The Cutaneous Reverse Arthus Reaction Requires Intercellular Adhesion Molecule 1 and L-Selectin Expression

This information is current as Yuko Kaburagi, Minoru Hasegawa, Tetsuya Nagaoka, Yuka of September 27, 2021. Shimada, Yasuhito Hamaguchi, Kazuhiro Komura, Eriko Saito, Koichi Yanaba, Kazuhiko Takehara, Takafumi Kadono, Douglas A. Steeber, Thomas F. Tedder and Shinichi Sato

J Immunol 2002; 168:2970-2978; ; Downloaded from doi: 10.4049/jimmunol.168.6.2970 http://www.jimmunol.org/content/168/6/2970

References This article cites 56 articles, 32 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/168/6/2970.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Cutaneous Reverse Arthus Reaction Requires Intercellular Adhesion Molecule 1 and L-Selectin Expression1

Yuko Kaburagi,* Minoru Hasegawa,* Tetsuya Nagaoka,* Yuka Shimada,* Yasuhito Hamaguchi,* Kazuhiro Komura,* Eriko Saito,* Koichi Yanaba,* Kazuhiko Takehara,* Takafumi Kadono,† Douglas A. Steeber,† Thomas F. Tedder,† and Shinichi Sato2*

The deposition of immune complexes (IC) induces an acute inflammatory response with tissue injury. IC-induced inflammation is mediated by inflammatory cell infiltration, a process highly regulated by expression of multiple adhesion molecules. To assess the role of L-selectin and ICAM-1 in this pathogenetic process, the cutaneous reverse passive Arthus reaction was examined in mice lacking ؊/؊ ؊/؊ ؊/؊

L-selectin (L-selectin ), ICAM-1 (ICAM-1 ), or both (L-selectin/ICAM-1 ). Edema and hemorrhage, which peaked 4 and 8 h Downloaded from after IC challenge, respectively, were significantly reduced in L-selectin؊/؊, ICAM-1؊/؊, and L-selectin/ICAM-1؊/؊ mice compared -with wild-type littermates. In general, edema and hemorrhage were more significantly inhibited in ICAM-1؊/؊ mice than in L-selec .tin؊/؊ mice, but were most significantly reduced in L-selectin/ICAM-1؊/؊ mice compared with ICAM-1؊/؊ or L-selectin؊/؊ mice Decreased edema and hemorrhage correlated with reduced neutrophil and mast cell infiltration in all adhesion molecule-deficient mice, but leukocyte infiltration was most affected in L-selectin/ICAM-1؊/؊ mice. Reduced neutrophil and mast cell infiltration was also observed for all mutant mice in the peritoneal Arthus reaction. Furthermore, cutaneous TNF-␣ production was inhibited in each http://www.jimmunol.org/ deficient mouse, which paralleled the reductions in cutaneous inflammation. These results indicate that ICAM-1 and L-selectin coop- eratively contribute to the cutaneous Arthus reaction by regulating neutrophil and mast cell recruitment and suggest that ICAM-1 and L-selectin are therapeutic targets for human IC-mediated disease. The Journal of Immunology, 2002, 168: 2970–2978.

