Journal of Immunological Methods, 131 (1990) 15-24 Elsevier
15
JIM 05611
Sugar competition assays reveal high affinity receptors for Erythrina cristigalli lectin on feline monocytes C.E. Whitehurst 1,2, N.K. D a y 1 and N. Gengozian 1 1 Department of Pediatrics, All Children's Hospital, University of South Florida, St. Petersburg FL 33701, U.S.A., and 2 Department of Biology, University of South Florida, St. Petersburg FL 33701, U.S.A. (Received 19 December 1989, accepted 16 March 1990)
An examination of fluorescein-labelled Erythrina cristigalli lectin (FITC-ECL) staining on feline mononuclear cells (MNC) using fluorescent microscopy and a novel sugar titration competition assay revealed that monocytes (MO) were stained brighter by FITC-ECL than were lymphocytes (LYM). When MNC were stained with FITC-ECL in the presence of 400 mM or greater o-galactose, analysis by flow cytometry revealed continued MO staining while LYM were negative. MO expressed a larger quantity of carbohydrate receptors (CHO-R) for ECL than did LYM. The CHO-R expressed on MO were mostly protease-insensitive and uncapped by sialic acid residues. All of the CHO-R on LYM were protease-sensitive and many were capped by sialic acid residues. A combined labelling of MNC for non-specific esterase staining, latex bead ingestion and FITC-ECL staining in the presence of 400 mM o-galactose confirmed that FITC-ECL specifically stains MO in the presence of high sugar competitor concentrations. Key words: Mononuclear cell, feline; Monocyte; Carbohydrate receptor; Erythrina cristigalli lectin; Flow cytometry
Introduction
Research involving the feline immune system has been hindered by a lack of molecular markers to identify and allow the fractionation of mono-
Correspondence to: N. Gengozian, Department of Pediatrics, All Children's Hospital, University of South Florida, St. Petersburg, FL 33701, U.S.A. Abbreviations: MO, monocytes; LYM, lymphocytes; BTMP, bitmap; ECL, Erythrina cristigalli lectin; FITC, fluorescein isothiocyanate; MNC, mononuclear cells; CHO-R, carbohydrate receptors; BSA, bovine serum albumin; NES, non-specific esterase stain; VCN, Vibrio cholerae neuraminidase; lacNac, N-acetyllactosamine; p-npg, p-nitrophenyl-fl-Dgalactoside; galNac, N-acetyl-D-galactosamine;iHBSS, incomplete Hanks' balanced salt solution; mPBS, modified phosphate-buffered saline.
nuclear cell subclasses. For a decade the characterization of feline lymphocytes was restricted to surface IgG and complement C3 receptorbeating B lymphocytes and guinea pig erythrocyte-rosetting T lymphocytes (Mackey et al., 1975; Taylor et al., 1975; Cockerell et al., 1976; Taylor and Siddiqui, 1977; Rojko et al., 1982). Only recently have feline T lymphocytes been characterized into differing functional subclasses, with guinea pig erythrocyte-rosetting T cells and gerbil erythrocyte-rosetting T cells exhibiting helper and suppressor activities, respectively, upon polyclonal B cell responses (Gengozian et al., 1988). To date, no molecular markers exist to identify the feline monocyte. Lectins are useful tools for characterizing and fractionating leukocyte subclasses (Sharon, 1983; Lis and Sharon, 1986a). ECL, a 57 kDa lectin derived from the seeds of Erythrina cristigalli,
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
16 binds strongly to N-acetyUactosamine and weakly to other galactosyl-related haptens (Iglesias et al., 1982; Kalades et al., 1982). In this report we describe the distribution of ECL-binding carbohydrate receptors (CHO-R) among feline mononuclear cells (MNC) using a novel sugar titration competition assay and flow cytometry. We show that due to the differing properties of these receptors among lymphocytes (LYM) and monocytes (MO), ECL can be used to specifically identify feline MO.
Materials and methods
Isolation of mononuclear cells Peripheral blood mononuclear cells (MNC) were obtained by centrifuging defibrinated whole blood from healthy adult cat donors on a FicollHypaque gradient. MNC were washed with incomplete Hanks' balanced salt solution (iHBSS), then resuspended in iHBSS containing 0.5% bovine serum albumin (iHBSS-BSA). As revealed by Wright's stain analyses, MNC always contained less than 5% polymorphonuclear cells.
Lectins and sugars Lyophilized fluorescein isothiocyanate-labelled
(Erythrina cristigalli lectin (FITC-ECL) and Erythrina corralodendrum lectin (ECorL) were purchased from Sigma Chemical Company (St. Louis, MO). All lectins were suspended to 1 m g / m l in 0.15 M NaC1 containing 10 m M Hepes, 0.08% NAN3, and p H 7.5. D-galactose was purchased from Gibco (Grand Island, NY); 13lactose, N-acetyl-D-galactosamine (galNAc), pnitrophenyl-/3-D-galactoside (p-npg), and Nacetyl-lactosamine (lacNac) were purchased from Sigma. All sugars were dissolved in modified PBS containing 20 mM EDTA, 0.02% N a N 3, p H 7.2 (mPBS).
Latex bead labelling of monocytes In a 12 × 75 polystyrene culture tube 6 × 10 6 M N C were resuspended to 3 x 106/ml in complete Hanks' balanced salt solution (HBSS) containing Ca z+ and Mg 2+ and 10% heat-inactivated fetal calf serum. To this, 20 btl of 0.81 /~m latex
beads (Bacto, Difco Laboratories, Detroit, MI) were added and the tube rotated in a 3 7 ° C incubator for 90 rain. The cells were washed in mPBS and then resuspended in iHBSS-BSA for analysis by fluorescent microscopy. A cell was scored as a MO if three or more beads were localized internally within that cell.
Sugar titration competition assays Microtiter plate wells were pre-coated with PBS 5% BSA and washed with PBS. Aliquots of 100/xl of M N C (5 x 106/ml) were added to each well. After one wash with mPBS, the cells were washed once with respective serial dilutions of the sugar solutions made in mPBS and then resuspended in 50 /~1 of the same respective sugar dilution or mPBS as a control. To each well 0.5/~1 of FITCECL (1 m g / m l ) was added and cells were incubated on ice for 10 rain and then washed twice with 150 txl of mPBS. The percentage of fluorescing cells and peak channel values representative of fluorescence intensity were determined using a Coulter Epics-C flow cytometer. Quantification parameters for viable LYM and MO were based on cell granularity and size using the 90 ° light scatter versus forward light scatter histogram to gate the respective M N C populations. Gains were adjusted against controls to eliminate the quantification of background fluorescence. A minimum of 1000 cells were counted from each value obtained.
FITC-ECL staining of cat MNC by a cytocentrifugation procedure Normal or latex bead-labelled M N C resuspended in iHBSS-BSA were cytocentrifuged onto a glass slide. While still wet and covered with a residual film of media, 10 #1 of FITC-ECL staining reagent was added. Slides were incubated for 5 min in a humidified petri plate at room temperature and then washed with a gentle rinse of cold PBS using a pasteur pipette. While still wet, coverslips were immediately added and the slides analyzed by fluorescent microscopy using a Zeiss Universal microscope equipped with a Xenon lamp and F I T C filter. The percentage of viable membranal fluorescing and beads-positive M N C were quantitated. At least 500 cells were counted for each experiment.
