Blood vessels of the Peyer's patch in the mouse: II. In vivo observations

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THE ANATOMICAL RECORD 206:403-417 (1983) Blood Vessels of the Peyer’s Patch in the Mouse: 11 . In Vivo Observations K. YAMAGUCHI AND G.I. SCHOEFL Department of Experimental Pathology, John Curtin School of Medical Research, Australian National Uniuersity, Canberra, ACT 2601, Australia ABSTRACT A technique was developed that allowed the in vivo observation of Peyer’s patches in the mouse for several hours. Untreated animals and animals depleted of lymphocytes were used. In this species, blood vessels associated with the lymphoid nodules are readily visible through the thin serosal muscle coat. High-endothelium venules are recognized by the large number of refractile cells that adhere to the luminal surface. A colloidal carbon suspension injected intra- venously labeled high-endothelium venules and was only rarely seen in arterial and capillary segments or in venules of the gut parenchyma. When fluorescein isothiocyanate-labeled (FITC-labeled) syngeneic spleen cells were injected, they appeared in vessels of the Peyer’s patch within a few seconds and began to adhere to the luminal surface of high-endothelium venules. In untreated animals, peak numbers of fluorescent cells were reached after about 20 min. Many adhered but some were swept away. In lymphocyte-depleted animals, however, peak numbers were reached after only a few minutes and most cells remained attached. In the preceding paper (Yamaguchi and Schoefl, 1983a), we examined the topographic distribution of blood vessels in Peyer’s patches of the mouse. In this tissue, lymphoid nodules are aligned in one plane and blood vessels sup- plying them form a characteristic pattern around each nodule. Of particular interest is a conspicuous venous network on the side fac- ing the serosa. These vessels, referred to as high-endothelium venules (HEV), have long been known to be sites where large numbers of lymphocytes migrate across the vascular wall (Gowans and Knight, 1964).Although they have been examined repeatedly (Marchesi and Go- wans, 1964; Sugimura et al., 1964; Soderstrom, 1967; Rbpke et al, 1972; Schoefl and Miles, 1972; Claesson and Jdrgensen, 1974; Wenk et al., 1974; Farr and DeBruyn, 1975; Anderson and Anderson, 1975; Umetani, 19771, we are not aware of any attempts to observe them in vivo. In the mouse, they are readily seen through the thin outer muscle coat of the intestine. They are thus well suited for observation in the liv- ing animal, and a technique was developed that allows observation of a Peyer’s patch for sev- of labeling lymphocytes with fluorescein iso- thiocyanate (Butcher and Weissman, 1980), we were able to observe labeled spleen cells when they were injected into the circulation and to follow their preferential localization in the high- endothelium venules. Observations were also made on mice that had been intravenously in- jected with a colloidal carbon suspension. MATERIALS AND METHODS Outbred male mice, of 25-30 gm body weight, and 6- to 10-week-oldinbred male BALBic mice were used for in vivo observation of blood ves- sels in Peyer’s patches. Some of the animals were injected intravenously with a colloidal carbon suspension (C11/1431a, Pelikanwerke, Hannover, Germany). In others, the behavior of intravenously injected fluorescein isothio- cyanate-labeled (FITC-labeled) spleen cells was monitored. Both untreated mice and lympho- cyte-depleted mice were used. Table 1 lists the number of animals used and their treatment. An additional seven outbred males were in- jected intravenously with 0.2 mlilO0 gm body era1 hours. we reDort here observations on untreated Received December 15, 1982; accepted April 18. 1983 mice and On mice Of lymphocytes’ K Yamaguchi’s present address Institute of Laboratory Animals, Making use of a recently developed technique Yamaguchi University, School of Medicine, Ube 755, Japan 0 1983 ALAN R. LISS, INC

Transcript of Blood vessels of the Peyer's patch in the mouse: II. In vivo observations

Page 1: Blood vessels of the Peyer's patch in the mouse: II. In vivo observations

THE ANATOMICAL RECORD 206:403-417 (1983)

Blood Vessels of the Peyer’s Patch in the Mouse: 11. In Vivo Observations

K. YAMAGUCHI AND G.I. SCHOEFL Department of Experimental Pathology, John Curtin School of Medical Research, Australian National Uniuersity, Canberra, ACT 2601, Australia

ABSTRACT A technique was developed that allowed the in vivo observation of Peyer’s patches in the mouse for several hours. Untreated animals and animals depleted of lymphocytes were used. In this species, blood vessels associated with the lymphoid nodules are readily visible through the thin serosal muscle coat. High-endothelium venules are recognized by the large number of refractile cells that adhere to the luminal surface. A colloidal carbon suspension injected intra- venously labeled high-endothelium venules and was only rarely seen in arterial and capillary segments or in venules of the gut parenchyma. When fluorescein isothiocyanate-labeled (FITC-labeled) syngeneic spleen cells were injected, they appeared in vessels of the Peyer’s patch within a few seconds and began to adhere to the luminal surface of high-endothelium venules. In untreated animals, peak numbers of fluorescent cells were reached after about 20 min. Many adhered but some were swept away. In lymphocyte-depleted animals, however, peak numbers were reached after only a few minutes and most cells remained attached.

In the preceding paper (Yamaguchi and Schoefl, 1983a), we examined the topographic distribution of blood vessels in Peyer’s patches of the mouse. In this tissue, lymphoid nodules are aligned in one plane and blood vessels sup- plying them form a characteristic pattern around each nodule. Of particular interest is a conspicuous venous network on the side fac- ing the serosa. These vessels, referred to as high-endothelium venules (HEV), have long been known to be sites where large numbers of lymphocytes migrate across the vascular wall (Gowans and Knight, 1964). Although they have been examined repeatedly (Marchesi and Go- wans, 1964; Sugimura et al., 1964; Soderstrom, 1967; Rbpke et al, 1972; Schoefl and Miles, 1972; Claesson and Jdrgensen, 1974; Wenk et al., 1974; Farr and DeBruyn, 1975; Anderson and Anderson, 1975; Umetani, 19771, we are not aware of any attempts to observe them in vivo.

In the mouse, they are readily seen through the thin outer muscle coat of the intestine. They are thus well suited for observation in the liv- ing animal, and a technique was developed that allows observation of a Peyer’s patch for sev-

of labeling lymphocytes with fluorescein iso- thiocyanate (Butcher and Weissman, 1980), we were able to observe labeled spleen cells when they were injected into the circulation and to follow their preferential localization in the high- endothelium venules. Observations were also made on mice that had been intravenously in- jected with a colloidal carbon suspension.

MATERIALS AND METHODS

Outbred male mice, of 25-30 gm body weight, and 6- to 10-week-old inbred male BALBic mice were used for in vivo observation of blood ves- sels in Peyer’s patches. Some of the animals were injected intravenously with a colloidal carbon suspension (C11/1431a, Pelikanwerke, Hannover, Germany). In others, the behavior of intravenously injected fluorescein isothio- cyanate-labeled (FITC-labeled) spleen cells was monitored. Both untreated mice and lympho- cyte-depleted mice were used. Table 1 lists the number of animals used and their treatment. An additional seven outbred males were in- jected intravenously with 0.2 mlilO0 gm body

era1 hours. we reDort here observations on untreated Received December 15, 1982; accepted April 18. 1983

mice and On mice Of lymphocytes’ K Yamaguchi’s present address Institute of Laboratory Animals, Making use of a recently developed technique Yamaguchi University, School of Medicine, Ube 755, Japan

0 1983 ALAN R. LISS, INC

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404 K. YAMAGUCHI AND G.I. SCHOEFL

TABLE 1. Animals used and their treatment

I.V. syngeneic Pretreatment I.V. carbon spleen cells

Outbred None 5 Outbred TD 3 days 1 BALBic None 4 BALB/c TD 1 hr 1' BALBIc TD 3 days 4 BALBic Cortisone 4

Columns 3 and 4 indicate the number of animals used. 'Lymph was collected up to 30 h r following the injection of spleen cells.

weight of the colloidal carbon suspension and allowed to survive 0.5, 2, and 6 hr and 1, 2, 7, and 28 days.

Lymphocyte-Depleted Animals Two methods were used to reduce the num-

ber of circulating lymphocytes: 1) Four mice were injected subcutaneously with 2 mg of hy- drocortisone 21-sodium succinate (Sigma Chemical Corporation) in 0.1 ml of saline on four consecutive days. They were used for in vivo observations 24 hr after the last injection. 2) The thoracic duct of four mice was canulated (Gesner and Gowans, 1962) with SP 10 or SP 31 tubing and lymph was allowed to drain €or a period of 3 days. During that time, the ani- mals were restrained in a Bollman cage mod- ified by an insert to accommodate mice. The animals were fed a commercial pellet diet and water ad libitum. To prevent dehydration and to augment lymph flow, they were injected in- traperitoneally or subcutaneously with 2 ml of sterile saline, containing 5 unitdm1 heparin, two to three times a day. Clots that occasion- ally formed in the canula were dislodged with a horse hair. The animals were housed in a room held a t about 20-25C0, and additional warmth was provided by 60-W carbon-thread lamps suspended above them. Lymph was col- lected into sterile Erlenmayer flasks contain- ing 5 ml of heparinized saline and assessed for recovered lymphocytes a t 12-hr intervals. Over the 3-day period, the total number of lympho- cytes recovered ranged from 9.4 x lo7 to

Preparation of FITC-Labeled Spleen Cells The method followed was essentially that of

Butcher and Weissman (1980). Briefly, spleens of male BALBic mice, of the same age and sex

21.3 x 107.

as the intended hosts, were minced, pressed gently through a 100-gauge stainless steel mesh, and suspended in a sterile solution containing equal parts of medium 199 and phosphate-buff- ered saline and 1% heat-inactivated fetal calf serum a t pH 7.4. The suspension was filtered through several thicknesses of surgical gauze to remove tissue debris. Cell viability was as- sessed by exclusion of Trypan blue.

Stock solutions of fluorescein isothiocyanate (FITC, isomer I, Sigma Chemical Corporation) at concentrations of -300 pgiml were prepared and stored a t -2OC". For use, the concentra- tion of FITC was determined spectrophoto- metrically and an aliquot was added to 5 x lo7 cells in 1 ml suspension medium to give a final concentration of 30 pgiml. After allowing 20 min a t room temperature for labeling, the cells were centrifuged through a cushion of heat- inactivated fetal calf serum (4.5-5 cm in depth) and washed twice with cold suspension me- dium. The cells were suspended in 0.2 ml me- dium and were used within 20 min; viability was 85-96%.

Surgical Preparation for I n Vivo Observation The animal was anesthetized with Nembu-

tal (30 mgilOO gm body weight) and taped to a thin glass plate, and a loop of intestine con- taining a Peyer's patch was eased through a midline incision. For epiillumination, the in- tact gut was positioned on a microscope slide as shown in Diagram 1. For transillumination, a short segment of the gut was cut open near the mesenteric attachment after major vas- cular trunks on the outer gut wall had been ligated. The intestine was gently flattened on a microscope slide with the serosal surface fac- ing up (Diagram 1). The exposed intestine was covered with gauze moistened with saline and the area of the Peyer's patch was covered with a cover slip. After the animal had been intra- muscularly injected with atropine (0.12 mgi 100 gm body weight) to suppress peristaltic movement, it was transferred to the micro- scope stage.

I n Viuo Observation A Leitz Biomed microscope, equipped with a

xenon lamp and with a warm stage (37C") was used. The temperature near the area of the Peyer's patch was -3OC" when measured with a YSI telethermorneter (model 46TUC, Yellow Springs Instruments, Yellow Springs, Ohio). The outer muscle coat of the mouse intestine is very thin, so that blood vessels of the Peyer's patch can be readily seen through this layer

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IN VIVO OBSERVATIONS OF PEYER’S PATCHES

Glass Plate

405

I I Intestine intact Intestine cut open

Epi - illumination Trans - illumination

( normal / carbon ) ( FITC )

Diagram 1. Surgical preparation of the mice for in vivo observations. For details see text.

in the living animal. This allowed examination of the microcirculation near the serosal sur- face; deeper in the tissue, vessels may be par- tially or totally obscured. No observations were made on vascular segments that face the in- testinal lumen. Although some blood vessels had to be ligated so that the intestinal wall could be flattened out for transillumination, there was no apparent change in blood flow through vessels of the Peyer’s patch. For ex- periments involving FITC-labeled spleen cells, the microscope was fitted with a Leitz Ploe- mopak (filter K, blue, narrow band, FITC) for epifluorescence.

Suitable areas of the microcirculation in the Peyer’s patch were selected and photographed before the animal was injected with either the colloidal carbon suspension (1 m1/100 gm body weight) or with 5 x lo7 spleen cells in 0.2 ml suspension medium via the tail vein (see Table 1). Observation of the selected area was then continued for up to 3 hr following the injection

of carbon and up to 2 hr after an inoculum of FITC-labeled cells. In one animal (see Table 11, the thoracic duct was canulated before the cells were injected and lymph was collected up to 30 hr.

Processing of Tissues At the end of the experiment, the area of the

Peyer’s patch was cut out, pinned onto a plastic sheet, fixed, dehydrated, and mounted in toto in Gurr’s neutral mounting medium. In ex- periments with FITC-labeled spleen cells, the blood vessels were filled with a 5% carbon-gel- atin solution (Yamaguchi and Schoefl, 1983a) before the patch was excised and processed as above. Some of the Peyer’s patches in the car- bon-injected animals (Table 1) were embedded in epoxy resin and sectioned for light micros- copy. Mice that had been intravenously in- jected with the colloidal carbon and had been allowed to survive for various periods were per-

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fused with saline to wash out the blood, and the Peyer’s patches were then pinned out, fixed, and mounted.

RESULTS General Observations

Untreated mice Figure 1 shows a general view of a Peyer’s

patch in an anesthetized mouse. Arterial ves- sels are generally narrower and straighter than vessels of the venous limb. Small arteries en- tering the Peyer’s patch tend to follow a course between the nodules. They give off lateral ar- teriolar branches that follow the surface of the nodule towards its serosal pole; there they break up into capillary vessels and penetrate the lymphoid tissue or communicate with venules via thoroughfares (Figs. 2, 8, 14). Arterioles may enter the nodule on the lateral surface and they may subdivide before they enter the lymphoid tissue. Sphincters are commonly seen where arterioles branch off their parent vessels (Fig. 3).

The venous system is more extensive and vessels take an undulating course (Fig. 2). Sev- eral venules drain the capillary meshwork of the lymphoid tissue on the serosal surface. They descend along the lateral surfaces of the nodule and join larger vessels deeper in the tissue. The transition from capillary to venule is usually signaled by an abrupt increase in vessel di- ameter (Fig. 2). Most of these venules have a modified high endothelium that is permeated by large numbers of lymphocytes. The irreg- ularities of the intimal surface can be appre- ciated a t higher magnifications (Fig. 41, though cytological details cannot be resolved in these preparations. Many refractile cells adhere to the luminal surface of these venules or they roll along it.

Lymphocyte-depleted mice In mice depleted of lymphocytes via a tho-

racic duct fistula, the nodules, which are nor- mally 1-1.5 mm in diameter, had shrunken to about half or two-thirds that size (Figs. 5 , 6). The reduction in lymphoid tissue made visible vascular segments that are normally obscured in untreated animals. Thus, although the vas- cular distribution was the same, there was an apparent increase in the vascular density and many vessels were tortuous. The diameter of high-endothelial venules appeared reduced.

Vascular Permeability to Colloidal Carbon Suspensions

In untreated mice injected with the colloidal carbon suspension, carbon began to localize in

high-endothelium venules as early as 3 min (Fig. 7) and continued to accumulate for about 1 hr. Carbon was not seen in arterioles and only rarely in capillary segments. Localization was primarily restricted to high-endothelium venules, where carbon deposited in patches (Figs. 9,lO). Initially, some carbon may merely adhere to the intimal surface, but extravasa- tion was suggested since the marker had spread some distance from the blood column 3 hr after the injection (Fig. 10). Penetration of the vas- cular wall by this marker was confirmed in 1- K r n resin sections. With time, carbon that ini- tially localized in the high-endothelium ven- ules dispersed. At 6 hr, some of the carbon was in perivascular macrophages; by 28 days, most of the carbon was scattered in the spaces be- tween the nodules (Figs. ll, 12).

Carbon was also injected into one mouse from which 12.2 x l o7 lymphocytes had been re- covered via a thoracic duct fistula over a period of 3 days. As in untreated animals, carbon lo- calized preferentially in the wall of high-en- dothelium venules (Figs. 13, 14) but the la- beling appeared less extensive. observations in Animals Injected With FITC-

Labeled Spleen Cells

Untreated animals A faint nonspecific yellow autofluorescence

is normally seen throughout the nodules of the Peyer’s patches (Fig. 15). It is not related to the blood vessels and could be distinguished easily from the bright green color of fluorescein isothiocyanate. Fluorescent cells appeared in the circulation within seconds following their injection into the tail vein, and some of them began to adhere to the luminal surface of high- endothelium venules. By 10 min (Fig. 161, large numbers of labeled cells were associated with these vessels; the maximum was reached a t -20 min. Some cells remained attached; others adhered briefly and were subsequently swept away, or rolled along the luminal surface. La- beled cells did not adhere to either capillaries or arterioles, though a few adhered to venules elsewhere in the intestine.

After -30 min, the brightness of labeled cells began to decline (Fig. 17); by 2 hr, the labeling was faint (Fig. 18). Approximately 60 min after the injection, elongated or arrowhead-shaped cells were, a t times, seen outside the vessel or in the lymphoid parenchyma. These fluoresced less brightly than cells adhering to the luminal surface. Few labeled cells were circulating when the experiment was terminated a t 2 hr. The thoracic duct of one animal was canulated be- fore the injection of FITC-labeled spleen cells.

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IN VIVO OBSERVATIONS OF PEYER’S PATCHES 407

Fig. 1. Peyer’s patch in an untreated mouse. The gut has been cut open (see Materials and Methods) and the tissue is transilluminated. x 60.

Fig. 2. Enlargement of an area shown in the upper left ofFigure 1. An arteriole (A), arteriovenous communications (AVC), and several high-endothelium venules (HV) are il- lustrated. x 170.

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Fig. 3. Example of arterial sphincters (arrows) in a mouse depleted of lymphocytes via a thoracic duct fistula. Loss of cellularity has increased the visibility of blood vessels in this animal, which had been injected intravenously with carbon ( 4 2 min). A venule with some extravasated carbon

is partially visible below the arterial twig. x 300. Detail of a high-endothelium venule. Refractile

cells (arrows) can be seen on the luminal surface of the endothelium. Double arrows mark the outer limit of the vascular wall. x 500.

Fig. 4.

Fluorescent cells began to appear in the lymph approximately 70 min after their intravenous injection.

Lymphocyte-depleted animals

cells localized very rapidly in mice that had

been depleted of lymphocytes via a thoracic duct fistula. Within a few minutes following the injection, many labeled cells adhered to the luminal surface of high-endothelium venules (Fig. 19), and in contrast to untreated animals

Intravenously injected FITC-labeled spleen they appeared to remain firmly attached. Eventually, the walls of these vessels were cov-

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Figs. 5 , 6 . Comparison of nodules (N) of the Peyer’s patch in an untreated mouse (Fig. 5) and in a mouse with a tho- racic duct fistula of 3 days‘ duration (Fig. 6). In these prep-

ered by large aggregates of labeled cells; clus- ters of labeled cells also adhered to the surface of larger venous vessels (Fig. ZO), which could be seen more easily in these animals. More labeled cells adhered to venules of the general intestinal circulation than in untreated ani- mals.

Although many FITC-labeled spleen cells lo- calized in high-endothelium venules of corti- sone-treated mice (Fig. 21), the number of ad- hering cells was not as large as that seen in animals with a thoracic duct fistula. Individual nodules appeared reduced in size, and locali- zation of labeled cells in areas other than the Peyer’s patches was similar to that in canu- lated mice.

DISCUSSION

The anatomical arrangement of Peyer’s patches in the wall of the intestine is well suited for examination of the intact vascular bed. In this tissue, lymphoid nodules are spread out in one plane and, in the mouse, many of the blood vessels supplying them can be seen through the thin outer muscle coat. The intestine can be exteriorized in the anesthetized animal for several hours without any overt signs of in- flammation. Although some vessels of the gut wall had to be ligated in segments prepared for transillumination, blood flow through the Peyer’s patch area was not noticeably dis- turbed. This emphasizes the anastomotic na-

arations, the blood vessels have been filled with a carbon- gelatin solution. x 45.

ture of the vascular network into which the Peyer’s patch is intercalated (Yamaguchi and Schoefl, 1983a).

The general distribution of blood vessels in these in vivo preparations was as described in carbonlgelatin-injected specimens and resin corrosion casts (Yamaguchi and Schoefl, 1983a). Arterial sphincters and arteriovenous com- munications that had been noted in the fixed preparations were confirmed in the live ani- mal. Under the conditions of Nembutal anes- thesia and atropine administration to suppress peristalsis, these thoroughfares were open and no marked variations were noted. In rat lymph nodes, Anderson and Anderson (1975) describe a widening of arteriovenous communications after nerve resection and with epinephrine. The latter also caused constriction of venous sphincters, structures that we were not able to identify in the Peyer’s patches.

High-endothelium venules were recognized readily by the irregular contours of the vessel wall and the presence of refractile cells. Mar- gination and sticking of these cells did not ap- pear to be a consequence of a mild acute in- flammatory response caused by the surgical procedure and by exteriorizing the gut, since refractile cells were seen only occasionally in other venous vessels of the gut wall.

The transition from capillary to high-endo- thelium venule was abrupt. In lymph nodes, Anderson and Anderson (1975) found that cap-

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410 K. YAMAGUCHI AND G.I. SCHOEFL

Fig. 7. High-endothelium venule with some carbon ex- travasated (arrow) 2 min after the injection, when most of the carbon particles are still circulating. This is part of an area that is also shown in Figure 14 1 hr later, when carbon has been cleared from the blood stream. x 350.

Fig. 8. An area encompassing arterial and venous seg- ments linked by an arteriovenous communication (AVO, illustrating carbon deposition in the high-endothelium ven- ules (arrow) after 2 hr. x 170.

illaries did not directly terminate in high-en- dothelium venules. In the Peyer’s patches, cap- illaries leading directly into high-endothelium venules or communicating with them via ven- ules lined by flat endothelium were seen fre-

quently both in resin casts (Yamaguchi and Schoefl, 1983a) and in the in vivo preparations.

In mice that had been depleted of lympho- cytes, individual lymphoid nodules were re- duced in size. This was especially the case in

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Fig. 9. Two nodules of a Peyer’s patch in an untreated mouse 3 hr after the intravenous injection of carbon parti- cles. Carbon has labeled the high-endothelium venules (HV). Arterioles (A) and capillaries are not affected. x 60.

Fig. 10. Higher power of the area marked by a rectangle in Figure 9. Carbon has deposited in patches in the vascular wail and some appears to have spread beyond it (arrow). x 500.

animals with a thoracic duct fistula. The effect was less marked in those injected with hydro- cortisone (Abe and Ito, 1978). As noted by An- derson et al. (1976) in lymph nodes of rats with thoracic duct fistulae, loss of cellularity re- sulted in most vessels becoming more tortuous. There was some decrease in the diameter of high-endothelium venules but not of arterial

and capillary vessels. The height of endothe- lium in these venules has been reported to be diminished when lymphocyte traffic is re- duced, for example, after treatment with an- tilymphocyte serum (Ropke, 1973; Syrjanen, 19781, in tumour-bearing animals (Kriiger, 19681, after neonatal thymectomy (Gold- schneider and McGregor, 1968; Jdrgensen and

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412 K. YAMAGUCHI AND G.I. SCHOEFL

Figs. 11, 12. Whole mounts of Peyer's patches 2 hr (Fig. 11) and 28 days (Fig. 12) after an intravenous injection of

colloidal carbon. Blood was washed out of the circulation to accentuate the localization of carbon. x 20.

Claesson, 19721, or in GvH reactions (Kotani et al., 1980).

An increase in endothelial height has been reported when lymphocyte traffic is aug- mented during an immune response (Borum and Claesson, 1971; Anderson et al., 1975) or in chronically inflamed tissue (Nightingale and Hurley, 1978). On the other hand, Anderson et al. (1976) describe typical high-endothe- lium venules even after prolonged thoracic duct drainage. This agrees with our own findings (Yamaguchi and Schoefl, 1983b). In mice with thoracic duct fistulae, the endothelial lining in high-endothelium venules was still composed of rather tall and plump endothelial cells, though the number of lymphocytes in the vas- cular wall was greatly reduced and resulted in a reduction of the overall thickness of the vas- cular wall.

When colloidal carbon was injected intra- venously as a permeability probe, it localized preferentially in high-endothelium venules. The distribution was patchy, and histological sec- tions confirmed that carbon penetrated the vascular wall and was not merely deposited on the luminal surface. Although rather large doses of the colloidal carbon1 were injected for in vivo observations, intravascular clumping such as described by Bribemark e t al. (1968) or Steh- bens and Florey (1960) in rabbits was not seen. High-endothelium venules have previously been reported to be more permeable than other blood vessels of lymphoid tissue (Schulze, 1925; Schoefl, 1970; Mikata and Niki, 1971; Nopa- jaroonsri et al., 1974; Anderson and Anderson, 1975; Van Deurs et al., 1975; Blau, 19781, and an association of marker particles with mi-

grating lymphocytes has been noted. In ani- mals with a thoracic duct fistula, where the number of circulating lymphocytes is greatly reduced, leakage of carbon appeared less. In these drained animals, some carbon also leaked from other blood vessels, indicating that this procedure is not without a general or systemic effect on the animal.

Unlike carbon deposits in labeled vessels of an inflammatory focus (Majno et al., 1961), which persisted for long periods, carbon that had leaked into the wall of high-endothelium venules began to spread into the surrounding area within a very short time. This was also the case in rat lymph nodes (Nopajaroonsri et al., 1974). Presumably, the sustained migra- tion of lymphocytes across the vascular basal lamina allows the marker to escape more eas- ily from the vascular wall and to spread into the extravascular space. Cellular movement within lymphoid tissue may contribute to the dispersion.

Of particular interest in high-endothelium venules is the preferential localization of blood- borne lymphocytes (Gowans and Knight, 1964). Attachment of the lymphocyte to the endothe- lium is the first step in their migration across the vascular wall, and many studies have been concerned with this process in the intact ani- mal and in isolated organs (Ford et al., 1978)

'Rabbits appear to be particularly sensitive to an endotoxin-like material that may be present in these preparations (Hanna and Wat- son, 19651. The carbon suspension is well tolerated by rats and mice (Cotran et al., 1967) though some bottles of the suspension had to be discarded when they were found to cause blackening of the lungs, which was used as a rough index of toxicity.

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Figs. 13, 14. Peyer’s patch in an animal drained of lym- phocytes for 3 days and injected with colloidal carbon 1 hr previously. A high-endothelium venule (HV), enlarged in Figure 14, illustrates preferential deposition of carbon in

these vessels. This field also illustrates a n arteriovenous communication (AVO between a n artery (A) and high-en- dothelium venules. Figure 13, X 55; Figure 14, X 200.

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4 14 K. YAMAGUCHI AND G.I. SCHOEFL

Figs. 15-18. Small area of a Peyer’s patch observed with epifluorescence. Figure 15 illustrates the extent of autoflu- orescence before the injection of FITC-labeled spleen cells.

Figures 16-18 show the same field, 10 min, 1 hr, and 2 hr later. Labeled cells adhere preferentially to high-endothe- lium venules. x 45.

and also in in vitro systems (Stamper and Woodruff, 1976, 1977; De Bono, 1977). A sul- phated compound synthesized by the endothe- lium (Andrews et al., 1980) and an “adherence- enhancing factor” isolated from thoracic duct lymph (Carey et al., 1981) have been impli- cated in attracting lymphocytes. For lympho- cytes to adher to the endothelium, they must be metabolically active (Woodruff and Kutt- ner, 1980). Treatment of the lymphocytes with

cytochalasin B inhibits adherence to the en- dothelium (Smith and Ford, 1979; Woodruff et al., 1977; Woodruff and Kuttner, 19801, sug- gesting that microfilaments may be involved in the process. This agrees with reports that lymphocytes make contact with the endothe- lial cell via microvilli (van Ewijk et al., 1975). Lymphocytes can be removed from the luminal surface of high-endothelium venules with a perfusate containing trypsin (Anderson et al.,

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Figs. 19, 20. Examples of the localization of FITC-la- beled cells in lymphocyte-depleted mice (thoracic duct fis- tula for 3 days) a t 5 min (Fig. 19) and 45 min (Fig. 20). x 45. 45.

Fig. 21. From a mouse treated with cortisone. FITC-la- beled spleen cells have been circulating for 11 min and many of them have localized in the high-endothelium venules. X

1976), and lymphocytes treated with trypsin or with a calcium complexing agent do not ad- here to high-endothelium venules in vitro (Woodruff and Kuttner, 19801, suggesting in- terference with surface binding sites.

Butcher and Weissman (1980) have recently published a method of fluorescence labeling of lymphocytes. Such cells localized in high-en-

dothelium venules of lymphoid tissue and flu- orescent cells could be demonstrated in frozen sections up to 11 days (Butcher et al., 1980). We have used this method to label spleen cells that we observed in the circulation of the living animals. They appeared in the microcircula- tion within a few seconds after their injection into the tail vein and began to adhere prefer-

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416 K. YAMAGUCHI AND G.I. SCHOEFL

entially to the luminal surface of high-endo- thelium venules. Like the unlabeled refractile cells, some remained attached while others rolled along the surface and were eventually swept away. In lymphocyte-depleted mice, however, labeled cells peaked earlier and re- mained attached to the vessel wall. The reason for this may be that in these animals labeled cells have less competition with unlabeled cells for sites of attachment. Of a more speculative nature is the possibility that a substance at- tracting or binding lymphocytes, such as the sulphated compound isolated by Ford and his colleagues (Ford et al., 1978; Andrews et al., 1980) or the previously mentioned “adherence- enhancing factor” (Carey et al., 1981), may ac- cumulate when lymphocyte traffic is curtailed.

We had no way of gauging how many of the adhering lymphocytes actually migrated across the vascular wall. Fluorescent cells were no- ticed adjacent to the venules by 1 hr and some were seen in the thoracic duct lymph a t about the same time. Other lymphocytes still ad- hered to the luminal surface of the high-en- dothelium venules at the end of 2 hr when the experiment was terminated. We do not know whether these cells had remained in situ for that length of time. Some may have been se- questered in other vascular beds before they reached the Peyer’s patch, and others may in fact have returned to the general circulation via the thoracic duct.

The loss of brightness we noticed in labeled cells by about 1 hr agrees with the phase of rapid decay in fluorescence reported by Butcher and Weissman (1980). They noted that FITC- labeled B lymphocytes fluoresced more brightly than T lymphocytes. No difference in the brightness of fluorescence was observed in cells adhering to the luminal surface, but cells that had apparently migrated across the vascular wall and were located in the extravascular compartment were usually fainter.

ACKNOWLEDGMENTS

We thank Dr. W. Cliff for the use of his Leitz Biomed microscope, Ms. Marie Colvill for her excellent photographic assistance, and Miss Elaine Wyatt for drawing Diagram 1.

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