he formation and local deposition of immune complexes ies have addressed the contribution of leukocyte accumulation to (IC)3 induces an acute inflammatory response with sig- the effector phase of the reaction. T nificant tissue injury. IC injury is implicated in a variety Leukocyte recruitment into inflammatory sites is achieved using of human diseases, including vasculitis syndrome, systemic lupus distinct constitutive or inducible families of cell adhesion mole- by guest on September 27, 2021 erythematosus, rheumatoid arthritis, Goodpasture’s syndrome, and cules (11Ð13). L-selectin (CD62L) which primarily mediates leu- glomerulonephritis (1). The classical experimental model for IC- kocyte capture and rolling on the endothelium is constitutively mediated tissue injury is the Arthus reaction, which is character- expressed by most leukocytes (14, 15). In vitro, L-selectin binds ized by edema, hemorrhage, and neutrophil infiltration (2). Cell- several glycosylated mucin-like proteins expressed by high endo- ␥ bound Fc receptors for IgG (Fc Rs) play a central role in the thelial venules (15). Cytokine-inducible ligands for L-selectin have initiation of IC-triggered inflammation (3, 4). Specifically, the FcR also been described for peripheral endothelial cells, but their iden- type III (Fc␥RIII, CD16) on mast cells initiates the cutaneous Ϫ/Ϫ w W-v tity remains unknown (16Ð18). L-selectin mice demonstrate Arthus reaction since mast cell-deficient Kit /Kit mice and decreased trauma- and TNF-␣-induced rolling of leukocytes, de- Fc␥RIII-deficient (Fc␥RIIIϪ/Ϫ ) mice exhibit substantially reduced creased leukocyte recruitment into an inflamed peritoneum, de- inflammation (5Ð9). C5aR expression is also required for normal creased delayed-type hypersensitivity responses, delayed rejection neutrophil influx and edema formation during the cutaneous of allogeneic skin transplants, and resistance to LPS -induced sep- Arthus reaction (10). Although Fc␥R and C5aR are codominant tic shock (19Ð25). ICAM-1 (CD54) is constitutively expressed at receptors in the initiation of a cutaneous Arthus reaction, few stud- low levels by endothelial cells and is rapidly up-regulated during inflammation, resulting in increased leukocyte-endothelial cell ad- ␤ hesion (26). Leukocytes express 2 integrins, including LFA-1 *Department of Dermatology, Kanazawa University Graduate School of Medical Sci- (CD11a/CD18), which interact with ICAM-1. ICAM-1-␤ integrin ence, Kanazawa, Japan; and †Department of Immunology, Duke University Medical 2 Center, Durham, NC 27710 interactions promote leukocyte rolling, but also mediate firm ad- Received for publication October 16, 2001. Accepted for publication January hesion and the transmigration of leukocytes at sites of inflamma- 11, 2002. tion (13, 27). ICAM-1Ϫ/Ϫ mice have significantly reduced num- The costs of publication of this article were defrayed in part by the payment of page bers of infiltrating neutrophils during peritonitis, reduced charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. susceptibility to LPS-induced septic shock, delayed skin wound repair, and impaired delayed-type hypersensitivity reactions, al- 1 This work was supported by a Grant-in-Aid from the Ministry of Education, Sci- ence, and Culture of Japan (to S.S.) and National Institutes of Health Grants CA54464 though allogeneic skin graft rejection is normal (20, 28Ð30). Re- and CA81776 (to T.F.T.). cent studies using L-selectin/ICAM-1Ϫ/Ϫ mice demonstrate a di- 2 Address correspondence and reprint requests to Dr. Shinichi Sato, Department of rect role for ICAM-1 in leukocyte rolling as the frequency of Dermatology, Kanazawa University Graduate School of Medical Science, 13-1 Ϫ/Ϫ ␣ Takaramachi, Kanazawa, Ishikawa 920-8641, Japan. E-mail address: s-sato@ rolling leukocytes in L-selectin mice treated with TNF- is med.kanazawa-u.ac.jp decreased significantly by the additional loss of ICAM-1 expres- 3 Abbreviation used in this paper: IC, immune complex. sion (27). Furthermore, the loss of both L-selectin and ICAM-1

Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 The Journal of Immunology 2971 expression reduces leukocyte recruitment into sites of inflamma- tissue section was weighed. The amount of hemorrhage was assessed 8 h tion beyond what is observed with loss of either receptor alone (30, after IC challenge by direct macroscopic measurement of the purpuric spot. 31). Therefore, L-selectin and ICAM-1 mediate optimal leukocyte The diameter of the major and minor axis of the blue spot was averaged for analysis. accumulation during inflammation through overlapping as well as synergistic functions. Histological examination and immunohistochemical staining Neutrophils and mast cells are the primary effector cells in the Tissues were fixed in 3.5% paraformaldehyde and then paraffin embedded. cutaneous Arthus reaction (1, 7, 8, 32). Despite this, studies in- Six-micrometer sections were stained using H&E for neutrophil evaluation vestigating the role of adhesion molecules mediating IC-induced and toluidine blue for mast cell staining. Extravascular neutrophils and neutrophil migration are limited. P-selectin expression is detected mast cells were counted in the entire section. For immunohistochemistry, along vessel walls before neutrophil accumulation in the rat cuta- tissue sections of skin biopsies were acetone fixed and then incubated with neous Arthus reaction, with neutrophil accumulation inhibited by 10% normal rabbit serum in PBS (10 min, 37¡C) to block nonspecific staining. Sections were then stained with rat mAbs specific for mouse mAb against P-selectin (33). Similarly, pretreatment with anti- ICAM-1 (Beckman Coulter, Miami, FL) as described previously (30). Rat CD18 mAb inhibits neutrophil influx in the cutaneous Arthus re- IgG (Southern Biotechnology Associates, Birmingham, AL) was used as a action of rat and rabbit (34, 35). Although a critical role for L- control for nonspecific staining. Sections were then incubated sequentially selectin and ICAM-1 has been demonstrated in various (20 min, 37¡C) with biotinylated rabbit anti-rat IgG secondary Abs (Vec- tastain avidin-biotin complex method; Vector Laboratories, Burlingame, inflammatory models (27, 30, 31), it is unknown whether L-selec- CA), then HRP-conjugated avidin-biotin complexes (Vectastain ABC tin and ICAM-1 contribute to the cutaneous Arthus reaction by method; Vector Laboratories). Sections were finally developed with 3,3Ј- mediating leukocyte recruitment in vivo. For this purpose, we an- diaminobenzidine tetrahydrochloride and hydrogen peroxide and counter- alyzed inflammation induced by IC in mice lacking either L-se- stained with methyl green. Downloaded from lectin, ICAM-1, or both receptors. The results demonstrate that Flow cytometric analysis ICAM-1 and L-selectin cooperatively contribute to IC-induced skin injury by regulating the accumulation of mast cells and After lavage fluid recovery, it was immediately placed on ice to inhibit the neutrophils. endoproteolytic release of cell surface L-selectin. Isolated peritoneal lavage cells (0.5 ϫ 106) were stained using predetermined optimal concentrations of either anti-c-Kit-FITC Ab (CD117, clone 2B8; BD PharMingen, San Materials and Methods Diego, CA) or anti-Gr-1-FITC Ab (clone RB6-8C5; BD PharMingen) plus http://www.jimmunol.org/ Mice either anti-CD18-PE Ab (clone C71/16; Beckman Coulter) or anti- L-selectin-PE Ab (clone MEL-14; Beckman Coulter) for 20 min at 4¡Cas L-selectinϪ/Ϫ mice were produced as described previously (19). ICAM- Ϫ/Ϫ described elsewhere (37, 38). Cells were washed and analyzed on a FAC- 1 mice (28) expressing residual amounts of ICAM-1 splice variants in Scan flow cytometer (BD PharMingen) by gating on c-Kit-positive mast the thymus and spleen but not in other organs, including skin (36), were cells or Gr-1-positive granulocytes. Positive and negative populations of obtained from The Jackson Laboratory (Bar Harbor, ME). Mice lacking cells were determined using unreactive isotype-matched mAbs (Beckman both L-selectin and ICAM-1 were generated as described elsewhere (27). Coulter) as controls for background staining. All mice were healthy, fertile, and did not display evidence of infection or disease. All mice were backcrossed between 5 and 10 generations onto the RT-PCR and real-time PCR C57BL/6 genetic background. Mice used for experiments were 12Ð16 wk old. Age-matched wild-type littermates and C57BL/6 mice (The Jackson Total RNA was isolated from frozen skin tissues using a RNA PCR kit by guest on September 27, 2021 