17
FITC-ECL staining reagent The FITC-ECL staining reagent used to specifically stain monocytes by the cytocentrifuge procedure was prepared by diluting in a microtiter plate 1/zl of FITC-ECL (1 mg/ml) in 20/~1 of a sugar competitor. Optimal sugar competitor concentrations were determined for each new lot of FITC-ECL by making serial dilutions of the sugar in mPBS and observing the quality of MO-specific FITC-ECL staining. These same optimal sugar concentrations could also be used to specifically stain MO when MNC were stained in larger volumes in microtiter plates. MNC (0.5 x 106) in 50/zl of the respective sugar solution were stained with 2/~1 of FITC-ECL (1 mg/ml). Following an incubation for 10 rain on ice, the cells were washed with the same sugar solution and resuspended in mPBS containing 0.5% BSA. Protease treatment of feline MNC MNC (20 x 106) were washed in iHBSS, resuspended in 3 ml of M199 containing 1 mg/ml of Pronase E from Streptomyces griseus (Sigma Chemical) and rotated in a 37 °C incubator for 2 h. Afterwards, the cells were washed with iHBSS and then resuspended in iHBSS-BSA. Cell viability, determined by trypan blue exclusion, was always greater than 95%. Control and proteasetreated MNC were stained with FITC-ECL or rabbit anti-cat IgG (control) as described above. The percentage of fluorescing cells and peak channel values were determined by EPICS-C flow cytometry. Non-specific esterase staining of feline MNC Normal or latex bead-labelled MNC were stained for non-specific esterase activity (NES) using the procedure of Yam et al. (1971). To enhance the staining quality, the reaction buffer included 10 mM of Ca 2+ and Mg 2÷, and after pH adjustment (6.2-6.4) and filtering, 1.5 ml of propylene glycol was added for every 50 ml of reaction buffer. The percentage of beads-positive and NES-positive cells were determined by oil immersion light microscopy. At least 200 cells were counted for each experiment. F1TC-ECL additive immunofluorescence of feline MNC Gengozian et al. (1988) has shown that feline T
lymphocytes which express suppressor activity upon the polyclonal B cell response are capable of forming rosettes with gerbil erythrocytes. A monoclonal antibody, CT87, was also reported to bind specifically to the gerbil erythrocyte receptor on these T suppressor cells (Gengozian et al., 1988). Because of this property, CT87 was used as a marker to distinguish T suppressor cells from other cells by using indirect immunofluorescence. In a microtiter plate 100 /~1 of MNC (10 x 106/ ml)/well were treated with either 50/zl of CT87 or iHBSS-BSA. After incubation on ice and washing with mPBS, cells were stained with 50 /zl of a 1/10 dilution of goat F(ab')2 anti-mouse IgG + IgM (Tago, Burlingame, CA) a n d / o r 50 #1 of a 1/20 dilution of FITC-labelled rabbit anti-cat IgG antiserum (Cooper Biomedical, Malvern, PA) or iHBSS-BSA. After washing with mPBS the cells were stained with 50 /xl of FITC-ECL diluted to 20 # g / m l in 4 mM p-npg or iHBSS-BSA. Cells were washed twice in cold 400 mM D-galactose, and given a final wash in mPBS. The cells were resuspended in mPBS with 0.5% BSA for analysis of the fluorescing MNC by EPICS-C flow cytometry.
Neuraminidase treatment of feline MNC MNC resuspended in HBSS were treated with 500 U / m l of Vibrio cholerae neuraminidase (Gibco) for 60 min in a 37°C waterbath with occasional mixing. Afterwards, the cells were washed with iHBSS and resuspended in iHBSSBSA. Results
To investigate the general staining properties of FITC-ECL on feline MNC, cells from several cats were stained with FITC-ECL by the cytocentrifugation procedure. Preliminary cytocentrifugation analyses revealed that 100% of the MNC were stained at FITC-ECL concentrations as low as 10 /~g/ml (data not shown). A subclass of larger cells comprising 5-15% of MNC, was consistently stained brighter than all other cells. When MNC were stained in the presence of up to 800 mM D-galactose, most cells were completely inhibited from staining; the large cells previously noted, however, continued to stain in a specific and re-
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Fig. 1. Flow cytometric analysis of feline MNC stained with FITC-ECL. A: the MNC-LYM- and MO-BTMP positions; B: the percentage of fluorescing cells and peak channel values (in brackets) for the MNC-gated BTMP; C: the LYM-BTMP; and D: the MO-BTMP.
producible manner. In D-galactose concentrations of less than 400 mM all of the feline MNC resumed staining. Thus, to specifically stain the large MNC, a D-galactose concentration of greater than 400 mM was required in the staining reagent. Feline MNC were next stained with a subagglutinating concentration of FITC-ECL and analyzed by flow cytometry. Cells having a MOlike morphology as gauged by the 90 o light scatter (granularity) and forward light scatter (cell size) parameters were stained significantly brighter than cells having a typical LYM morphology (Fig. 1), The flow cytometer was then used to measure the
relative percentages and fluorescent intensities of MNC stained by sugar titration competition assays. In the presence of 400 mM to 4 mM Dgalactose a higher percentage of cells within the MO-gated bitmaps (BTMP) were stained with FITC-ECL than in the LYM-BTMP (Fig. 2A). The fluorescent intensities of cells within both BTMP were approximately the same (Fig. 2B). In the presence of low D-galactose concentrations nearly 100% of the cells from both the LYM- and MO-BTMP were stained by FITC-ECL (Fig. 2A); however, the fluorescent intensities of the cells monitored in the LYM-BTMP remained con-
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Fig. 2. Differential FITC-ECL and FITC-ECorL staining of feline MNC as determined by sugar titration competition assays. A: the percentage of cells stained at decreasing D-galactose concentrations; B: the relative intensities of cells stained at decreasing D-galactose concentrations.
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Fig. 3. Inhibitive properties of differing sugars on the FITC-ECL staining of feline MNC as determine by sugar titration competition assays. The percentages (A) and fluorescent intensities (B) of cells stained at decreasing sugar concentrations.
sistently lower than that of cells monitored in the MO-BTMP (Fig. 2B). These results suggest that feline MO bind FITC-ECL with a higher affinity than do LYM. To discern if the differential staining of M N C was a property unique to ECL, the staining property of another Erythrina-derived lectin, Erythrina corralodendrum lectin (ECorL), was analyzed by sugar titration competition assays run parallel with FITC-ECL. Fig. 2 shows that FITC-ECorL did not stain MNC in the same manner as ECL. The MO- and LYM-BTMP showed identical titration profiles when M N C were stained with FITCECorL. These results suggest that the preferential staining of MO at higher D-galactose concentration is a property unique to ECL, and not other Erythrina-derived lectin species such as ECorL. Other saccharides are known to compete for the CHO-binding sites of ECL (Iglesias et al., 1982). t)-galactose is a relatively weak competitor for ECL binding. To determine if other sugar competitors would cause the same FITC-ECL staining phenomenon on feline MNC as that caused by D-galactose, four other sugar competitors (lacNAc, p-npg, galNAc and lactose) were analyzed by the cytocentrifugation staining procedure. Like D-galactose, the four sugars inhibited the staining of all M N C except the large MO-like
cells. The optimal concentrations of the different sugars needed to induce this differential staining were 100 /zM lacNAc, 4 mM p-npg, 20 mM galNAc, 400 m M lactose and 400 m M galactose. When the four sugars were analyzed in parallel with D-galactose by sugar titration competition assays and flow cytometry, they caused similar titration profiles. As shown in Fig. 3, the MOBTMPS have higher percentages of FITC-ECL stained cells which show higher average fluorescent intensities than those cells monitored within the LYM-BTMPS (Fig. 3). The relative inhibitory capacities of the five sugars on the FITC-ECL staining of cells quantitated in the L Y M - B T M P were lacNAc > p-npg > lactose > galNAc > galactose. These inhibitory capacities are in concordance with the relative binding affinity of each sugar for ECL as determined by immunoprecipitation analyses (Kaladas et al., 1982) (Fig. 3A). This observation suggests that the binding of FITC-ECL to feline LYM is CHO-specific. Given that the relative inhibitory capacities of the five sugars were not as clearly defined for cells monitored in the MO-BTMP (Fig. 3A), it can be suggested that FITC-ECL may bind MO in a non-specific manner, independent of a CHO-R. This possibility, however, is somewhat negated by the large decreases in the fluorescent intensities
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Fig. 4. Effects of neuraminidase treatment on the FITC-ECL staining of feline MNC as determined by sugar titration competition assays. The percentages (A) and fluorescent intensities (B) of cells stained at decreasing D-galactose concentrations.
for cells in the M O - B T M P at the higher sugar concentrations (Fig. 3B). To further define differential F I T C - E C L staining properties of cells monitored in the LYM- and MO-BTMP, sugar titration competition assays were performed on feline M N C which were treated with neuraminidase (VCN). As shown by the titration profiles in Fig. 4, when compared to control MNC, the F I T C - E C L staining of L Y M was more VCN-sensitive than that of MO. When compared to controls, the cells monitored in the M O - B T M P showed only a slight increase in the percentage and fluorescent intensities of F I T C - E C L stained cells at the higher D-galactose concentrations (Figs. 4A and 4B). In contrast, both the percentages and fluorescent intensities of cells monitored in the L Y M - B T M P were significantly increased over that of control cells at the higher D-galactose concentrations (Figs. 4A and 4B). These observations suggest that on LYM, many carbohydrate receptors (CHO-R) for ECL are capped or sterically hindered by sialic acid residues. The titration curves in Fig. 4B also suggest that the VCN-treated L Y M have fewer C H O - R for ECL than do control or VCN-treated MO, since their F I T C - E C L fluorescent intensities level off to about half that of
the cells monitored in the M O - B T M P as the Dgalactose concentrations decrease. Taken together, the above findings suggest that feline MO are stained brighter by F I T C - E C L than are L Y M due to the expression of a higher number of C H O - R for ECL on their m e m b r a n a l surface. To further define the physical properties of the ECL-binding C H O - R on cells monitored by the LYM- versus MO-BTMP, M N C were treated with pronase E and analyzed for F I T C - E C L staining in the presence or absence of 4-mM p-npg by flow cytometry. As shown in Fig. 5, the pronase Etreated M N C monitored in the L Y M - B T M P show a significant decrease (75%) in the percentage of F I T C - E C L fluorescing cells, whereas pronase Etreated M N C of the M O - B T M P do not show such a decrease. This data suggests that on LYM, the C H O - R which bind ECL are physically different from those expressed on MO. Thus, the differences in the F I T C - E C L binding properties of the two cell types appears to be attributed not only to differences in the relative quantities of CHO-R, but also to differences in the protein content or structure of these CHO-R. In contrast to the above, when pronase E-treated M N C were stained with F I T C - E C L in the presence of 4 m M
21 TABLE I ADDITIVE IMMUNOFLUORESCENCE OF MNC STAINED WITH ECL, CT87 AND RABBIT ANTI-CAT IgG Experiment number
% membranal fluorescing mononuclear cells ECL a IgG b IgG + ECL
CT87 ¢
CT87 + ECL
CT87 + IgG + ECL
1 2 3 4
30 21 9 29
7 14 22 13
52 34 30 49
31 22 38 28
74 39 49 62
80 53 65 75
Mean± SD
22.3 ± 9.7
14.0± 6.2
41.3 ± 10.9
29.8 ±6.7
56.0± 15.3
68.3 ± 11.9
a FITC-ECL staining was in the presence of 400 mM D-galactose. b FITC-labelled MNC were stained with rabbit anti-cat IgG antiserum. Indirect MNC were stained with monoclonal antibody CT87 and FITC-labelled goat anti-mouse IgG + IgM F(ab')2.
p - n p g , the p e r c e n t a g e of fluorescing cells in b o t h the L Y M - a n d M O - B T M P were n o t significantly less than t h a t of the n o n - p r o n a s e E - t r e a t e d cells (Fig. 5). T h e p r o n a s e E - t r e a t e d M N C did, however, show a large r e d u c t i o n (over 50%) in the
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IgG
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Fig. 5. Effects of pronase E treatment on the percentage of FITC-ECL staining on feline MNC as determined by flow cytometry. Mean percentages and standard deviations (error bars) were calculated from triplicate assays.
fluorescent intensities for b o t h the L Y M - a n d M O - B T M P ( d a t a n o t shown). This o b s e r v a t i o n i n d i c a t e s t h a t the E C L b i n d i n g C H O - R expressed on M O a r e m o r e p r o n a s e E insensitive t h a n those which are e x p r e s s e d o n L Y M . T o further c h a r a c t e r i z e the p u t a t i v e M O - l i k e cells s t a i n e d b y F I T C - E C L in the p r e s e n c e o f high c o n c e n t r a t i o n s of s u g a r c o m p e t i t o r s , M N C were first s t a i n e d with F I T C - E C L in the p r e s e n c e o f 4 m M p - n p g to exclude L Y M staining, a n d then with r a b b i t a n t i - c a t I g G a n d / o r CT87, the l a t t e r a m o n o c l o n a l a n t i b o d y w h i c h b i n d s specifically to feline T - s u p p r e s s o r - L Y M . In all c o m b i n a t i o n s , F I T C - E C L s t a i n e d cells such that a n a d d i t i v e effect on the p e r c e n t a g e o f F I T C - E C L fluorescing cells o c c u r r e d with the o t h e r r e a g e n t s ( T a b l e I). This f i n d i n g c o n f i r m e d t h a t the M N C s t a i n e d b y F I T C - E C L in the p r e s e n c e of 4 m M p - n p g are unique from B-LYM and T suppressor-LYM. L a t e x b e a d i n g e s t i o n is a f u n c t i o n specific to M O in a M N C p o p u l a t i o n ( W i n c h e s t e r a n d Ross, 1986). T o d i r e c t l y d e t e r m i n e if M O were i n d e e d the large M N C specifically s t a i n e d b y F I T C - E C L in the p r e s e n c e o f high sugar c o m p e t i t o r concentrations, M N C were t r e a t e d with 0.81-#m latex beads. A s a control, l a t e x b e a d ingestion was c o r r e l a t e d with the p r e s e n c e of n o n - s p e c i f i c esterase s t a i n i n g ( N E S ) , a h i s t o c h e m i c a l stain u n i q u e to M O w h i c h allows their d i s t i n c t i o n f r o m L Y M ( Y a m et al., 1971). A s shown in T a b l e II, for the c o m b i n e d e x p e r i m e n t s , over 95% of all F I T C E C L positive staining M N C in the p r e s e n c e o f 400 m M D-galactose c o n t a i n e d i n g e s t e d latex beads.
22 TABLE II MICROSCOPIC EXAMINATION CORRELATING FITC-ECL STAINING WITH NES AND BEAD INGESTING ACTIVITY Exp.
% Mononuclear Cells 1
no.
Beads ( + ) NES (+)
Control ECL2 (+)
Beads ( - ) ECL ( - )
Beads ( - ) ECL (+)
Beads ( + ) ECL ( - )
Beads ( + ) ECL (+)
1
4
3
97
0
0
3
2 3 4 5
14 7 4 8
12 8 2 7
88 90 95 94
2 3 0 0
1 1 0 0
9 6 4 6
Mean±SD
ECL + beads ECL 95 96 92 93 95
NES + beads NES 97 97 90 95 99
(94.2±1.6)
(95.6± 3.4)
a At least 300 cells were counted for each experiment. b FITC-ECL staining was in the presence of 400 mM o-galactose.
Also, in each e x p e r i m e n t , the p e r c e n t a g e of F I T C E C L positive cells c o r r e s p o n d e d well with the p e r c e n t a g e s of N E S staining a n d b e a d ingesting cells ( T a b l e II). T h e s e d a t a c o n f i r m that feline
M O are p r e f e r e n t i a l l y s t a i n e d b y F I T C - E C L in the p r e s e n c e of high c o n c e n t r a t i o n s of sugar competitors. T h e a b o v e results show that F I T C - E C L c a n b e used as an i m m u n o f l u o r e s c e n t reagent to specifically i d e n t i f y feline M O . A p h o t o m i c r o g r a p h of feline M N C s t a i n e d with F I T C - E C L in the presence of 400 m M o - g a l a c t o s e is shown in Fig. 6. T h e M O , those ceils w i t h i n g e s t e d latex beads, are s t a i n e d in the p r e s e n c e o f 400 m M D-galactose, while the L Y M are negative.
Discussion
Fig. 6. Photomicrograph of FITC-ECL staining on feline MNC in the presence of 400 mM o-galactose. The top panel shows a field of MNC. The monocytes have ingested latex beads. The bottom panel shows monocytes stained by FITC-ECL while lymphocytes are negative for staining.
I n this report, we h a v e shown that E C L c a n be used to specifically i d e n t i f y feline M O . By h a v i n g a high e n o u g h c o n c e n t r a t i o n of a sugar c o m p e t i t o r in the F I T C - E C L i m m u n o f l u o r e s c e n t reagent, the staining of L Y M is i n h i b i t e d , while M O r e m a i n b r i g h t l y stained. T h e s t a i n i n g p r o p e r t i e s of the two cell t y p e s are a t t r i b u t a b l e to differences in the p r o p e r t i e s of their respective C H O - R which b i n d ECL. T h e sugar t i t r a t i o n c o m p e t i t i o n assay develo p e d for this s t u d y s h o w e d t h a t relative to feline M O , L Y M have a l o w e r affinity for E C L . T h e F I T C - E C L s t a i n i n g of L Y M was p r o t e a s e sensitive, i n d i c a t i n g t h a t their C H O - R were m o s t likely g a l a c t o s y l o l i g o s a c c h a r i d e c o n s t i t u e n t s of m e m branal glycoproteins. The VCN treatment of LYM e x p o s e d m o r e C H O - R for E C L , suggesting the p r e s e n c e of c r y p t i c g a l a c t o s y l b i n d i n g sites for
23
ECL which were blocked by sialic acid residues. The blocking effect may be direct, whereby sialic acids are the terminal saccharide residues covalently linked to penultimate galactosyl residues, or could be indirect, where due to their highly negative charge, sialic acid residues sterically hinder the binding of ECL to adjacent CHO (Sharon and Lis, 1975). In support of the former is the common occurrence of the oligosaccharide NeuNAc a2,3-D-Gal-fll,4-D-GlcNAc-fll---, present in complex asparagine-linked heteropolysaccharide units of a wide range of membranat glycoproteins (Spiro, 1970; Hughes, 1983). When desialylated, these oligosaccharides have lacNAc as their terminal disaccharide residue (D-Gal-fll, 4-GlcNAc-fll--). This lacNAc residue is the terminal saccharide conformation to which ECL binds with highest affinity. This is due to the presence of an extended hydrophobic binding site on ECL specific for penultimate ---4-D-GlcNAc-fll--- residue (Iglesias et al., 1982; Kaladas et al., 1982; De Boeck et al., 1984). The sugar titration competition assays showed that in contrast to feline LYM, MO express a larger quantity of CHO-R for ECL. These CHO-R are more protease insensitive and few are capped by sialic acid residues. A possible explanation for the observed partial protease insensitivity is that a fraction of the ECL CHO-R expressed on MO may be unsialylated glycolipids. One family of neutral glycosphingolipids, the paraglobosides (or lactoneotetraglycosylceramides), do have terminal lacNAc residues to which ECL can bind with high affinity (D-Gal-fll,4-D-GlcNAc-fll,3-D-Gal-fll,4D-Glc-fll,l' ceramide). Such paraglobosides have been characterized on human polymorphonuclear leukocytes (Wherret, 1973) and in bovine and human spleen extracts (Wiegandt, 1971). Sialosyl derivatives have also been shown to form a large fraction of the ganglioside content of human splenic tissues (Sweeley and Siddiqui, 1977). Why feline MO have such a higher affinity for ECL than do LYM could not be determined by the results of this study. Perhaps MO express high quantities of oligosaccharides having terminal unsialylated lacNAc residues, whereas LYM express other unsialylated terminal galactosyl residues. Such residues are known to compete for the ECL CHO-binding site, but with lesser efficiency than
terminal lacNAc residues (Kaladas et al., 1982). Also, it is possible that multivalent versus univalent lectin-CHO interactions could be involved (Sharon and Lis, 1975). Alternatively, a secondary non-specific interaction may be occurring such as hydrophobic bonds involving the cell surface (Lis and Sharon, 1986b). If MO express more glycolipid receptors for ECL than do LYM, the increased hydrophobicity of these receptors could be envisaged as increasing the likelihood of a hydrophobic interaction when ECL binds to its CHO ligand. Although speculative, this might explain why D-galactose concentrations as high as 800 mM did not inhibit the FITC-ECL staining of MO. Such an interaction as envisaged above would not be unique to ECL. Many other lectins have been noted to have secondary binding sites which are specific for a non-CHO ligand (Barondes, 1988). The observed staining property of FITC-ECL on feline MNC was unique, and not a characteristic of ECorL, another Erythrina-derived lectin. It should be noted, however, that seven other Erythrina-derived lectins, all having slightly different galactosyl oligosaccharide binding properties, have been characterized (Lis et al., 1985). Whether any of these lectins bind feline MNC in a similar manner as ECL is unknown. In conclusion, as a MO-specific marker, we believe ECL may prove useful for isolating feline MO and characterizing their role in various immune interactions and immunopathologies.
References Barondes, S.H. (1988) Bifunctional properties of lectins: lectins redefined. Trends Biochem. Sci. 13, 480. Cockerell, G.L., Drakauka, S., Hoover, E.A., Olsen, R.G. and Yohn, D.S. (1976) Characterization of feline T- and Blymphocytes and identification of an experimentally induced T-cell neoplasm in the cat. J. Natl. Cancer Inst. 57, 907. DeBoeck, H.D., Loontiens, F.G., Lis, H. and Sharon, N. (1984) Binding of simple carbohydrates and some N-acetyllactosamine-containing oligosaccharides to Erythrina cristigalli agglutinin as followed with a fluorescent indicator ligand. Arch. Biochem. Biophys. 234, 297. Gengozian, N., Good, R.A. and Day, N.K. (1988) Guinea pig and gerbil erythrocytes rosette with different cells in the blood, bone marrow and thymus of the cat. Cell. Immunol. 112, 1.
24 Hughes, R.C. (1983) Glycoproteins. Chapman and Hall, New York, p. 11. Iglesias, J.I., Lis, H. and Sharon, N. (1982) Purification and properties of a D-galactose/N-acetyl-D-galactosaminespecific lectin from Erythrina cristigalli. Eur. J. Biochem 123, 247. Kaladas, P.M., Kabat, E.A., Iglesias, J.I., Lis, H. and Sharon, N. (1982) Immunochemical studies on the combining site of the D-galactose/N-acetyl-D-galactosamine specific lectin from Erythrina cristigalli seeds. Arch. Biochem. Biophys. 217, 624. Lis, H. and Sharon, N. (1986a) Applications of lectins. In: I.E. Liener, N. Sharon and I.J. Goldstein (Eds.), The lectins: Properties, functions and applications in biology and medicine. Academic Press, New York, p. 293. Lis, H. and Sharon, N. (1986b) Lectins as molecules and as tools. Ann. Rev. Biochem. 55, 35-67. Lis, H., Jaubert, F.J. and Sharon, N. (1985) Isolation and properties of N-acetyllactosamine-specific lectins from nine Erythrina species. Phytochemistry 24, 2803. Mackey, L., Jarrett, W., Jarrett, O. and Wilson, L. (1975) B and T cells in a cat with thymic lymphosarcoma. J. Natl. Cancer Inst. 54, 1483. Rojko, J.L., Hoover, E.A., Finn, B.L. and Olsen, R.G. (1982) Characterization and mitogenesis of feline lymphocyte populations. Int. Arch. Allergy Appl. Immunol. 68, 226. Sharon, N. (1983) Lectin receptors as lymphocyte surface markers. Adv. Immunol. 34, 213.
Sharon, N. and Lis, H. (1975) Use of lectins for the study of membranes. Methods Membr. Biol. 3, 147. Spiro, R.G. (1970) Glycoproteins. Ann. Rev. Biochem. 39, 599. Sweeley, C.C. and Siddiqui, B. (1977) In: M.I. Horowitz and W. Pigman (Eds.), The Glycoconjugates, Vol. 1, Chemistry of Mammalian Glycolipids. Academic Press, New York, p. 459. Taylor, D.W. and Siddiqui, W.A. (1977) Responses of enriched populations of feline T and B lymphocytes to mitogen stimulation. Am. J. Vet. Res. 38, 1969. Taylor, D., Hokama, Y. and Peril, S.F. (1975) Differentiating T and B lymphocytes by rosette formation. J. Immunol. 115, 862. Wellman, M.L., Kociba, G.J. and Rojko, J.L. (1986) Guinea pig erythrocyte rosette formation as a non-specific cell surface marker assay in the cat. Am. J. Vet. Res. 47, 433. Wherret, J.R. (1973) Characterization of the major ganglioside in human red cells a n d / o r a related tetrahexosyl ceramide in white cells. Biochim. Biophys. Acta 326, 63. Wiegandt, H. (1971) Glycosphingolipids. Adv. Lipid Res. 9, 249. Winchester, R.J. and Ross, G.D. (1986) in: N.R. Rose, H. Friedman, J.L. Friedman and J.L. Fahey (Eds.) Manual of Clinical Laboratory Immunology. American Association of Microbiologists, Washington, DC, p. 215. Yam, L.T., Li, C.Y. and Crosby, W.H. (1971) Cytochemical identification of monocytes and granulocytes. Am. J. Clin. Pathol. 55, 283.
15
JIM 05611
Sugar competition assays reveal high affinity receptors for Erythrina cristigalli lectin on feline monocytes C.E. Whitehurst 1,2, N.K. D a y 1 and N. Gengozian 1 1 Department of Pediatrics, All Children's Hospital, University of South Florida, St. Petersburg FL 33701, U.S.A., and 2 Department of Biology, University of South Florida, St. Petersburg FL 33701, U.S.A. (Received 19 December 1989, accepted 16 March 1990)
An examination of fluorescein-labelled Erythrina cristigalli lectin (FITC-ECL) staining on feline mononuclear cells (MNC) using fluorescent microscopy and a novel sugar titration competition assay revealed that monocytes (MO) were stained brighter by FITC-ECL than were lymphocytes (LYM). When MNC were stained with FITC-ECL in the presence of 400 mM or greater o-galactose, analysis by flow cytometry revealed continued MO staining while LYM were negative. MO expressed a larger quantity of carbohydrate receptors (CHO-R) for ECL than did LYM. The CHO-R expressed on MO were mostly protease-insensitive and uncapped by sialic acid residues. All of the CHO-R on LYM were protease-sensitive and many were capped by sialic acid residues. A combined labelling of MNC for non-specific esterase staining, latex bead ingestion and FITC-ECL staining in the presence of 400 mM o-galactose confirmed that FITC-ECL specifically stains MO in the presence of high sugar competitor concentrations. Key words: Mononuclear cell, feline; Monocyte; Carbohydrate receptor; Erythrina cristigalli lectin; Flow cytometry
Introduction
Research involving the feline immune system has been hindered by a lack of molecular markers to identify and allow the fractionation of mono-
Correspondence to: N. Gengozian, Department of Pediatrics, All Children's Hospital, University of South Florida, St. Petersburg, FL 33701, U.S.A. Abbreviations: MO, monocytes; LYM, lymphocytes; BTMP, bitmap; ECL, Erythrina cristigalli lectin; FITC, fluorescein isothiocyanate; MNC, mononuclear cells; CHO-R, carbohydrate receptors; BSA, bovine serum albumin; NES, non-specific esterase stain; VCN, Vibrio cholerae neuraminidase; lacNac, N-acetyllactosamine; p-npg, p-nitrophenyl-fl-Dgalactoside; galNac, N-acetyl-D-galactosamine;iHBSS, incomplete Hanks' balanced salt solution; mPBS, modified phosphate-buffered saline.
nuclear cell subclasses. For a decade the characterization of feline lymphocytes was restricted to surface IgG and complement C3 receptorbeating B lymphocytes and guinea pig erythrocyte-rosetting T lymphocytes (Mackey et al., 1975; Taylor et al., 1975; Cockerell et al., 1976; Taylor and Siddiqui, 1977; Rojko et al., 1982). Only recently have feline T lymphocytes been characterized into differing functional subclasses, with guinea pig erythrocyte-rosetting T cells and gerbil erythrocyte-rosetting T cells exhibiting helper and suppressor activities, respectively, upon polyclonal B cell responses (Gengozian et al., 1988). To date, no molecular markers exist to identify the feline monocyte. Lectins are useful tools for characterizing and fractionating leukocyte subclasses (Sharon, 1983; Lis and Sharon, 1986a). ECL, a 57 kDa lectin derived from the seeds of Erythrina cristigalli,
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
16 binds strongly to N-acetyUactosamine and weakly to other galactosyl-related haptens (Iglesias et al., 1982; Kalades et al., 1982). In this report we describe the distribution of ECL-binding carbohydrate receptors (CHO-R) among feline mononuclear cells (MNC) using a novel sugar titration competition assay and flow cytometry. We show that due to the differing properties of these receptors among lymphocytes (LYM) and monocytes (MO), ECL can be used to specifically identify feline MO.
Materials and methods
Isolation of mononuclear cells Peripheral blood mononuclear cells (MNC) were obtained by centrifuging defibrinated whole blood from healthy adult cat donors on a FicollHypaque gradient. MNC were washed with incomplete Hanks' balanced salt solution (iHBSS), then resuspended in iHBSS containing 0.5% bovine serum albumin (iHBSS-BSA). As revealed by Wright's stain analyses, MNC always contained less than 5% polymorphonuclear cells.
Lectins and sugars Lyophilized fluorescein isothiocyanate-labelled
(Erythrina cristigalli lectin (FITC-ECL) and Erythrina corralodendrum lectin (ECorL) were purchased from Sigma Chemical Company (St. Louis, MO). All lectins were suspended to 1 m g / m l in 0.15 M NaC1 containing 10 m M Hepes, 0.08% NAN3, and p H 7.5. D-galactose was purchased from Gibco (Grand Island, NY); 13lactose, N-acetyl-D-galactosamine (galNAc), pnitrophenyl-/3-D-galactoside (p-npg), and Nacetyl-lactosamine (lacNac) were purchased from Sigma. All sugars were dissolved in modified PBS containing 20 mM EDTA, 0.02% N a N 3, p H 7.2 (mPBS).
Latex bead labelling of monocytes In a 12 × 75 polystyrene culture tube 6 × 10 6 M N C were resuspended to 3 x 106/ml in complete Hanks' balanced salt solution (HBSS) containing Ca z+ and Mg 2+ and 10% heat-inactivated fetal calf serum. To this, 20 btl of 0.81 /~m latex
beads (Bacto, Difco Laboratories, Detroit, MI) were added and the tube rotated in a 3 7 ° C incubator for 90 rain. The cells were washed in mPBS and then resuspended in iHBSS-BSA for analysis by fluorescent microscopy. A cell was scored as a MO if three or more beads were localized internally within that cell.
Sugar titration competition assays Microtiter plate wells were pre-coated with PBS 5% BSA and washed with PBS. Aliquots of 100/xl of M N C (5 x 106/ml) were added to each well. After one wash with mPBS, the cells were washed once with respective serial dilutions of the sugar solutions made in mPBS and then resuspended in 50 /~1 of the same respective sugar dilution or mPBS as a control. To each well 0.5/~1 of FITCECL (1 m g / m l ) was added and cells were incubated on ice for 10 rain and then washed twice with 150 txl of mPBS. The percentage of fluorescing cells and peak channel values representative of fluorescence intensity were determined using a Coulter Epics-C flow cytometer. Quantification parameters for viable LYM and MO were based on cell granularity and size using the 90 ° light scatter versus forward light scatter histogram to gate the respective M N C populations. Gains were adjusted against controls to eliminate the quantification of background fluorescence. A minimum of 1000 cells were counted from each value obtained.
FITC-ECL staining of cat MNC by a cytocentrifugation procedure Normal or latex bead-labelled M N C resuspended in iHBSS-BSA were cytocentrifuged onto a glass slide. While still wet and covered with a residual film of media, 10 #1 of FITC-ECL staining reagent was added. Slides were incubated for 5 min in a humidified petri plate at room temperature and then washed with a gentle rinse of cold PBS using a pasteur pipette. While still wet, coverslips were immediately added and the slides analyzed by fluorescent microscopy using a Zeiss Universal microscope equipped with a Xenon lamp and F I T C filter. The percentage of viable membranal fluorescing and beads-positive M N C were quantitated. At least 500 cells were counted for each experiment.
17
FITC-ECL staining reagent The FITC-ECL staining reagent used to specifically stain monocytes by the cytocentrifuge procedure was prepared by diluting in a microtiter plate 1/zl of FITC-ECL (1 mg/ml) in 20/~1 of a sugar competitor. Optimal sugar competitor concentrations were determined for each new lot of FITC-ECL by making serial dilutions of the sugar in mPBS and observing the quality of MO-specific FITC-ECL staining. These same optimal sugar concentrations could also be used to specifically stain MO when MNC were stained in larger volumes in microtiter plates. MNC (0.5 x 106) in 50/zl of the respective sugar solution were stained with 2/~1 of FITC-ECL (1 mg/ml). Following an incubation for 10 rain on ice, the cells were washed with the same sugar solution and resuspended in mPBS containing 0.5% BSA. Protease treatment of feline MNC MNC (20 x 106) were washed in iHBSS, resuspended in 3 ml of M199 containing 1 mg/ml of Pronase E from Streptomyces griseus (Sigma Chemical) and rotated in a 37 °C incubator for 2 h. Afterwards, the cells were washed with iHBSS and then resuspended in iHBSS-BSA. Cell viability, determined by trypan blue exclusion, was always greater than 95%. Control and proteasetreated MNC were stained with FITC-ECL or rabbit anti-cat IgG (control) as described above. The percentage of fluorescing cells and peak channel values were determined by EPICS-C flow cytometry. Non-specific esterase staining of feline MNC Normal or latex bead-labelled MNC were stained for non-specific esterase activity (NES) using the procedure of Yam et al. (1971). To enhance the staining quality, the reaction buffer included 10 mM of Ca 2+ and Mg 2÷, and after pH adjustment (6.2-6.4) and filtering, 1.5 ml of propylene glycol was added for every 50 ml of reaction buffer. The percentage of beads-positive and NES-positive cells were determined by oil immersion light microscopy. At least 200 cells were counted for each experiment. F1TC-ECL additive immunofluorescence of feline MNC Gengozian et al. (1988) has shown that feline T
lymphocytes which express suppressor activity upon the polyclonal B cell response are capable of forming rosettes with gerbil erythrocytes. A monoclonal antibody, CT87, was also reported to bind specifically to the gerbil erythrocyte receptor on these T suppressor cells (Gengozian et al., 1988). Because of this property, CT87 was used as a marker to distinguish T suppressor cells from other cells by using indirect immunofluorescence. In a microtiter plate 100 /~1 of MNC (10 x 106/ ml)/well were treated with either 50/zl of CT87 or iHBSS-BSA. After incubation on ice and washing with mPBS, cells were stained with 50 /zl of a 1/10 dilution of goat F(ab')2 anti-mouse IgG + IgM (Tago, Burlingame, CA) a n d / o r 50 #1 of a 1/20 dilution of FITC-labelled rabbit anti-cat IgG antiserum (Cooper Biomedical, Malvern, PA) or iHBSS-BSA. After washing with mPBS the cells were stained with 50 /xl of FITC-ECL diluted to 20 # g / m l in 4 mM p-npg or iHBSS-BSA. Cells were washed twice in cold 400 mM D-galactose, and given a final wash in mPBS. The cells were resuspended in mPBS with 0.5% BSA for analysis of the fluorescing MNC by EPICS-C flow cytometry.
Neuraminidase treatment of feline MNC MNC resuspended in HBSS were treated with 500 U / m l of Vibrio cholerae neuraminidase (Gibco) for 60 min in a 37°C waterbath with occasional mixing. Afterwards, the cells were washed with iHBSS and resuspended in iHBSSBSA. Results
To investigate the general staining properties of FITC-ECL on feline MNC, cells from several cats were stained with FITC-ECL by the cytocentrifugation procedure. Preliminary cytocentrifugation analyses revealed that 100% of the MNC were stained at FITC-ECL concentrations as low as 10 /~g/ml (data not shown). A subclass of larger cells comprising 5-15% of MNC, was consistently stained brighter than all other cells. When MNC were stained in the presence of up to 800 mM D-galactose, most cells were completely inhibited from staining; the large cells previously noted, however, continued to stain in a specific and re-
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producible manner. In D-galactose concentrations of less than 400 mM all of the feline MNC resumed staining. Thus, to specifically stain the large MNC, a D-galactose concentration of greater than 400 mM was required in the staining reagent. Feline MNC were next stained with a subagglutinating concentration of FITC-ECL and analyzed by flow cytometry. Cells having a MOlike morphology as gauged by the 90 o light scatter (granularity) and forward light scatter (cell size) parameters were stained significantly brighter than cells having a typical LYM morphology (Fig. 1), The flow cytometer was then used to measure the
relative percentages and fluorescent intensities of MNC stained by sugar titration competition assays. In the presence of 400 mM to 4 mM Dgalactose a higher percentage of cells within the MO-gated bitmaps (BTMP) were stained with FITC-ECL than in the LYM-BTMP (Fig. 2A). The fluorescent intensities of cells within both BTMP were approximately the same (Fig. 2B). In the presence of low D-galactose concentrations nearly 100% of the cells from both the LYM- and MO-BTMP were stained by FITC-ECL (Fig. 2A); however, the fluorescent intensities of the cells monitored in the LYM-BTMP remained con-
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sistently lower than that of cells monitored in the MO-BTMP (Fig. 2B). These results suggest that feline MO bind FITC-ECL with a higher affinity than do LYM. To discern if the differential staining of M N C was a property unique to ECL, the staining property of another Erythrina-derived lectin, Erythrina corralodendrum lectin (ECorL), was analyzed by sugar titration competition assays run parallel with FITC-ECL. Fig. 2 shows that FITC-ECorL did not stain MNC in the same manner as ECL. The MO- and LYM-BTMP showed identical titration profiles when M N C were stained with FITCECorL. These results suggest that the preferential staining of MO at higher D-galactose concentration is a property unique to ECL, and not other Erythrina-derived lectin species such as ECorL. Other saccharides are known to compete for the CHO-binding sites of ECL (Iglesias et al., 1982). t)-galactose is a relatively weak competitor for ECL binding. To determine if other sugar competitors would cause the same FITC-ECL staining phenomenon on feline MNC as that caused by D-galactose, four other sugar competitors (lacNAc, p-npg, galNAc and lactose) were analyzed by the cytocentrifugation staining procedure. Like D-galactose, the four sugars inhibited the staining of all M N C except the large MO-like
cells. The optimal concentrations of the different sugars needed to induce this differential staining were 100 /zM lacNAc, 4 mM p-npg, 20 mM galNAc, 400 m M lactose and 400 m M galactose. When the four sugars were analyzed in parallel with D-galactose by sugar titration competition assays and flow cytometry, they caused similar titration profiles. As shown in Fig. 3, the MOBTMPS have higher percentages of FITC-ECL stained cells which show higher average fluorescent intensities than those cells monitored within the LYM-BTMPS (Fig. 3). The relative inhibitory capacities of the five sugars on the FITC-ECL staining of cells quantitated in the L Y M - B T M P were lacNAc > p-npg > lactose > galNAc > galactose. These inhibitory capacities are in concordance with the relative binding affinity of each sugar for ECL as determined by immunoprecipitation analyses (Kaladas et al., 1982) (Fig. 3A). This observation suggests that the binding of FITC-ECL to feline LYM is CHO-specific. Given that the relative inhibitory capacities of the five sugars were not as clearly defined for cells monitored in the MO-BTMP (Fig. 3A), it can be suggested that FITC-ECL may bind MO in a non-specific manner, independent of a CHO-R. This possibility, however, is somewhat negated by the large decreases in the fluorescent intensities
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Fig. 4. Effects of neuraminidase treatment on the FITC-ECL staining of feline MNC as determined by sugar titration competition assays. The percentages (A) and fluorescent intensities (B) of cells stained at decreasing D-galactose concentrations.
for cells in the M O - B T M P at the higher sugar concentrations (Fig. 3B). To further define differential F I T C - E C L staining properties of cells monitored in the LYM- and MO-BTMP, sugar titration competition assays were performed on feline M N C which were treated with neuraminidase (VCN). As shown by the titration profiles in Fig. 4, when compared to control MNC, the F I T C - E C L staining of L Y M was more VCN-sensitive than that of MO. When compared to controls, the cells monitored in the M O - B T M P showed only a slight increase in the percentage and fluorescent intensities of F I T C - E C L stained cells at the higher D-galactose concentrations (Figs. 4A and 4B). In contrast, both the percentages and fluorescent intensities of cells monitored in the L Y M - B T M P were significantly increased over that of control cells at the higher D-galactose concentrations (Figs. 4A and 4B). These observations suggest that on LYM, many carbohydrate receptors (CHO-R) for ECL are capped or sterically hindered by sialic acid residues. The titration curves in Fig. 4B also suggest that the VCN-treated L Y M have fewer C H O - R for ECL than do control or VCN-treated MO, since their F I T C - E C L fluorescent intensities level off to about half that of
the cells monitored in the M O - B T M P as the Dgalactose concentrations decrease. Taken together, the above findings suggest that feline MO are stained brighter by F I T C - E C L than are L Y M due to the expression of a higher number of C H O - R for ECL on their m e m b r a n a l surface. To further define the physical properties of the ECL-binding C H O - R on cells monitored by the LYM- versus MO-BTMP, M N C were treated with pronase E and analyzed for F I T C - E C L staining in the presence or absence of 4-mM p-npg by flow cytometry. As shown in Fig. 5, the pronase Etreated M N C monitored in the L Y M - B T M P show a significant decrease (75%) in the percentage of F I T C - E C L fluorescing cells, whereas pronase Etreated M N C of the M O - B T M P do not show such a decrease. This data suggests that on LYM, the C H O - R which bind ECL are physically different from those expressed on MO. Thus, the differences in the F I T C - E C L binding properties of the two cell types appears to be attributed not only to differences in the relative quantities of CHO-R, but also to differences in the protein content or structure of these CHO-R. In contrast to the above, when pronase E-treated M N C were stained with F I T C - E C L in the presence of 4 m M
21 TABLE I ADDITIVE IMMUNOFLUORESCENCE OF MNC STAINED WITH ECL, CT87 AND RABBIT ANTI-CAT IgG Experiment number
% membranal fluorescing mononuclear cells ECL a IgG b IgG + ECL
CT87 ¢
CT87 + ECL
CT87 + IgG + ECL
1 2 3 4
30 21 9 29
7 14 22 13
52 34 30 49
31 22 38 28
74 39 49 62
80 53 65 75
Mean± SD
22.3 ± 9.7
14.0± 6.2
41.3 ± 10.9
29.8 ±6.7
56.0± 15.3
68.3 ± 11.9
a FITC-ECL staining was in the presence of 400 mM D-galactose. b FITC-labelled MNC were stained with rabbit anti-cat IgG antiserum. Indirect MNC were stained with monoclonal antibody CT87 and FITC-labelled goat anti-mouse IgG + IgM F(ab')2.
p - n p g , the p e r c e n t a g e of fluorescing cells in b o t h the L Y M - a n d M O - B T M P were n o t significantly less than t h a t of the n o n - p r o n a s e E - t r e a t e d cells (Fig. 5). T h e p r o n a s e E - t r e a t e d M N C did, however, show a large r e d u c t i o n (over 50%) in the
100"
13= CONTROL CELLS [ ] = PROTEASE-TREATED CELLS
~,,'~NOCYTES
LYMPHOCYTES 90"
80-
70. uJ (.3 60"
o
50
40
30.
20.
10.
IgG
ECL
ECL + 4mM p-npg
ECL
ECL 44rnM p-npg
Fig. 5. Effects of pronase E treatment on the percentage of FITC-ECL staining on feline MNC as determined by flow cytometry. Mean percentages and standard deviations (error bars) were calculated from triplicate assays.
fluorescent intensities for b o t h the L Y M - a n d M O - B T M P ( d a t a n o t shown). This o b s e r v a t i o n i n d i c a t e s t h a t the E C L b i n d i n g C H O - R expressed on M O a r e m o r e p r o n a s e E insensitive t h a n those which are e x p r e s s e d o n L Y M . T o further c h a r a c t e r i z e the p u t a t i v e M O - l i k e cells s t a i n e d b y F I T C - E C L in the p r e s e n c e o f high c o n c e n t r a t i o n s of s u g a r c o m p e t i t o r s , M N C were first s t a i n e d with F I T C - E C L in the p r e s e n c e o f 4 m M p - n p g to exclude L Y M staining, a n d then with r a b b i t a n t i - c a t I g G a n d / o r CT87, the l a t t e r a m o n o c l o n a l a n t i b o d y w h i c h b i n d s specifically to feline T - s u p p r e s s o r - L Y M . In all c o m b i n a t i o n s , F I T C - E C L s t a i n e d cells such that a n a d d i t i v e effect on the p e r c e n t a g e o f F I T C - E C L fluorescing cells o c c u r r e d with the o t h e r r e a g e n t s ( T a b l e I). This f i n d i n g c o n f i r m e d t h a t the M N C s t a i n e d b y F I T C - E C L in the p r e s e n c e of 4 m M p - n p g are unique from B-LYM and T suppressor-LYM. L a t e x b e a d i n g e s t i o n is a f u n c t i o n specific to M O in a M N C p o p u l a t i o n ( W i n c h e s t e r a n d Ross, 1986). T o d i r e c t l y d e t e r m i n e if M O were i n d e e d the large M N C specifically s t a i n e d b y F I T C - E C L in the p r e s e n c e o f high sugar c o m p e t i t o r concentrations, M N C were t r e a t e d with 0.81-#m latex beads. A s a control, l a t e x b e a d ingestion was c o r r e l a t e d with the p r e s e n c e of n o n - s p e c i f i c esterase s t a i n i n g ( N E S ) , a h i s t o c h e m i c a l stain u n i q u e to M O w h i c h allows their d i s t i n c t i o n f r o m L Y M ( Y a m et al., 1971). A s shown in T a b l e II, for the c o m b i n e d e x p e r i m e n t s , over 95% of all F I T C E C L positive staining M N C in the p r e s e n c e o f 400 m M D-galactose c o n t a i n e d i n g e s t e d latex beads.
22 TABLE II MICROSCOPIC EXAMINATION CORRELATING FITC-ECL STAINING WITH NES AND BEAD INGESTING ACTIVITY Exp.
% Mononuclear Cells 1
no.
Beads ( + ) NES (+)
Control ECL2 (+)
Beads ( - ) ECL ( - )
Beads ( - ) ECL (+)
Beads ( + ) ECL ( - )
Beads ( + ) ECL (+)
1
4
3
97
0
0
3
2 3 4 5
14 7 4 8
12 8 2 7
88 90 95 94
2 3 0 0
1 1 0 0
9 6 4 6
Mean±SD
ECL + beads ECL 95 96 92 93 95
NES + beads NES 97 97 90 95 99
(94.2±1.6)
(95.6± 3.4)
a At least 300 cells were counted for each experiment. b FITC-ECL staining was in the presence of 400 mM o-galactose.
Also, in each e x p e r i m e n t , the p e r c e n t a g e of F I T C E C L positive cells c o r r e s p o n d e d well with the p e r c e n t a g e s of N E S staining a n d b e a d ingesting cells ( T a b l e II). T h e s e d a t a c o n f i r m that feline
M O are p r e f e r e n t i a l l y s t a i n e d b y F I T C - E C L in the p r e s e n c e of high c o n c e n t r a t i o n s of sugar competitors. T h e a b o v e results show that F I T C - E C L c a n b e used as an i m m u n o f l u o r e s c e n t reagent to specifically i d e n t i f y feline M O . A p h o t o m i c r o g r a p h of feline M N C s t a i n e d with F I T C - E C L in the presence of 400 m M o - g a l a c t o s e is shown in Fig. 6. T h e M O , those ceils w i t h i n g e s t e d latex beads, are s t a i n e d in the p r e s e n c e o f 400 m M D-galactose, while the L Y M are negative.
Discussion
Fig. 6. Photomicrograph of FITC-ECL staining on feline MNC in the presence of 400 mM o-galactose. The top panel shows a field of MNC. The monocytes have ingested latex beads. The bottom panel shows monocytes stained by FITC-ECL while lymphocytes are negative for staining.
I n this report, we h a v e shown that E C L c a n be used to specifically i d e n t i f y feline M O . By h a v i n g a high e n o u g h c o n c e n t r a t i o n of a sugar c o m p e t i t o r in the F I T C - E C L i m m u n o f l u o r e s c e n t reagent, the staining of L Y M is i n h i b i t e d , while M O r e m a i n b r i g h t l y stained. T h e s t a i n i n g p r o p e r t i e s of the two cell t y p e s are a t t r i b u t a b l e to differences in the p r o p e r t i e s of their respective C H O - R which b i n d ECL. T h e sugar t i t r a t i o n c o m p e t i t i o n assay develo p e d for this s t u d y s h o w e d t h a t relative to feline M O , L Y M have a l o w e r affinity for E C L . T h e F I T C - E C L s t a i n i n g of L Y M was p r o t e a s e sensitive, i n d i c a t i n g t h a t their C H O - R were m o s t likely g a l a c t o s y l o l i g o s a c c h a r i d e c o n s t i t u e n t s of m e m branal glycoproteins. The VCN treatment of LYM e x p o s e d m o r e C H O - R for E C L , suggesting the p r e s e n c e of c r y p t i c g a l a c t o s y l b i n d i n g sites for
23
ECL which were blocked by sialic acid residues. The blocking effect may be direct, whereby sialic acids are the terminal saccharide residues covalently linked to penultimate galactosyl residues, or could be indirect, where due to their highly negative charge, sialic acid residues sterically hinder the binding of ECL to adjacent CHO (Sharon and Lis, 1975). In support of the former is the common occurrence of the oligosaccharide NeuNAc a2,3-D-Gal-fll,4-D-GlcNAc-fll---, present in complex asparagine-linked heteropolysaccharide units of a wide range of membranat glycoproteins (Spiro, 1970; Hughes, 1983). When desialylated, these oligosaccharides have lacNAc as their terminal disaccharide residue (D-Gal-fll, 4-GlcNAc-fll--). This lacNAc residue is the terminal saccharide conformation to which ECL binds with highest affinity. This is due to the presence of an extended hydrophobic binding site on ECL specific for penultimate ---4-D-GlcNAc-fll--- residue (Iglesias et al., 1982; Kaladas et al., 1982; De Boeck et al., 1984). The sugar titration competition assays showed that in contrast to feline LYM, MO express a larger quantity of CHO-R for ECL. These CHO-R are more protease insensitive and few are capped by sialic acid residues. A possible explanation for the observed partial protease insensitivity is that a fraction of the ECL CHO-R expressed on MO may be unsialylated glycolipids. One family of neutral glycosphingolipids, the paraglobosides (or lactoneotetraglycosylceramides), do have terminal lacNAc residues to which ECL can bind with high affinity (D-Gal-fll,4-D-GlcNAc-fll,3-D-Gal-fll,4D-Glc-fll,l' ceramide). Such paraglobosides have been characterized on human polymorphonuclear leukocytes (Wherret, 1973) and in bovine and human spleen extracts (Wiegandt, 1971). Sialosyl derivatives have also been shown to form a large fraction of the ganglioside content of human splenic tissues (Sweeley and Siddiqui, 1977). Why feline MO have such a higher affinity for ECL than do LYM could not be determined by the results of this study. Perhaps MO express high quantities of oligosaccharides having terminal unsialylated lacNAc residues, whereas LYM express other unsialylated terminal galactosyl residues. Such residues are known to compete for the ECL CHO-binding site, but with lesser efficiency than
terminal lacNAc residues (Kaladas et al., 1982). Also, it is possible that multivalent versus univalent lectin-CHO interactions could be involved (Sharon and Lis, 1975). Alternatively, a secondary non-specific interaction may be occurring such as hydrophobic bonds involving the cell surface (Lis and Sharon, 1986b). If MO express more glycolipid receptors for ECL than do LYM, the increased hydrophobicity of these receptors could be envisaged as increasing the likelihood of a hydrophobic interaction when ECL binds to its CHO ligand. Although speculative, this might explain why D-galactose concentrations as high as 800 mM did not inhibit the FITC-ECL staining of MO. Such an interaction as envisaged above would not be unique to ECL. Many other lectins have been noted to have secondary binding sites which are specific for a non-CHO ligand (Barondes, 1988). The observed staining property of FITC-ECL on feline MNC was unique, and not a characteristic of ECorL, another Erythrina-derived lectin. It should be noted, however, that seven other Erythrina-derived lectins, all having slightly different galactosyl oligosaccharide binding properties, have been characterized (Lis et al., 1985). Whether any of these lectins bind feline MNC in a similar manner as ECL is unknown. In conclusion, as a MO-specific marker, we believe ECL may prove useful for isolating feline MO and characterizing their role in various immune interactions and immunopathologies.
References Barondes, S.H. (1988) Bifunctional properties of lectins: lectins redefined. Trends Biochem. Sci. 13, 480. Cockerell, G.L., Drakauka, S., Hoover, E.A., Olsen, R.G. and Yohn, D.S. (1976) Characterization of feline T- and Blymphocytes and identification of an experimentally induced T-cell neoplasm in the cat. J. Natl. Cancer Inst. 57, 907. DeBoeck, H.D., Loontiens, F.G., Lis, H. and Sharon, N. (1984) Binding of simple carbohydrates and some N-acetyllactosamine-containing oligosaccharides to Erythrina cristigalli agglutinin as followed with a fluorescent indicator ligand. Arch. Biochem. Biophys. 234, 297. Gengozian, N., Good, R.A. and Day, N.K. (1988) Guinea pig and gerbil erythrocytes rosette with different cells in the blood, bone marrow and thymus of the cat. Cell. Immunol. 112, 1.
24 Hughes, R.C. (1983) Glycoproteins. Chapman and Hall, New York, p. 11. Iglesias, J.I., Lis, H. and Sharon, N. (1982) Purification and properties of a D-galactose/N-acetyl-D-galactosaminespecific lectin from Erythrina cristigalli. Eur. J. Biochem 123, 247. Kaladas, P.M., Kabat, E.A., Iglesias, J.I., Lis, H. and Sharon, N. (1982) Immunochemical studies on the combining site of the D-galactose/N-acetyl-D-galactosamine specific lectin from Erythrina cristigalli seeds. Arch. Biochem. Biophys. 217, 624. Lis, H. and Sharon, N. (1986a) Applications of lectins. In: I.E. Liener, N. Sharon and I.J. Goldstein (Eds.), The lectins: Properties, functions and applications in biology and medicine. Academic Press, New York, p. 293. Lis, H. and Sharon, N. (1986b) Lectins as molecules and as tools. Ann. Rev. Biochem. 55, 35-67. Lis, H., Jaubert, F.J. and Sharon, N. (1985) Isolation and properties of N-acetyllactosamine-specific lectins from nine Erythrina species. Phytochemistry 24, 2803. Mackey, L., Jarrett, W., Jarrett, O. and Wilson, L. (1975) B and T cells in a cat with thymic lymphosarcoma. J. Natl. Cancer Inst. 54, 1483. Rojko, J.L., Hoover, E.A., Finn, B.L. and Olsen, R.G. (1982) Characterization and mitogenesis of feline lymphocyte populations. Int. Arch. Allergy Appl. Immunol. 68, 226. Sharon, N. (1983) Lectin receptors as lymphocyte surface markers. Adv. Immunol. 34, 213.
Sharon, N. and Lis, H. (1975) Use of lectins for the study of membranes. Methods Membr. Biol. 3, 147. Spiro, R.G. (1970) Glycoproteins. Ann. Rev. Biochem. 39, 599. Sweeley, C.C. and Siddiqui, B. (1977) In: M.I. Horowitz and W. Pigman (Eds.), The Glycoconjugates, Vol. 1, Chemistry of Mammalian Glycolipids. Academic Press, New York, p. 459. Taylor, D.W. and Siddiqui, W.A. (1977) Responses of enriched populations of feline T and B lymphocytes to mitogen stimulation. Am. J. Vet. Res. 38, 1969. Taylor, D., Hokama, Y. and Peril, S.F. (1975) Differentiating T and B lymphocytes by rosette formation. J. Immunol. 115, 862. Wellman, M.L., Kociba, G.J. and Rojko, J.L. (1986) Guinea pig erythrocyte rosette formation as a non-specific cell surface marker assay in the cat. Am. J. Vet. Res. 47, 433. Wherret, J.R. (1973) Characterization of the major ganglioside in human red cells a n d / o r a related tetrahexosyl ceramide in white cells. Biochim. Biophys. Acta 326, 63. Wiegandt, H. (1971) Glycosphingolipids. Adv. Lipid Res. 9, 249. Winchester, R.J. and Ross, G.D. (1986) in: N.R. Rose, H. Friedman, J.L. Friedman and J.L. Fahey (Eds.) Manual of Clinical Laboratory Immunology. American Association of Microbiologists, Washington, DC, p. 215. Yam, L.T., Li, C.Y. and Crosby, W.H. (1971) Cytochemical identification of monocytes and granulocytes. Am. J. Clin. Pathol. 55, 283.
