performed in that study. It is possible that thenumber of neutrophils present was very low. Clawson et al . (1966) could haveobserved what have been referred to here as the granulocyte. Additionally, thatstudy did not include staining with SBB. It is possible that the cell typedesignated a neutrophil by comparative anatomical features would have beenSBB negative. That report also stated that the granules observed in the Americanpaddlefish neutrophils were much larger than those in mammalian neutrophilswere. They may have been describing the cells referred to here as EGCs.

The leukocyte differential in American paddlefish had a much greater per-centage of monocytes than the 0 to 4 % reported for other fish species (Bullis,1993), and a lower granulocyte percentage. In bowfin Amia calva L. and north-ern longnose gar Lepisosteus osseus (L.) eosinophils were seen in high numbersin tissues, but were present in small numbers in blood smears (Scharrer, 1944).Additional differential leukocyte counts should be performed during differenttimes of the year to confirm these paddlefish findings.

L E UK O CY T E D I S TR I BU T IO N I N LY MP H O ID T I SS U ES

The juvenile American paddlefish kidney was similar to previously describedteleost kidney tissue (Ellis, 1977; Georgi & Beedle, 1978; Fange 1986; Petrie-Hanson & Ainsworth, 2000). Teleost RHT is believed to have an equivalentfunction to mammalian bone marrow; the haematopoietic tissue generates stemcells and the renal elements filter blood and regulate water, electrolyte andprotein balance (Ellis, 1977; Zapata & Cooper, 1990; Castillo et al ., 1993;Petrie-Hanson & Ainsworth, 2000). The renal interstitial tissue was previouslydescribed as granulopoietic in chondrosteans (Fange, 1982). The presence of immature erythrocytes and lymphocytes substantiates that the American pad-dlefish RHT functions similarly. The presence of anatomically immature gran-ulocytes and lymphocytes in the MMT and CMT, however, suggests thesetissues are also granulopoietic and leukopoietic in American paddlefish.

The cortical and medullary organization of the juvenile American paddlefishthymus appeared to be very similar to that described in other fish species. Incontrast, the thick, connective tissue capsule and trabeculae and lobulatedparenchyma were similar to the mammalian thymus. The large multi-layereddegenerated epithelial cell cysts were interpreted as Hassalls corpuscles. Hassallsbodies were previously reported in the American paddlefish thymus (Goodet al ., 1966). Epithelial cysts are a standard thymic feature of ectotherms(Zapata & Cooper, 1990). Presumptive Hassalls bodies were described in theGobiesocidae thymus (Gorgollon, 1983). In the elasmobranch thymus, fibrousepithelial aggregates have been described which can be interpreted as rudimen-

tary Hassalls corpuscles (Zapata et al ., 1996 b). Hassalls corpuscles are believedto play a role in thymic hormone (Cruse & Lewis, 1995) and humoral factorproduction that may play roles in T cell differentiation (Burkitt et al ., 1993).



8/2/2019 j.0022-1112.2005.00668.x

12/15

Monocytes, macrophages, lymphocytes, EGCs and myoid cells were observed inthe juvenile American paddlefish thymus. The American paddlefish thymus maybe used as a model to define the roles of myoid cells and reticular epithelial cellsof Hassalls corpuscles in positive and negative selection of developing T cells infishes. The sturgeon thymus is similar, but Hassalls corpuscles were notreported (Fange, 1986). The thymus has been characterized in many fish species(Zapata & Cooper, 1990; Castillo et al ., 1991; Chilmonczyk, 1992; Black, 1994;Petrie-Hanson & Ainsworth, 2000) and is histologically and functionally com-parable to the mammalian thymus.

Splenic organization demonstrated erythrocytic and leukocytic compartmen-talization, or red and white pulp areas, and vascularization patterns similar tothose described in other fish species (Petrie-Hanson & Ainsworth, 2000). Thelocation of types A and B lymphocytes suggest that American paddlefish splenicperiarteriole lymphocytes process antigens and macrophages adjacent to thelymphocytes function to trap and present antigen to the lymphocytes. Similarsites have been suggested to function in the same manner for other fish species(Manning & Mughal, 1985; Ellis, 1988; Zapata & Cooper, 1990; Secombes &Fletcher, 1992; Pulsford et al ., 1994; Petrie-Hanson & Ainsworth, 2000), and arecomparable to mammalian splenic architecture and function.

Although MMT was present in three locations, the cellular composition wasthe same: monocytes, macrophages and densely packed lymphocytes. A verysimilar distribution of MMT is described in ganoids (Scharrer, 1944) andsturgeons (Fange, 1986). Scharrer (1944) concluded that this tissue is bonemarrow, and the first vertebrate appearance of bone marrow occurred in theganoids (bowfins, gars, sturgeons and paddlefish) (Scharrer, 1944).

The myeloid tissue associated with the heart was well developed, though notextensive, and was predominately type B lymphocytes. This tissue was noted tohave a lymph node like appearance in sturgeons (Fange, 1986), but that type of tissue organization was not observed in the 18 mph American paddlefish. Fange(1986) examined adult specimens with extensive heart associated myeloid tissue.Examination of adult American paddlefish may allow more accurate compar-isons of this tissue.

The Peyers patches were very similar to mammalian Peyers patches. Promi-nent Peyers patches were seen in the spiral valve of older, wild specimens(Weisel, 1971), and dense lymphoid accumulations were observed near the

ileocecal valve (Good et al ., 1966). Specific type A or B lymphocyte zonescould not be discerned with special stains. It is probable that in older orantigenically challenged fish, these areas would develop. The plasma cell popu-lation of the CMT and spleen expanded rapidly after antigenic challenge (Goodet al ., 1966), and it is possible that this tissue will respond as well.

The large cells that contain eosinophilic granules appear similar to Panethcells found in the human small intestine, but those in the American paddlefishare not concentrated in crypts. This may represent an early phylogeneticappearance of these cells. Paneth cells are characterized as exocrine proteinsecreting cells (Burkitt et al ., 1993); one of the secretions is lysozyme. Lysozyme

is a bactericidal enzyme that is an integral component of non-specific immunityin vertebrates. It is usually associated with leukocytes. The large cells observedare most likely a type of EGC. These cells can be found in high numbers in gill



8/2/2019 j.0022-1112.2005.00668.x

13/15

and gut tissues, and play a role in non-specific cellular immunity (Secombes,1996). Similar cells were described by Weisel (1971) and referred to as rodletcells. This referral may have been mistaken. Rodlet cells do not occur in all fishspecies. Briefly, they measure 812 mm, and contain long, narrow eosinophilicrods, and are usually associated with epithelial and endothelial cells. TheAmerican paddlefish intestinal cells referred to are larger, with round or squar-ish granules, and do not resemble rodlet cells described by other authors(Ferguson, 1992; Koponen & Myers, 2000; Kramer & Potter, 2003; Dezfuliet al ., 2003).

This study focused on leukocytes and their distribution in lymphoid tissues.American paddlefish leukocyte AP, NBE and BG staining characteristics arevery similar to those observed in mammals. The lymphoid tissues of these fishhave leukocyte distributions very similar to analogous mammalian lymphoidtissues, and include some structures not present in teleosts. This providesmorphological evidence that Chondrostei may better represent the ancestralform of fishes that evolved into modern day tetrapods. The location of specificleukocyte generation and maturation has yet to be determined. A more detailedanalysis of American paddlefish leukocytes is needed to conclusively identify Tand B lymphocytes. Specific gene expression and the production of antibodiesfor cell surface markers are needed to accomplish this. Studies of the finestructure of primary and secondary lymphoid tissues are currently being per-formed.

This research was funded by the College of Veterinary Medicine at Mississippi StateUniversity. G. Glenney and R. Mackey provided technical assistance. Paddlefish used in

this study were reared from ova collected with S. Mims at Kentucky State University.Thanks to M. Adkison and R. Hedrick, University of California, for providing the twomonoclonal antibodies.

References

Acton, R. T., Weinheimer, P. F., Dupree, H. K., Russell, T. R., Wolcott, M., Evans, E.E., Schrohenloher, R. E. & Bennett, J. C. (1971). Isolation and characterization of the immune macroglobulin from the paddlefish, Polyodon spathula . Journal of Biological Chemistry 246, 67906769.

Adkison, M. A., Basurco, B. & Hedrick, R. P. (1996). Humoral immunoglobulins of thewhite sturgeon, Acipenser transmontanus : partial characterization of and recogni-tion with monoclonal antibodies. Developmental and Comparative Immunology 20,285298.

Ainsworth, A. J. (1992). Fish granulocytes: morphology, distribution, and function.Annual Review of Fish Diseases 2, 123148.

Bevan, A., Burns, G. F., Gray, L. & Cawley, L. C. (1980). Cytochemistry of human T-cellsubpopulations. Scandinavian Journal of Immunology 11, 223233.

Black, S. (1994). Morphology of the lymphoid organs of channel catfish: ontogeny of thethymus, anterior kidney, and spleen; histology and fine structure of the thymus;and thymic involution. PhD dissertation, College of Veterinary Medicine. Stark-ville, Mississippi State University.

Bullis, R. A. (1993). Clinical pathology of temperate freshwater and estuarine fishes. InFish Medicine (Stoskopf, M. K., ed.), pp. 232239. Philadelphia, PA: W. B.Saunders.



8/2/2019 j.0022-1112.2005.00668.x

14/15

Burkitt, H. G., Young, B. & Heath, J. W. (1993). Wheaters Functional Histology .Edinburgh: Churchill Livingstone.

Castillo, A., Lopez-Fierro, P., Zapata, A., Villena, A. & Razquin, B. (1991). Post-hatching development of the thymic epithelial cells in the rainbow trout Salmo gairdneri : an ultrastructural study. The American Journal of Anatomy , 190, 299307.

Castillo, A., Sanchez, C., Dominguez, J., Kaattari, S. L. & Villena, A. J. (1993). Ontogenyof IgM and IgM-bearing cells in rainbow trout. Developmental and ComparativeImmunology 17, 419424.

Catovsky, D. & Enno, A. (1977). Morphological and cytochemical identification of lymphoid cells. Lymphology , 10, 7784.

Chilmonczyk, S. (1992). The thymus in fish: development and possible function in theimmune response. Annual Review of Fish Diseases 2, 181200.

Clawson, C. C., Finstad, J. & Good, R. A. (1966). Evolution of the immune response V.Electron microscopy of plasma cells and lymphoid tissue of the paddlefish. Labora-tory Investigations 15, 18301847.

Cruse, J. M. & Lewis, R. E. (1995). Illustrated Dictionary of Immunology . Boca Raton,FL: CRC Press.

Dezfuli, B. S., Giari, L., Simoni, E., Palazzi, D. & Manera, M. (2003). Alteration of rodletcells in chub caused by the herbicide Stam 1 M-4 (Propanil). Journal of FishBiology 63, 232239. doi: 10.1046/j.1095-8649.2003.00151.x

Ellis, A. E. (1977). Ontogeny of the immune response in Salmo salar . Histogenesis of thelymphoid organs and appearance of membrane immunoglobulin and mixed leuco-cyte reactivity. In Developmental Immunology (Solomon, J. B. & Horton, J. D.,eds), pp. 225231. Amsterdam: Elsevier/North Holland Biomedical Press.

Ellis, A. E. (1988). Ontogeny of the immune system in teleost fish. In Fish Vaccination(Ellis, A. E., ed.), pp 2031 London: Academic Press.

Fange, R. (1982). A comparative study of lymphomyeloid tissue in fish. Developmental and Comparative Immunology , Supplement 2 , 2333.

Fange, R. (1986). Lymphoid organs in sturgeons (Acipenseridae). Veterinary Immunologyand Immunopathology 12, 153161.

Ferguson, H. W. (1992). Systemic Pathology of Fish . Ames, IA: Iowa State University Press.Georgi, T. A. & Beedle, D. (1978). The histology of the excretory kidney of the paddlefish,

Polyodon spathula . Journal of Fish Biology 13, 587590.Good, R. A., Finstad, J., Pollara, B. & Gabrielsen, A. E. (1966). Morphological studies

on the lymphoid tissues among the lower vertebrates. In Phylogeny of Immunity(Smith, R. T., Miescher, P. A, & Good, R. A., eds), pp. 149167. Gainesville, FL:University of Florida Press.

Gorgollon, P. (1983). Fine structure of the thymus in the adult cling fish Sicyasessanguineus (Pisces Gobiesocidae). Journal of Morphology 177, 2540.

Hine, P. M. (1992). The granulocytes of fish. Fish and Shellfish Immunology 2, 7998.Hine, P. M. & Wain, J. M. (1988). Ultrastructural and cytochemical observations on the

granulocytes of the sturgeon, Acipenser brevirostrum (Chondrostei). Journal of Fish

Biology 33, 235245.Kaattari, S. L. (1992). Fish B lymphocytes: defining their form and function. Annual Review of Fish Diseases 2, 161180.

Koponen, K. & Myers, M. S. (2000). Seasonal changes in intra- and interorgan occur-rence of rodlet cells in freshwater bream. Journal of Fish Biology 56, 250263.doi:10.1006/jfbi.1999.1169

Kramer, C . R. & Potter, H. (2003). Rodlet cells in the posterior intestine of embryos andneonates of two poeciliid species. Journal of Fish Biology 62, 12111216. doi:10.1046/j.1095-8649.2003.00087.x

Machin, G. A., Halper, J. P. & Knowles, D. M. (1980). Cytochemically demonstratableB-glucuronidase activity in normal and neoplastic human lymphoid cells. Blood 56,11111119.

Manning, M. J. & Mughal, M. S. (1985). Factors affecting the immune responses of immature fish. In Fish and Shellfish Pathology (Ellis, A. E., ed.), pp. 2740.London: Academic Press.



8/2/2019 j.0022-1112.2005.00668.x

15/15

Petrie-Hanson, L. & Ainsworth, A. J. (2000). Differential cytochemical staining charac-teristics of channel catfish leukocytes identify cell populations in lymphoid organs.Veterinary Immunology and Immunopathology 73, 129144.

Pollara, B., Suran, A., Finstad, J. & Good, R. A. (1968). N-terminal amino acidsequences of immunoglobulin chains in Polyodon spathula . Proceedings of the

National Academy of Science 59, 13071312.Pulsford, A., Tomlinson, M. G., Lemaire-Gony, S. & Glynn, P. J. (1994). Developmentand immunocompetence of juvenile flounder Platichthys flesus , L. Fish and Shell- fish Immunology 4, 6378.

Razquin, B. E., Castillo, A., Lopez-Fierro, P., Alvarez, F., Zapata, A. & Villena, A. J.(1990). Ontogeny of IgM-producing cells in the lymphoid organs of rainbow trout,Salmo gairdneri Richardson: an immuno- and enzyme-histochemical study. Journal of Fish Biology 36, 159173.

Reite, O. B. (1998). Mast cells/eosinophilic granule cells of teleostean fish: a reviewfocusing on staining properties and functional responses. Fish and Shellfish Immu-nology 8, 489513.

Sanchez, C., Alvarez, A., Castillo, A., Zapata, A., Villena, A. & Dominguez, J. (1995).Two different subpopulations of Ig-bearing cells in lymphoid organs of rainbowtrout. Developmental and Comparative Immunology 19, 7986.

Scharrer, E. (1944). The histology of the meningeal myeloid tissue in the ganoids Amiaand Lepisosteus . Anatomical Record 88, 291310.

Secombes, C. J. (1996). The nonspecific immune system: cellular defenses. In The FishImmune System, Organism, Pathogen, Environment (Iwama, G. & Nakanishi, T.,eds), pp. 63104. New York: Academic Press.

Secombes, C. J. & Fletcher, T. C. (1992). The role of phagocytes in the protectivemechanisms of fish. Annual Review of Fish Diseases 2, 5371.

Secombes, C. J., van Groningen, J. J. M., van Muiswinkel, W. B. & Egberts, E. (1983).Ontogeny of the immune system in carp ( Cyprinus carpio L .). The appearance of antigenic determinants on lymphoid cells detected by mouse anti-carp thymocytemonoclonal antibodies. Developmental and Comparative Immunology 7, 455464.

Shen, L., Stuge, T. B., Zhou, H., Khayat, M., Barker, K. S., Quiniou, S. M. A., Wilson,M., Bengten, E., Chinchar, V. G., Clem, L. W. & Miller, N. W. (2002). Channelcatfish cytotoxic cells: a mini-review. Developmental and Comparative Immunology26, 141149.

Weisel, G. (1971). Anatomy and histology of the digestive system of the paddlefish(Polyodon spathula ). Journal of Morphology 140, 243256.

Zapata, A. G. & Cooper, E. L. (1990). The Immune System: Comparative Histophysiology .Chichester: John Wiley and Sons.

Zapata, A. G., Chiba, A. & Varas, A. (1996 a). Cells and tissues of the immune system of fishes. In The Fish Immune System, Organism, Pathogen, Environment (Iwama, G.& Nakanishi, T., eds), pp. 153. New York: Academic Press.

Zapata, A. G., Torroba, M., Sacedon, R., Varas, A. & Vicente, A. (1996 b). Structure

of the lymphoid organs of elasmobranchs. Journal of Experimental Zoology 275,125143.



Documents

j.0022-1112.2005.00668.x