Laboratory) were used as controls with equivalent results so all control according to the manufacturer’s instructions (Promega, Madison, WI). results were pooled. All mice were housed in a specific pathogen-free RNA yield and purity were determined by spectrophotometry. RNA was barrier facility and screened regularly for pathogens. All studies and pro- then reverse transcribed into cDNA and amplified. Amplification was per- cedures were approved by the Committee on Animal Experimentation of formed in a PCR thermal cycler MP (Takara, Kusatsu, Japan) for 30 cycles Kanazawa University School of Medicine. of denaturation at 94¡C for 30 s, annealing at 60¡C for 45 s, and extension at 72¡C for 60 s. The final extension was performed for 10 min and then Reverse passive Arthus reactions for 5 min at 5¡C. The sense primer for mouse TNF-␣ was 5Ј-AGC CCA CGT AGC AAA CCA CCA A-3Ј and the antisense primer was 5Ј-ACA For cutaneous Arthus reactions, mice anesthetized by inhalation of diethyl Ј ether were shaved on their dorsal skin and wiped with 70% alcohol. Rabbit CCC ATT CCC TTC ACA GAG CAA T-3 (Bex, Tokyo, Japan). The ␮ ␮ sense primer for ␤-actin was 5Ј-GTG GGG CGC CCC AGG CAC CA-3Ј IgG anti-chicken egg albumin Abs (60 g/30 l; Cappel, Aurora, OH) Ј Ј were injected intradermally with a 29-gauge needle, followed immediately and the antisense primer was 5 -GCT CGG CCG TGG TGG TGA AGC-3 thereafter by i.v. injection of chicken egg albumin (20 mg/kg; Sigma-Al- (Bex). The PCR products were electrophoresed on a 2% agarose gel and drich, St. Louis, MO) (10). The intradermal injection of purified polyclonal stained with ethidium bromide. rabbit IgG (60 ␮g/30 ␮l; Sigma-Aldrich) followed by i.v. installation of Real-time PCR was performed as previously described (39). Briefly, chicken egg albumin served as a control. The solution of chicken egg RNA was isolated and reverse transcribed as described above. The level of TNF-␣ mRNA was determined by real-time PCR using the predeveloped albumin contained 1% Evans blue dye (Sigma-Aldrich). Tissues were har- ␣ vested 4 or 8 h later and assessed for edema, hemorrhage, and numbers of TaqMan probe and primers to TNF- (Applied Biosystems, Foster City, infiltrating neutrophils and mast cells. CA) on a model 7700 Applied Biosystems Prism Sequence Detector (Ap- plied Biosystems). The 18S rRNA endogenous control (Applied Biosys- The peritoneal reverse passive Arthus reaction was initiated by the i.v. ␣ injection of chicken egg albumin at 20 mg/kg, followed immediately by the tems) was used to normalize RNA. The TNF- mRNA level in wild-type i.p. injection of 800 ␮g of rabbit IgG anti-chicken egg albumin Ab or littermates was used as the calibrator. Real-time RT-PCR assays were con- control purified rabbit polyclonal IgG in a volume of 400 ␮l. Four or 8 h ducted in triplicate for each sample. later, the peritoneum was exposed by a midline abdominal incision, and 5 ml of ice-cold PBS containing 0.1% BSA was injected into the peritoneal Statistical analysis cavity via a 27-gauge needle. Cells in the recovered lavage fluid were The Mann-Whitney U test was used for determining the level of signifi- centrifuged onto glass slides and stained with Giemsa for light microscopic cance of differences in sample means and Bonferroni’s test was used for examination to quantify neutrophil and mast cell numbers. multiple comparisons. Quantitation of edema and hemorrhage Results Edema was evaluated by two methods 4 h after IC challenge (10). First, the diameter of extravascular Evans blue dye on the reverse side of the injec- Edema and hemorrhage in the cutaneous reverse passive Arthus tion site was measured directly. Evans blue dye binds to serum proteins and reaction thereby can be used to quantify alterations in vascular permeability. The diameter of the major and minor axis of the blue spot was averaged for Cutaneous inflammation induced by an Arthus reaction can be sep- analysis. Second, the injection area or a control site was removed using a arated into two distinct responses: edema, which reaches a maxi- disposable sterile 6-mm punch biopsy (Maruho, Osaka, Japan), and each mum at 3Ð4 h after IC challenge, and hemorrhage, which peaks in 2972 ICAM-1 AND L-SELECTIN IN THE ARTHUS REACTION intensity at 8 h (3). Therefore, edema and hemorrhage were eval- p Ͻ 0.05), ICAM-1Ϫ/Ϫ (43Ð54%, p Ͻ 0.05), and L-selectin/ uated 4 and 8 h after IC challenge, respectively, in L-selectinϪ/Ϫ, ICAM-1Ϫ/Ϫ mice (54%, p Ͻ 0.005) compared with wild-type ICAM-1Ϫ/Ϫ, and L-selectin/ICAM-1Ϫ/Ϫ mice compared with mice. The added loss of L-selectin in ICAM-1Ϫ/Ϫ mice did not wild-type littermates. When edema was assessed by measuring the dramatically affect neutrophil accumulation compared with ICAM- diameter of Evans blue dye in the extravascular space, edema was 1Ϫ/Ϫ mice at either time point. Mast cell numbers were also as- significantly reduced in L-selectinϪ/Ϫ (31% decrease, p Ͻ sessed in skin tissue sections stained with toluidine blue (Figs. 2A 0.0001), ICAM-1Ϫ/Ϫ (43%, p Ͻ 0.0001), and L-selectin/ICAM- and 4). Before IC challenge, there were no significant differences 1Ϫ/Ϫ mice (51%, p Ͻ 0.0001) compared with wild-type littermates in mast cell numbers between mutant and wild-type littermates. By (Fig. 1A). ICAM-1Ϫ/Ϫ mice exhibited significant inhibition of dye contrast, 4 h after IC challenge, mast cell numbers were signifi- vascular leak when compared with L-selectinϪ/Ϫ mice ( p Ͻ 0.01), cantly reduced in L-selectinϪ/Ϫ (43% decrease, p Ͻ 0.0001), whereas the loss of both ICAM-1 and L-selectin resulted in a sig- ICAM-1Ϫ/Ϫ (52%, p Ͻ 0.0001), and L-selectin/ICAM-1Ϫ/Ϫ (60%, nificant further reduction of dye vascular leak relative to the L- p Ͻ 0.0001) mice compared with wild-type littermates. Similar selectin loss alone ( p Ͻ 0.001). Similar results were obtained results were obtained after8hofICformation. Mast cell numbers when edema was evaluated as the wet weight of skin biopsies from did not significantly increase in L-selectin/ICAM-1Ϫ/Ϫ mice after the site of IC formation (Fig. 1B). No edema was detected in mu- IC challenge: the additional loss of ICAM-1 in L-selectinϪ/Ϫ mice tant mice or their wild-type littermate controls following intrader- resulted in significantly reduced mast cell numbers relative to mal injection of rabbit polyclonal IgG with systemic chicken egg L-selectinϪ/Ϫ mice after both 4 and8h(p Ͻ 0.05), while mast cell albumin (Fig. 1A and data not shown). Thus, L-selectin loss re- accumulation was significantly diminished in L-selectin/ICAM- duced the early cellular response characterized by edema, with 1Ϫ/Ϫ mice compared with ICAM-1Ϫ/Ϫmice ( p Ͻ 0.01) after 8 h. Downloaded from ICAM-1 deficiency inhibiting edema beyond that found with L- Thus, the loss of either L-selectin or ICAM-1 expression signifi- selectin deficiency. cantly reduced leukocyte accumulation, but the combined loss of Hemorrhage was macroscopically quantitated after8hbymea- L-selectin and ICAM-1 led to greater reductions in leukocyte ac- suring the size of the purpuric spot. Hemorrhage was significantly cumulation than the loss of each molecule alone. inhibited in L-selectinϪ/Ϫ (45% decrease, p Ͻ 0.002), ICAM-1Ϫ/Ϫ Ϫ/Ϫ (48%, p Ͻ 0.0005), and L-selectin/ICAM-1 mice (64%, p Ͻ Leukocyte infiltration in the peritoneal Arthus reaction http://www.jimmunol.org/ 0.0001) compared with wild-type littermates (Fig. 1C). L-selectin/ The i.p. injection of Ab with the i.v. injection of Ag elicits a re- Ϫ Ϫ ICAM-1 / mice exhibited significantly reduced hemorrhage verse passive Arthus reaction characterized by leukocyte influx Ϫ Ϫ Ϫ Ϫ compared with both ICAM-1 / ( p Ͻ 0.05) and L-selectin / into the peritoneal cavity (1). After4hofICchallenge, neutrophil mice ( p Ͻ 0.05). Hemorrhage was not detected in mutant mice or numbers in the peritoneal cavity were significantly reduced in their wild-type littermate controls following intradermal injection L-selectinϪ/Ϫ (67% decrease, p Ͻ 0.05), ICAM-1Ϫ/Ϫ (91%, p Ͻ of rabbit polyclonal IgG with systemic chicken egg albumin (Fig. 0.05), and L-selectin/ICAM-1Ϫ/Ϫ mice (67%, p Ͻ 0.01) relative to 1C and data not shown). Therefore, the combined loss of L-selec- wild-type littermates (Fig. 2B). After 8 h, neutrophil influx re- tin and ICAM-1 resulted in a greater inhibition of edema and hem- mained significantly inhibited in both ICAM-1Ϫ/Ϫ (67%, p Ͻ by guest on September 27, 2021 orrhage than the loss of each adhesion molecule alone. 0.02) and L-selectin/ICAM-1Ϫ/Ϫ mice (53%, p Ͻ 0.01) compared with wild-type littermates. Mast cell recruitment was also signif- Leukocyte infiltration in the cutaneous Arthus reaction icantly reduced in L-selectinϪ/Ϫ (42% decrease, p Ͻ 0.01), ICAM- Extravascular neutrophils were assessed in skin tissue sections af- 1Ϫ/Ϫ (64%, p Ͻ 0.0001), and L-selectin/ICAM-1Ϫ/Ϫ mice (68%, ter4and8hofICformation (Figs. 2A and 3). Neutrophil numbers p Ͻ 0.0001) after4hofICchallenge relative to wild-type litter- were significantly reduced in L-selectinϪ/Ϫ (33Ð34% decrease, mates (Fig. 2B). Similar differences were detected after 8 h. The

FIGURE 1. Edema and hemorrhage in the cutaneous reverse passive Arthus reaction. Mice were injected intradermally with rabbit IgG anti-chicken egg albumin Ab, followed by systemic chicken egg albumin and 1% Evans blue dye. Dorsal skins were harvested from mutant mice and wild-type (WT) littermates after 4 or 8 h. Edema was evaluated as the diameter of the Evans blue spot (A) as well as the wet weight of a 6-mm punch biopsy (B). Wild-type littermates that received an intradermal injection of polyclonal rabbit IgG followed by i.v. installation of chicken egg albumin served as controls. Hemorrhage after 8 h was assessed as the diameter of the purpuric spot (C). Edema and hemorrhage were significantly inhibited in L-selectinϪ/Ϫ, ICAM-1Ϫ/Ϫ, and L-selectin/ICAM-1Ϫ/Ϫ mice compared with wild-type littermates for all panels (p Ͻ 0.005). Horizontal bars indicate mean values for each group of mice. The Journal of Immunology 2973

FIGURE 2. Arthus reaction-induced recruit- ment of neutrophils and mast cells in the skin (A) and the peritoneum (B) from mutant and wild- type littermates at 4 and 8 h after IC challenge. Numbers of neutrophils and mast cells per skin section were determined by counting H&E- and toluidine blue-stained skin sections, respectively. The peritoneal reverse passive Arthus reaction was induced by the i.v. injection of chicken egg Downloaded from albumin, followed immediately by the i.p. injec- tion of rabbit IgG anti-chicken egg albumin Ab. Cells in the recovered lavage fluid were then cen- trifuged onto glass slides and stained with Giemsa to quantify neutrophil and mast cell numbers. All values represent the mean Ϯ SEM of results ob-

tained from 5 to 10 mice in each group. Statistical http://www.jimmunol.org/ analysis is provided in Results. by guest on September 27, 2021

additional loss of ICAM-1 in L-selectinϪ/Ϫ mice led to signifi- detected after4hinmice injected with control Ab (Fig. 5A and cantly reduced mast cell numbers compared with L-selectinϪ/Ϫ data not shown). Thus, reduced cutaneous inflammatory responses mice after 4 and8h(p Ͻ 0.05). By contrast, there was no leu- in each adhesion molecule-deficient mouse correlated with re- kocyte influx in mutant mice or their control littermates following duced TNF-␣ gene transcription. i.p. injection of rabbit polyclonal IgG with systemic chicken egg albumin (data not shown). Thus, the effect of the loss of each adhesion molecule on leukocyte recruitment in the peritoneal Expression of L-selectin and CD18 on peritoneal mast cells Arthus reaction was similar to that observed in the cutaneous Reduced Arthus reaction-induced mast cell accumulation in adhe- Arthus reaction. sion molecule-deficient mice suggests a role for L-selectin and ICAM-1 in mast cell recruitment. Therefore, mouse peritoneal ␣ TNF- production mast cells expressing c-Kit (38, 41) were analyzed for cell surface ␣ ␤ IC stimulate the production and release of TNF- from infiltrating L-selectin and/or CD18 ( 2 integrin) expression by flow cytom- leukocytes (8, 10, 32, 40), which is detected 1Ð5 h after the initi- etry. Significant L-selectin expression on the surface of c-Kit-pos- ation of a peritoneal Arthus reaction but not after 6 h (40). To itive mast cells from wild-type mice was detected when compared assess the involvement of TNF-␣ in the cutaneous Arthus reaction, with mast cells from L-selectinϪ/Ϫ mice (Fig. 6A) or staining using TNF-␣ mRNA levels were examined in the skin after4hbyRT- an unreactive isotype-matched control mAb (data not shown). PCR (Fig. 5A) and were quantitated by real-time PCR (Fig. 5B). CD18 was also expressed on mast cells from wild-type mice com- TNF-␣ mRNA levels were up-regulated in skin from wild-type pared with staining using an unreactive isotype-matched mAb and in each adhesion molecule-deficient mouse after 4 h (Fig. 5A). (Fig. 6A). As a positive control, granulocytes expressed significant However, TNF-␣ mRNA levels were significantly decreased in levels of both L-selectin and CD18 (Fig. 6B). Deficiency of L-selectinϪ/Ϫ, ICAM-1Ϫ/Ϫ, and L-selectin/ICAM-1Ϫ/Ϫ mice rel- ICAM-1 did not influence L-selectin or CD18 expression by mast ative to their wild-type littermates ( p Ͻ 0.0001, Fig. 5B). ICAM- cells or granulocytes (data not shown). Similarly, the loss of L- 1Ϫ/Ϫ and L-selectin/ICAM-1Ϫ/Ϫ mice exhibited similar TNF-␣ selectin did not alter CD18 expression on either of these cell pop- mRNA levels that were significantly lower than those of L-selec- ulations (data not shown). Thus, L-selectin and CD18 were both tinϪ/Ϫ mice ( p Ͻ 0.001). By contrast, TNF-␣ production was not highly expressed on the surface of mouse peritoneal mast cells. 2974 ICAM-1 AND L-SELECTIN IN THE ARTHUS REACTION Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Histologic tissue sections showing neutrophil infiltration in the skin of mutant and wild-type littermates at4h(A) and 8 h (B) after IC challenge. Neutrophils were revealed by H&E staining. Original magnification, ϫ100.

ICAM-1 expression mice (data not shown). In addition, the loss of L-selectin expres- ICAM-1 expression on various types of cells, including keratino- sion did not affect ICAM-1 expression in either the intact or in- cytes and fibroblasts, is induced by stimulation with proinflamma- flamed skin (data not shown). Thus, ICAM-1 was predominantly tory cytokines in vitro (26, 42). Thus, the loss of ICAM-1 expres- expressed on cutaneous endothelial cells. sion on fibroblasts and keratinocytes may contribute to the reduced inflammation observed in ICAM-1Ϫ/Ϫ mice. To assess this, cuta- neous ICAM-1 expression during the Arthus reaction was exam- Discussion ined immunohistochemically. In normal skin, ICAM-1 was de- In the present study, the loss of ICAM-1, L-selectin, or both sig- tected exclusively on endothelial cells (Fig. 7A), with up-regulated nificantly inhibited edema and vascular hemorrhage after IC chal- ICAM-1 expression by endothelial cells 4 h after IC induction lenge (Fig. 1). The extent that L-selectin and/or ICAM-1 deficien- (Fig. 7B). However, ICAM-1 expression could not be accurately cies influenced inflammation varied at different time points, with assessed on endothelial cells after 8 h since large numbers of in- each cell type, and in different tissues (Figs. 1 and 2). This suggests flammatory cells infiltrating the vessel wall obscured ICAM-1 that the repertoire of functional adhesion molecules mediating IC- staining (Fig. 7C). ICAM-1 expression was not detected on kera- mediated inflammation changes during the course of inflammation tinocytes, fibroblasts, or infiltrating inflammatory cells after 4 or and is influenced by tissue site. Edema and hemorrhage were gen- 8 h (Fig. 7, B and C, and data not shown). ICAM-1 expression was erally inhibited to a larger extent by ICAM-1 deficiency after IC not detected in either the intact or inflamed skin from ICAM-1Ϫ/Ϫ formation when compared with L-selectin deficiency (Fig. 1). The Journal of Immunology 2975 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Histologic tissue sections showing mast cell accumulation in the skin of mutant and wild-type littermates at4h(A)and8h(B) after IC challenge. Mast cells (arrows) were detected as cells with metachromatic staining of granules in toluidine blue-stained sections. Original magnification, ϫ75.

Nonetheless, the added loss of L-selectin in ICAM-1Ϫ/Ϫ mice fur- dothelial cell interactions at peripheral sites of inflammation (15). ther diminished inflammatory responses relative to ICAM-1-defi- L-selectinϪ/Ϫ mice exhibit impaired peripheral inflammatory re- ciency alone (Fig. 1). This is consistent with the finding that L- sponses including delayed-type hypersensitivity responses and re- selectin and ICAM-1 function cooperatively to mediate optimal jection of allogeneic skin transplants (20, 21, 31). However, leukocyte rolling as well as to recruit leukocytes into inflammatory whether the observed reduction in Ag-induced inflammation is due sites (27, 30, 31). In addition to reduced inflammation in adhesion to disruption of early events in Ag sensitization or later effector molecule-deficient mice, neutrophil and mast cell accumulation in phases has been controversial. L-selectin deficiency does not in- the skin and peritoneum were significantly reduced during Arthus hibit the in vivo generation of effector T cells able to mount in vitro reactions (Figs. 2Ð4). Similarly, reduced cutaneous inflammatory proliferative responses to Ag or the generation of effector cytotoxic responses correlated with a significant decrease in TNF-␣ mRNA T cell responses in skin allograft recipients (20, 31, 43). In addi- levels within skin at sites of IC deposition (Fig. 5). Taken together, these results demonstrate that ICAM-1 and L-selectin coopera- tion, intravital microscopy studies have shown that leukocyte roll- Ϫ/Ϫ tively contribute to the development of the cutaneous Arthus re- ing is reduced in L-selectin mice in the cremasteric microvas- action by regulating neutrophil and mast cell accumulation at the culature during Ag challenge at this site (44). These results suggest inflammatory site. that L-selectin contributes directly to leukocyte rolling in the pe- L-selectin regulates lymphocyte migration into lymph nodes ripheral vasculature during immune responses to Ag (44). How- across high endothelial venules and is involved in leukocyte-en- ever, a requirement for L-selectin during the Ag sensitization 2976 ICAM-1 AND L-SELECTIN IN THE ARTHUS REACTION

FIGURE 6. Expression of L-selectin and CD18 on peritoneal mast cells and granulocytes. Peritoneal cells from untreated wild-type mice were stained with either anti-c-Kit-FITC Ab or anti-Gr-1-FITC Ab plus either anti-CD18-PE Ab or anti-L-selectin-PE Ab. Flow cytometric analysis was performed by gating on c-Kit-positive mast cells or Gr-1-positive granu- Downloaded from locytes. Representative histograms are shown for expression of L-selectin or CD18 on mast cells (A) or granulocytes (B). The dashed lines for L- selectin expression represent staining obtained using L-selectinϪ/Ϫ mice. The dashed lines for CD18 expression represent control staining using an unreactive isotype-matched mAb. These results represent those obtained with five wild-type mice. http://www.jimmunol.org/

FIGURE 5. TNF-␣ mRNA expression in the skin of mutant and wild- type littermates at 4 h after IC challenge. Total RNA was isolated from frozen skin tissues, reverse transcribed into cDNA, and then amplified lectin deficiency significantly reduced leukocyte accumulation using TNF-␣ and ␤-actin primers. The PCR products were electrophoresed during the passive cutaneous and peritoneal Arthus reactions ex- on an agarose gel and stained with ethidium bromide. Representative cludes the Ag sensitization process (Figs. 2Ð4). Thus, reduced Ag- mRNA expression of TNF-␣ and ␤-actin is shown (A). The amount of induced cellular hypersensitivity in L-selectinϪ/Ϫ mice results TNF-␣ mRNA was measured by real-time PCR and normalized to the 18S from interrupted leukocyte-endothelial cell interactions and not ␣ rRNA endogenous control (B). The TNF- mRNA level in wild-type lit- from reduced immune responses. Furthermore, these results fully

Ϯ by guest on September 27, 2021 termates was used as the calibrator. All values represent the mean SEM confirm the existence of a peripheral vascular endothelial ligand of results obtained with five mice of each group. The sample mean for each for L-selectin. group of mutant mice was significantly different from that of wild-type littermates (p Ͻ 0.0001). Mast cells derive from bone marrow progenitors that migrate through the circulation into tissues where they proliferate and ma- ture (45). