Evidence for conservation of peripheral lymphocyte homing receptors between the bovine, murine and human species

Evidence for conservation of peripheral lymphocyte homing receptors between the bovine, murine and human species

Veterinary Immunology and Immunopathology, 33 (1992) 79-88 Elsevier Science Publishers B.V., Amsterdam 79 Evidence for conservation of peripheral ly...

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Veterinary Immunology and Immunopathology, 33 (1992) 79-88 Elsevier Science Publishers B.V., Amsterdam

79

Evidence for conservation of peripheral lymphocyte homing receptors between the bovine, murine and human species B r a d T. B o s w o r t h a n d J a m e s A. H a r p u s Department of Agriculture, Agricultural Research Service, National Animal Disease Center. Metabolic Diseases and Immunology Research Unit, P.O. Box 70, Ames, IA 50010, USA (Accepted 13 September 1991 )

ABSTRACT Bosworth, B.T. and Harp, J.A., 1992. Evidence for conservation of peripheral lymphocyte homing receptors between the bovine, murine and human species. Vet. Immunol. Immunopathol., 33: 7988. In rodents and humans, lymphocytes circulate throughout the body and return preferentially to their tissues of origin via a process termed homing. The specificity of homing is controlled by the binding of tissue-specific receptors on lymphocytes to ligands on specialized high-walled endothelial venules (HEV) found in lymphoid tissue. The murine and human peripheral lymphocyte homing receptors (PLHR) have been characterized and shown to be similar to each other. We present evidence for a similar receptor in the bovine. Bovine peripheral blood lymphocytes (PBL) bind to the HEV of murine peripheral lymph node tissue in vitro. The same sugars that have been shown to decrease the binding of murine or human lymphocytes to murine HEV also decrease the binding of bovine PBL to murine HEV. Neuraminidase treatment affects lymphocyte binding in a similar manner in the bovine, murine and human species. Phorbol myristate acetate (PMA) stimulation, which has been shown to reduce the expression of murine and human PLHR, also reduces the binding of bovine PBL to murine HEV. These data suggest conservation of PLHR between these species.

ABBREVIATIONS HEV, high-walled endothelial venules; FITC, fluorescein isothiocyannate; PBL, peripheral blood lymphocytes; PLHR, peripheral lymphocyte homing receptors; PMA, phorbol myristate acetate; SAR, specific adherence ratio.

INTRODUCTION L y m p h o c y t e h o m i n g is t h e p r o c e s s b y w h i c h l y m p h o c y t e s r e c i r c u l a t e throughout the body and return to their tissues of origin (Gowans and Knight, Correspondence to: James A. Harp, US Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Metabolic Diseases and Immunology Research Unit, P.O. Box 70, Ames, IA 50010, USA

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1964; Stoolman et al., 1987; Butcher, 1990 ) In rodents and humans, the specificity of homing is controlled by adhesive interactions of homing receptors on lymphocytes with vascular addressins on high-walled endothelial venules (HEV) (Gallatin et al., 1983; Streeter et al., 1988a,b; Holzmann et al., 1989; Stoolman, 1989; Butcher, 1990). Murine and human lymphocyte homing receptors and vascular addressins have been studied and characterized using an in vitro HEV binding assay (Gallatin et al., 1983; Streeter et al., 1988a,b; Holzmann et al., 1989 ). In this HEV assay, peripheral lymphocytes bind preferentially to HEV in frozen tissue sections of peripheral lymph nodes and mucosal lymphocytes bind preferentially to HEV in frozen tissue sections of Peyer's patches (Gallatin et al., 1983; Streeter et al., 1988a,b; Holzmann et al., 1989). The murine and human peripheral lymphocyte homing receptors (PLHR) have been characterized and shown to be very similar to each other (Gallatin et al., 1983; Siegelman and Weismann, 1989; Bowen et al., 1989; Lasky et al., 1989; Kishimoto et al., 1990; Siegelman et al., 1990). Sequence analysis of the cDNA encoding for murine and human PLHR reveals an extracellular lectin domain (Siegelman and Weissman, 1989; Bowen et al., 1989; Lasky et al., 1989; Siegelman et al., 1990). Evidence that this lectin domain is involved in the homing of peripheral lymphocytes includes the ability of sugars, such as mannose-6-phosphate (M6P), fructose- l-phosphate (F 1P) and fucoidin, to inhibit the binding of lymphocytes to peripheral lymph node HEV (Stoolman et al., 1984, 1987; Stoolman and Rosen, 1985). The aforementioned sugars compete with each other for binding to purified PLHR (Imai et al., 1990 ). Also, the antibody MEL-14, which blocks the homing of murine peripheral lymphocytes, binds to the lectin domain of PLHR (Bowen et al., 1990). In this report, we present evidence for a receptor in the bovine that is similar to murine and human PLHR. Bovine peripheral blood lymphocytes (PBL) bind preferentially to HEV of murine peripheral lymph node tissue in vitro. Sugars that have been shown to decrease the binding of murine or human lymphocytes to murine HEV also decrease the binding of bovine PBL to murine HEV. Neuraminidase treatment affects lymphocyte binding in a similar manner in the bovine, murine and human species. Phorbol myristate acetate (PMA) stimulation, which has been shown to reduce the expression of murine and human PLHR (Huang et al., 1989; Jung and Dailey, 1990; Kishimoto et al., 1990), also reduces the binding of bovine PBL to murine HEV. These data suggest that the structure and function of PLHR are conserved between these species.

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MATERIALS A N D M E T H O D S

Cell preparation Bovine PBL were collected as previously described (Whitmire and Harp, 1991 ). Platelets were removed from the PBL by centrifugation through Percoil (specific gravity 1.029). The PBL were then washed in assay buffer (RPMI 1640 with 5% fetal bovine serum; Gibco Laboratories, Grand Island, NY). Bovine PBL in assay buffer were then labeled with Hoechst 33342 (Sigma Chemical Co., St. Louis, MO; 0.6 #g ml-l, 30 min at 39°C). Murine splenocytes and Peyer's patch lymphocytes from adult Balb/c mice were labeled with fluorescein isothiocyanate (FITC) (Sigma; 50 #g ml- 1, 30 min at 37°C). After labeling, lymphocytes were pelleted by centrifugation through fetal bovine serum to remove unbound label and dead cells, and then washed in assay buffer.

HE V assay Axillary, mesenteric and popliteal lymph nodes, and Peyer's patches were removed from adult Balb/c mice for use in the HEV binding assay described previously (Stamper and Woodruff, 1976; Butcher et al., 1979; Stoolman et al., 1987 ). Briefly, lymphoid tissue embedded in O.C.T. (Miles Inc, Elkhart, IN) was frozen on dry ice and sectioned with a cryostat. Tissue sections were placed on glass slides. The O.C.T. was removed with forceps and sections were overlaid with 100 gl of lymphocyte suspension in assay buffer. Sections were rotated at 60 rev min- l for 30 min at 5 ° C. The sections were then fixed in 3% glutaraldehyde in phosphate-buffered saline (PBS) for 45 min, rinsed in PBS and examined microscopicallywith darkfield/fluorescentillumination.

Tissue specificity of bovine PBL binding Hoechst-labeledbovine PBL (2 × l 0 7 ml-~ ) were mixed at a 10:1 ratio with FITC-labeled murine splenocytes or Peyer's patch lymphocytes (2× 10 6 ml- ~). Samples containing bovine PBL and murine splenocytes were used to overlay sections of murine peripheral lymph nodes (axiUary and popliteal). Samples containing bovine PBL and murine Peyer's patch lymphocytes were used to overlay sections of murine Peyer's patches. The numbers of bovine and murine lymphocytes bound to HEV were determined. The specific adherence ratio (SAR) was calculated as previously described by dividing the ratio of bovine to murine lymphocytes bound to HEV by the ratio of bovine to murine lymphocytes used to overlay the lymph node sections (Butcher et al., 1979).

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Neuraminidase, PMA and sugar treatment Bovine PBL in assay buffer ( 1 × 107 m l - 1 were incubated for 30 min at 39 °C with 0.5 U m l - ' neuraminidase, 1/tg m l - 1 PMA or m e d i u m alone. The bovine PBL were then washed in assay buffer and resuspended along with FITC-labeled murine splenocytes at a 10:1 ratio (2 × 107 m l - 1 bovine, 2 X 106 m l - ' murine ). The bovine and murine lymphocytes were overlaid on murine mesenteric lymph node sections and the SAR was determined (Butcher et al., 1979). Murine lymph node sections were pre-incubated for 30 min at 37 °C with either PBS alone or PBS containing 0.5 U m1-1 of neuraminidase as described previously (Rosen et all., 1985), washed in PBS and then overlaid with bovine PBL (2 X 107 m l - ~) in assay buffer. Murine lymphocytes were not used as an internal control in these experiments since the treatment of lymph node sections would have affected the binding of both murine and bovine lymphocytes. In experiments testing the effects of sugars, bovine PBL in assay buffer ( 2 X 10 7 m1-1 ) were incubated for 15 min on ice with 5 m M F1P, 5 m M fructose-6-phosphate (F6P), 1/~g m l - ' fucoidin or m e d i u m alone before being overlaid on mesenteric lymph node sections. The aforementioned sugars were included with the bovine PBL during the incubation on murine lymph node sections. Murine lymphocytes were not added as an internal control since the sugars would also have interfered with their binding to HEV. The neuraminidase, PMA, and sugars used in these experiments were obtained from Sigma Chemical Co (St. Louis, MO).

Experimental design and data analysis Experiments were performed on separate days for each treatment regimen or tissue-specific HEV assay. Each experiment consisted of an average of eight replicate sections. The binding of bovine PBL relative to the internal control (murine lymphocytes) was determined by calculating the SAR (Butcher et al., 1979). When murine lymphocytes could not be used as an internal control, consecutively cut tissue sections containing HEV of similar size and n u m b e r were used to assess lymphocyte binding. The total number of lymphocytes bound to HEV in all treated or control samples for each experiment was counted and compared to determine the percent binding in treated samples relative to the control. The standard error (SE) represents the variation between separate experiments carried out on different days. Treatments that the lymphocytes or lymph node sections had received were not known to the person determining the

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number of lymphocytes bound to HEV. Over 90% of the treated lymphocytes were viable, based on their ability to exclude trypan blue dye. RESULTS

Bovine PBL bound preferentially to murine peripheral node HEV compared with murine Peyer's patch HEV. The average SAR for binding to peripheral nodes and Peyer's patches was 0.83 and 0.13, respectively (Table 1 ). Bovine PBL bound primarily to HEV of lymph nodes with a limited amount of binding to medullary sinuses (Fig. 1 (a)). Hoechst 33342 did not affect the binding of murine or bovine lymphocytes to HEV. In one experiment, the labels were reversed, i.e. bovine PBL were FITC labeled and murine splenocytes were Hoechst labeled (Fig. 1 (b) and TABLE 1 Binding of bovine PBL to murine HEV Experiment No. Peripheral lymph node HEV t

Peyer's patch HEV 2

1 2 3

121/163 (0.76) 4 559/61 (0.92) 474/59 (0.80)

91/82 (0.11) 123/80 (0.15) ND 5

Average SAR + SE

0.83 _+0.08

0.13 + 0.03

iBovine PBL and murine splenocytes ( 10:1 ratio) were used to overlay peripheral lymph node sections. 2Bovine PBL and murine Peyer's patch lymphocytes ( 10: l ratio) were used to overlay Peyer's patch sections. 3Numbers of bovine PBL/murine lymphocytes bound to HEV. 4SAR, determined as previously described (Butcher et al., 1979 ). 5ND, not done.

TABLE 2 Effects of sugars on bovine PBL binding to murine HEV ~ Experiment No.

F 1P

F6P

Fucoidin

1 2

89/2482 (36%) 3 196/535 (37%)

320/317 (101%) 522/535 (98%)

267/732 (36%) 108/404 (32%)

Average % binding __SE

36 _+ 1%

99 _+2%

34 __3%

tBovine PBL were incubated with 5 mM FIP, 5 mM F6P, 1 #g ml -~ fucoidin or medium alone, and then overlaid on murine mesenteric lymph node sections. 2Numbers of treated bovine PBL/control bovine PBL bound to HEV. 3The percent binding of treated bovine PBL relative to the control.

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Fig. 1. (a) Bindingof bovine and murinelymphocytesto murineHEV. Hoechst-labeledbovine PBL (arrows) and FITC-labeled murine splenocytes (arrowheads) binding to murine HEV. The murine HEV is darker than the surroundinglymphoidtissue.

Table 1, Experiment 3 ). There were no differences in the SAR from this experiment compared with the SAR in experiments with Hoechst-labeled bovine PBL and FITC-labeled murine splenocytes (Fig. 1 (a) and Table 1, Experiments 1 and 2). Hoechst 33342 appeared to be a better label than FITC as the intensity was greater, the cells retained fluorescence for an extended period of time and a 100-fold increase in the concentration of Hoechst 33342 did not affect bovine PBL binding in the HEV assay (data not shown). The presence of F 1P and fucoidin in the HEV assay reduced the binding of bovine PBL to murine HEV to about one-third that of control levels (Table 2 ). Conversely, F6P did not affect the binding of bovine PBL as compared with controls (Table 2). Treatment of bovine PBL with 0.5 U m l - l neuraminidase caused a 150% increase in HEV binding relative to controls. Stimulation of bovine PBL with PMA decreased their binding to HEV (Table 3 ). The SAR of bovine PBL treated with PMA was reduced to 26% and 24% of the control SAR in Experiments 1 and 2, respectively.

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Fig. 1. (b) Binding of bovine and murine lymphocytes to murine HEV. FITC-labeled bovine PBL (arrows) and Hoechst-labeled murine splenocytes (arrowheads) binding to murine HEV. The reversal of Hoechst and FITC labels had no effect on the specificity of lymphocyte binding in the HEV assay.

TABLE3 EffectofPMAt~atmentonbovinePBLbindingto murineHEV t Experiment No.

Control

PMA

1 2

437/1032 (0.42) 3 400/81 (0.49)

86/79 (0.11) 84/67 (0.12)

Average SAR + SE

0.44 + 0.05

0.12 + 0.01

tBovine PBL were incubated with PMA or medium alone (control), mixed 10:1 with murine splenocytes and overlaid on murine mesenteric lymph node sections. 2Numbers of bovine PBL/murine splenocytes bound to HEV. 3SAR determined as described previously (Butcher et al., 1979 ).

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DISCUSSION

Murine and h u m a n P L H R are similar; both contain a lectin domain and the binding of lymphocytes to HEV in both species is inhibited by specific sugars (Stoolman et al., 1987; Siegelman and Weissman, 1989; Bowen et al., 1989; Lasky et al., 1989; Kishimoto et al., 1990; Siegelman et al., 1990). In the present study, F 1P and fucoidin inhibited bovine PBL binding to murine HEV. These same sugars inhibit the binding of murine peripheral lymphocytes to murine HEV (Stoolman et al., 1987 ). The sugar F6P, which does not affect the binding of murine lymphocytes to HEV (Stoolman et al., 1987), had no effect on bovine PBL binding in the present study. The similar effects of sugars on lymphocyte binding suggest conservation of a lectin domain in P L H R of all three species. Removal of sialic acid residues from bovine lymphocytes with neuraminidase increased binding to murine HEV. Previous studies examining the binding of h u m a n PBL to murine HEV also reported an increase in binding after neuraminidase treatment (Stoolman et al., 1987 ). This increased binding after sialic acid removal may be due to the decrease in negative charge on the lymphocyte which could enhance binding to HEV (Stoolman et al., 1987). Conversely, neuraminidase treatment of murine lymph node sections has been reported to decrease the binding of murine lymphocytes to peripheral HEV (Rosen et al., 1985). We saw a similar reduction in binding of bovine PBL following neuraminidase treatment of lymph node sections (data not presented). It has been suggested that sialic acid residues on murine peripheral HEV interact with P L H R and that neuraminidase treatment decreases lymphocyte binding by removing the sialic acid residues from the HEV vascular addressins (Rosen et al., 1985; True et al., 1990). The reduction in the binding of both bovine PBL and murine lymphocytes to murine HEV after neuraminidase treatment of the HEV suggests that PLHR of both species recognize the same vascular addressins. Murine and h u m a n P L H R are rapidly down-regulated by a variety of stimulatory agents, including PMA (Huang et al., 1989; Jung and Dailey, 1990; Kishimoto et al., 1990). In the present study, binding of bovine PBL to murine HEV was decreased by pre-treatment of the PBL with PMA. This suggests that the bovine receptor may also be down-regulated by PMA. In summary, the ability of bovine PBL to bind to murine HEV, and the similarities between the effects of sugars, neuraminidase and PMA on the binding of bovine, murine and h u m a n lymphocytes in the HEV assay, suggest that PLHR are conserved in all three species. Definitive identification of the bovine receptor will be facilitated by the isolation and sequencing of a cDNA encoding the bovine equivalent of the murine and h u m a n PLHR.

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ACKNOWLEDGEMENTS

The authors thank Aden Anderson, Sally Crandell, Nancy Eischen, Gary Fry and Bruce Pesch for technical assistance, and Kathleen Kelderman for assistance in manuscript preparation.

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