gro, a member of the interleukin-8 family, on leukocytes in microcirculation of the rat mesentery

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

56,60-69 (1992)

Effect of Rat CINC/gro, a Member of the Interleukin-8 Family, on Leukocytes in Microcirculation of the Rat Mesentery KAZUYOSHI WATANABE,’ MAKOTO SUEMATSU,* MEGUMI IIDA,’ KIYOSUMI TAKAISHI,’ YOSHIO IIZUKA,’ HIDEKAZU SUZUKI,* MASAYUKI SUZUKI,* MASAHARU TSUCHIYA,* AND SUSUMU TSURUFUJI’ ‘Institute of Cytosignal Research, Inc., 2-58 Hiromachi I-chome, Shinagawa-ku, Tokyo 140, Japan; and ‘Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan Received August 20, 1991 Rat cytokine-induced neutrophil chemoattractant (CINC) is a member of the IL-8 family, and its human counterpart is gro/MGSA but not IL-8. We ascertained that chemically synthesized CINC was comparable to native CINClgro with regard to chemotactic activity for rat neutrophils and studied the effect of synthesized CINClgro on circulating leukocytes in microvascular vessels of rat mesentery. Exposure of rat mesentery to 10-s M authentic CINC/gro induced neutrophil adherence to and extravasation from postcapillary venules (PCVs) but not from capillaries or arterioles. CINC/gro concentrations as low as lo-“’ M were effective in causing neutrophil adherence. Neutrophils adhered to thin PCVs (mean diameter, -25 km) after exposure to CINC/gro for 15 min. The mean diameters of the PCV with adherence of neutrophils after exposure to CINC/gro for 30 and 60 min were 37 and 43 km, respectively. The diameters of PCV with extravasation of neutrophils also increased in a time-dependent manner. The starting position of adherence of neutrophils was -25-50 pm away from the upper junction of two vessels and remained virtually unchanged during exposure to CINUgro for 60 min. However, the distance from the start to the end of neutrophil adherence increased in a time-dependent manner. The effect of CINC/gro on adherence and extravasation of leukocytes was neutrophil specific since other leukocytes such as lymphocytes and monocytes were not identified among the adherent and extravasated leukocytes. 0 wn Academic Press, Inc.

INTRODUCTION Previously, we showed that rat glomerular epithelial cells and the normal rat kidney epithelial cell line (NRK-52E), when treated with interleukin-I (IL-l), tumor necrosis factor, or lipopolysaccharide, produced a neutrophil chemoattractant. We designated this factor cytokine-induced neutrophil chemoattractant (CINC) (Watanabe et al., 1987, 1989a; Watanabe and Nakagawa, 1990). CINC is composed of 72 amino acid residues and its amino acid sequence shows that CINC is a member of the family which includes human interleukin-8 (IL-g), gro/MGSA, platelet basic protein, and platelet factor-4 (Watanabe er al., 1989b; Wolpe and Cerami, 1989). Human IL-8 is a chemoattractant for neutrophils and T-lymphocytes identified recently (Yoshimura ef al., 1987; Waltz ef al., 1987; Gregory et al., 1988; Larsen et al., 1989a,b). On the other hand, gro/melanoma growth-stimulating activity (MGSA) has been identified as a growth factor (Richmond and Thomas, 1988), and has a chemotactic activity for neutrophils comparable to that of IL-8 (Schroder et al., 1990). Rat CINC, however, has a closer sequence homology to gro/MGSA (69.4%) than to IL-8 (47.2%) among human peptides (Watanabe et al., 1989b). Therefore, CINC is the rat equivalent of human gro/MGSA but not of IL-S. 60 0014-4800192 $3.00 Copyright AU rights

0 1992 by Academic Press, Inc. of reproduction in any form reserved

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The chemotactic factors of the IL-8 family cause neutrophil infiltration in vivo (Schroder et al., 1990). For example, rat CINClgro and human IL-8 injected intradermally and subcutaneously, respectively, cause neutrophil infiltration in the rat. Adherence of leukocytes to the endothelium is a prerequisite for migration toward the extravascular space, and enhanced adhesion at sites of inflammation serves in the recruitment of cells to specific sites. However, the types of vessels involved in the adherence and extravasation of neutrophils and other phenomena caused by IL-g-related substances are not yet understood. Here, we report the changes induced by administration of rat CINC/ gro to the rat mesentery microvascular bed, and we compare the in vitro chemotactic activity for neutrophils of synthesized CINC/gro with that of native CINU gro. MATERIALS

AND METHODS

Materials

Rat CINC/gro, which consists of 72 amino acid residues, was synthesized chemically in the liquid phase at the Peptide Institute, Inc. (Osaka, Japan). Ten peptides of CINC/gro, corresponding residues 1-7, 8-17, 18-24, 25-31, 32-39, 40-47, 48-54, 55-57, 58-64, and 65-72, were prepared in advance. The detailed synthesis will be reported elsewhere. The authenticity of the synthesized CINCl gro was confirmed by mass spectrum analysis and amino acid composition. The retention time of synthetic CINC/gro from the reverse-phase HPLC C-18 column was consistent with that of native CINC/gro. Other chemicals were of the highest purity available from local commercial sources. Preparation

of Native

CINClgro

NRK-52E cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nissui Seiyaku Co., Tokyo), supplemented with 5% fetal calf serum until confluence, and then in DMEM containing 0.1% bovine serum albumin and 0.1 nM interleukin-1B for 2 days. CINC/gro was purified from the medium by ammonium sulfate fractionation, ion-exchange chromatography, and reverse-phase HPLC as described (Watanabe et al., 1989a). Neutrophil

Chemotaxis

Elicited rat neutrophils were collected 16 hr after intraperitoneal injection of 1% casein in Krebs-Ringer bicarbonate solution. In vitro neutrophil chemotaxis was determined by the procedure reported previously (Watanabe et al., 1985, 1989~) using a multiwell Boyden chamber. Briefly, 0.15 ml of neutrophil suspension (lo7 cells/ml of RPM1 1640 medium) was placed in the upper chamber; the lower chamber contained 0.2 ml of the sample to be tested. The upper and lower compartments were separated by a polycarbonate filter with 2-pm-diameter pores. The Boyden chamber was incubated for 80 min at 37°C in a CO, incubator. After incubation, the number of the PMNs in the lower chamber and the cells adhering to the underside of the filter were counted with a Coulter counter. The migration rate was expressed as the percentage of the total number of cells that had migrated through the filter to the number of PMNs applied to the upper chamber.

62 Treatment

WATANABE

of Rat Mesenteric

Microvascular

ET AL.

Beds with CINClgro

Removal of the rat mesentery from the intraperitoneal space and its unfolding on a plastic stage were performed according to the method of Chambers and Zweifach (1944). Male rats (Wistar, -200 g body wt) were anesthetized intraperitoneally with 30 mg/kg of sodium pentobarbital. The abdomen was opened via a midline incision. The mesentery near the cecum was carefully exposed and mounted on a plastic stage. The preparation was kept warm and continuously superfused with lipopolysaccharide-free saline supplemented with 0.1% human serum albumin. Histological

Study

At 15, 30, and 60 min after treatment with CINC/gro or vehicle, all organs and tissues from the intraperitoneal space were ligated close to the abdominal side and then cut. The organs and tissues were immediately soaked in absolute methanol (-40°C) and allowed to stand for -16-24 hr at -40°C. The mesentery was separated from the small intestine and cecum and soaked in distilled water to wash out debris. The mesentery was spread on a slide glass, dried for about 1 hr, stained with Mayer’s hematoxylin solution for 13 min, and histologically observed. Mayer’s hematoxylin solution stains nuclei of neutrophils and other mononuclear cells dark blue, but does not stain red blood cells having no nuclei. Therefore, blood vessels without adherent nucleated cells were orange and smooth. On the other hand, blood vessels with adherent neutrophils were stained with dark blue and were easily observed. Cells with segmental and polymorphic nuclei were identified as neutrophils. A microvessel with >I0 neutrophils infiltrated near the area (100 x 100 pm) was counted as infiltration. The diameter and length of the blood vessel before and after fixation in cold methanol were measured by a micrometer equipped with a microscope. RESULTS Chemotactic activities of chemically synthesized CINC/gro and purified native CINC/gro for rat neutrophils were investigated. The two types of CINC/gro were chromatographed by reverse-phase HPLC. Each peak area was monitored by absorbance at 220 nm and adjusted to identical concentrations. Synthetic CINC/ gro had a chemoattractant activity comparable to that of the native CINC/gro purified from NRK-52E cell culture medium (Fig. 1). Both CINCs exhibited maximal chemoattraction for neutrophils at a concentration of lo-* M as previously described (Watanabe et a/., 1991). Contraction of the rat mesentery after fixation by cold methanol was studied. The diameter and length of microvascular vessels of the mesentery were measured before and after fixation. The diameters of the microvascular blood vessels were virtually unchanged after fixation without any distinction of the arteriole and venule (Table IA). On the other hand, the length of microvascular vessels decreased slightly, to 88.6% of that before fixation. There was no obvious difference in the extent of contraction between arterioles and venules (Table 1B). We studied the effect of lo-’ M chemically synthesized CINC/gro, which caused maximal neutrophil chemotaxis in vitro (Fig. l), on the microvascular beds of rat mesentery according to the method described under Materials and Methods. As shown in Fig. 2A, the intravascular surfaces remained smooth, without ad-

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MESENTERY

01 0

0.1

1

Concentration,

10

100

loo0

nM

1. Chemotactic activities of synthesized CINC/gro (m) and purified native CINC/gro (Cl). Data are means 2 SE of six determinations. FIG.

hered or infiltrated neutrophils, after super-fusion for 15 min with the vehicle, lipopolysaccharide-free saline containing 0.1% human serum albumin. However, exposure of the mesentery to lo-’ A4 CINUgro for 15 min induced obvious neutrophil adherence to postcapillary venules. Neutrophils adhered to postcapillary venules of -20-30 pm in diameter, but not to arterioles, capillaries, and thick postcapillary venules (-40 pm) (Fig. 2B). Furthermore, the portions frequently adhered to were downstream from the junction of two postcapillary venules. Neutrophils extravasated were not yet observed. Super-fusion with lo-’ M CINUgro for 30 min caused more severe neutrophil adherence to postcapillary venules -40-50 Frn in diameter (Fig. 2C). As shown in TABLE I Changes in Diameter (A) and Length (B) of Microvessels in Mesentery after Fixation by Cold Methanol (A) Diameter (urn)

(B) Length (wd

Before fixation

After fixation

Before fixation

After fixation

Arteriole

20 20 20 45

25 20 20 40

400 200 175 200

350 180 100 180

Venules

35 35 25 30 40 35 30 20 15

35 35 25 30 35 40 35 20 15

100 800 470 120 200 200

80 600 470 120 220 170

Mean

28.5 p,rn

28.8 urn (102.5 f 2.9%)

324 km

288 urn (88.6 2 4.4%)

64

FIG. 2. The histological appearance of rat mesentery after sham or CINCigro treatment. The stimulants, exposure times, and magnifications of rat mesenteries were: (A) 0.1% HSA in saline, 15 min, X100; (B) lo-* M CINC/gro, 15 min, x100; (C) lo-’ M CINC/gro, 30 min, X100; (D) lo-’ M CINC/gro, 30 mitt, x400; (E) 0.1% HSA in saline, 30 mitt, x400; (F) lo-‘M CINC/gro, 30 mitt, x400; (G) 10-s M CINC/gro, 60 mitt, X100; (H) lo-* M CINC/gro, 60 min, x400; (I) 10m9 M CINUgro, 30 min. X100; (J) lo-” M CINC/gro, 30 min, x400. Marker equals 100 pm. cap, capillary; art, arteriole; pcv, postcapillary venule; ad, PCV with adherent neutrophils; arrow, blood vessel junction; open arrow, neutrophils.

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AL.

Fig. 2D, obvious infiltrated neutrophils were observed around the postcapillary venules neutrophils adhering. The intravascular surfaces of control rat mesentery remained smooth (Fig. 2E), although luminal surfaces of the postcapillary venules -40 pm in diameter of treated rat were occupied by numerous neutrophils, identifiable by their polymorphic nuclei (Fig. 2F). Mononuclear cells among adhering leukocytes are unidentifiable. Exposure of the microvascular vessel network of the rat mesentery to 10m8 M of CINC/gro for 60 min induced extravasation of enormous neutrophils. The postcapillary venules with adherent and infiltrated neutrophils extend over vessels of varying thicknesses (Figs. 2G and 2H). We also studied the effect of a lower CINC/gro concentration on the movement of neutrophils in the vascular circulation of the rat mesentery. As shown in Fig. 21, lop9 M CINC/gro caused weaker neutrophil adherence and infiltration than 10d8 M of CINC/gro. A slight adherence of neutrophils to thin postcapillary venules was induced by perfusion with lo- lo M CINC/gro (Fig. 2J), although no change was observed in microvascular beds of mesentery after treatment with 10-l’ M CINUgro for 30 min (data not shown). The diameters of postcapillary venules with adherent neutrophils increased in a time-dependent manner (Fig. 3). Fifteen minutes after the treatment with CINC/ gro, neutrophils adhered to thin postcapillary venules of 24.4 -+ 1.7 p.rn (n = 32) in diameter. These are the thinnest postcapillary venules in which blood cells pass through after passing in capillaries. At 30 and 60 min after exposure to CINC/gro, the mean diameters of the postcapillary venules to which neutrophils adhered increased to 37.3 + 2.1 p.rn (n = 69) and 43.3 f 3.4 pm (n = 93), respectively. On the other hand, few neutrophils were extravasated from or had infiltrated the vessels after exposure to CINUgro for 15 min. Prolonged exposure to CINC/

Time,

min.

FIG. 3. Diameters of the postcapillary venules with adherent or extravasated neutrophils. Diameters of the postcapillary venules with adherent (0) or extravasated (0) neutrophils after exposure of the mesentery to lo-* M CINC/gro for 15, 30, and 60 min.

EFFECT OF CINC/GRO ON LEUKOCYTES

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67

gro for 30 and 60 min caused neutrophil extravasation from the postcapillary venules with adherent neutrophils. The mean values were 27.7 ? 1.3 pm (n = 93) and 35.6 f 1.6 km (n = 82), respectively (Fig. 3). The mean values were significantly lower than those of vessels to which neutrophils had adhered within the same periods. The distances from the upper junctions of two blood vessels to the areas of neutrophil adherence were measured. The front end, the start of neutrophil adherence, was almost always about 25-50 km from the junction, regardless of the exposure time (Fig. 4). The tail end, which is the end of adherence, changed to downstream and the distances from the upper junction increased in a timedependent manner. The adherence center, the site of severest adhesion, gradually shifted downstream. DISCUSSION The chemotactic activity of synthesized CINC/gro for rat neutrophils was comparable to that of native CINC/gro purified from the conditioned medium of NRK52E cells (Fig. 1). These data support the accuracy of the amino acid sequence of CINC/gro reported previously (Watanabe et al., 1989b). There was little contraction of the rat mesentery after fixation in cold methanol. The thickness and the length of the vessels were almost unchanged and decreased by about 10% (Table I). Therefore, the thickness values and the distances from the upperstream junctions of postcapillary venules are comparable to those before fixation. In this study, we use the terms “adherence” and “adherent neutrophils” to indicate that neutrophils make contact with blood vessels. However, there may be also subendothelial lodging neutrophils, locating in a space between endothelial cell layer and basement membrane. It is very difficult to determine whether neutrophils stick to endothelium or locate in a subendothelial space from the obser-

/ /

,..” *....*

tail end

C

f

,..”

/ ,..” & adhering

-

P----,,l_ 0’ 0

15

o”“‘---------30 Time,

front 45

end

center

-4 60

min.

FIG. 4. Distances from the upper junction of two blood vessels to the site of neutrophil adherence. The front end (O), which is the start of adherence; rear end (A), which is the end point of adherence; and the adherence center (O), the site of the most severe adherence.

68

WATANABE

ET AL.

vation of mesentery with a microscope. Therefore, the term “adherent neutrophils” includes adherent and subendothelial lodging neutrophils in this study. Rat CINC/gro (lo-* M) caused neutrophil adherence to the postcapillary venules within 15 min. This concentration causes maximal neutrophil chemotaxis in vitro (Fig. 1). The thickness of the postcapillary venules with adherent neutrophils, although only about -20-30 pm in diameter (Fig. 3), increased in a timedependent manner. The mean value increased to 43.3 p,rn in diameter, after exposure for 60 min to CINC/gro. Such venules contained the thickest venules (-60-100 urn in diameter) in the rat mesentery. Extravasated neutrophils were not observed during treatment with CINC/gro for the first 15 min. But prolonged exposure to CINC/gro caused neutrophil extravasation from the postcapillary venules to which they had adhered. The mean diameter of postcapillary venules from which neutrophils had extravasated also increased in a time-dependent manner. The mean values were significantly lower than those of vessels with adherent neutrophils at the same period (Figs. 3 and 4). This is because the process of neutrophil extravasation after adherence to endothelial cells requires several minutes. The areas to which neutrophils easily adhered were -25-50 Frn away from the upperjunction of two vessels (Fig. 4). These sites were virtually unchanged during treatment with CINClgro for 60 min. The reason that neutrophils adhere first to such sites may be that blood flow is disturbed by the confluence of two streams. There are probably places where the blood flow slows locally and/or transiently, in which neutrophils can adhere to endothelial cells by overcoming shear stress. Why the area of neutrophil adherence increased in a time-dependent manner is yet unknown (Fig. 4). However, this phenomenon may be explained by the theory that neutrophils migrated into the subendothelial space and then expanded the space by degranulating type IV collagenase and elastase (Masure et al., 1991). Neutrophils did not adhere to arterioles and capillaries; therefore, the adherence caused by CINC/gro was specific for postcapillary venules in the same manner as in other chemotactic factors such as LTB, and C5a (Bray et al., 1981; Arfors et al., 1987). Furthermore, those leukocytes which adhered to venules and extravasated were solely neutrophils and not lymphocytes or monocytes. This is consistent with a previous report describing rat CINC/gro as a neutrophil-specific chemoattractant in vivo. However, Larsen et al. (1989) reported that human IL-8 attracts not only neutrophils but also T-lymphocytes. There are two kinds of human peptides with strong neutrophil chemoattractant properties, IL-8 and gro/MGSA, which have high sequence homology to each other. However, the expression of IL-8 is more prominent than that of gro/MGSA in human cells (Schriider et al., 1989, 1990). On the other hand, rat cells express CINCYgro, the rat equivalent of gro/MGSA, but a rat IL-8 counterpart has not yet been identified despite our efforts to do so (data not shown). It is therefore possible that the rat does not have an IL-8 counterpart but only CINC/gro, which correlates with human gro/MGSA. Rat CINUgro is a member of the family that includes human IL-8 and gro/ MGSA, and it has a high sequence homology to the two human peptides. The rat CINUgro receptor on neutrophils is probably very similar to that of human IL-8 and gro/MGSA, but the potency of human IL-8 and gro/MGSA to attract rat neutrophils is at least one order of magnitude less than that of rat CINC/gro.

EFFECT

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CINC/GRO

ON

Therefore, the best way to investigate member in rats is to use CINUgro.

LEUKOCYTES

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the pathologic

69

roles of an IL-8 family

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LARSEN,C.G., ANDERSON, A.O., APPELLA,E.,OPPENHEIM, J.J.,and MATSUSHIMA,K. (1989a). The neutrophil-activating protein (NAP-l) is also chemotactic for T lymphocytes. Science 243, 1464-1466. LARSEN, C. G., ANDERSON, A. O., OPPENHEIM, J. J., and MATSUSHIMA, K. (1989b). Production of interleukin-8 by human dermal tibroblasts and keratinocytes in response to interleukin-1 or tumor necrosis factor. Immunology 68, 31-36. MASURE, S., PROOST, P., DAMME, J. V., and OPDENAKKER, G. (1991). Purification and identification of 91-kDa neutrophil gelatinase. Release by the activating peptide interleukin-8. Eur. .Z. Biochem. 198, 391-398. RICHMOND, A., and THOMAS, H. G. (1988). Melanoma growth stimulatory activity: isolation from human melanoma tumors and characterization of tissue distribution. .l. Cell. Biochem. 36, 185-198. SCHR~DER, J-M., YOUNG, J., GREGORY, H., and CHRISTOPHERS, E. (1989). Amino acid sequence characterization of two structurally related neutrophil activating peptides obtained from lesional psoriatic scales. J. Invest. Dermatol. 92, 515 [Abstract]. SCHR~DER, J-M., PERSOON, N. L. M., and CHRISTOPHERS,E. (1990). Lipopolysaccharide-stimulated human monocytes secrete, apart from neutrophil-activating peptide-l/Interleukin 8, a second neutrophil-activating protein. J. Exp. Med. 171, 1091-1100. WALTZ, A., PEVERI, P., ASCHAUER, H., and BAGGIOLINI, M. (1987). Purification and amino acid sequencing of NAF, a novel neutrophil-activating factor produced by monocytes. Biochem. Biophys. Res. Common. 149, 755-761. WATANABE, K., NAKAGAWA, H., and TSURUFUJI, S. (1985). A new simple plastic chemotaxis device of the Boyden chamber type utilizing an immunoassay plate. Jpn. J. Pharmacol. 39, 102-104. WATANABE, K., and NAKAGAWA, H. (1987). Production of chemotactic factor for polymorphonuclear leukocytes by epithelioid cells from rat renal glomeruli in culture. Biochem. Biophys. Res. Commun. 149, 989-994. WATANABE, K., KINOSHITA, S., and NAKAGAWA, H. (1989a). Purification and characterization of cytokine-induced neutrophil chemoattractant produced by epithelioid cell line of normal rat kidney (NRK-52E cell). Biochem. Biophys. Res. Commun. 161, 1093-1099. WATANABE, K., KIYOSHI, K., FUJIOKA, M., KINOSHITA, S., and NAKAGAWA, H. (1989b). The neutrophil chemoattractant produced by the rat kidney epithelioid cell line NRK-52E is a protein related to the KClgro protein. J. Biol. Chem. 264, 19,55919,563. WATANABE, K., KINOSHITA, S., and NAKAGAWA, H. (1989~). Very rapid assay of polymorphonuclear leukocyte chemotaxis in vitro. J. Pharmacol. Methods 22, 13-18. WATANABE, K., and NAKAGAWA, H. (1990). Cytokines enhance the production of chemotactic factor for polymorphonuclear leukocytes by renal glomerular epithelioid cells. Nephron 54, 169-175. WATANABE, K., KOIZUMI, F., KURASHIGE, Y., TSURUFUJI, S., and NAKAGAWA, H. (1991). Rat CINC, a member of the interleukin-8 family, is a neutrophil-specific chemoattractant in Vivo. Exp. Mol.

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WOLPE, S. D., and CERAMI, A. (1989). Macrophage inflammatory proteins 1 and 2: Members of a novel superfamily of cytokines. FASEB 3, 2565-2573.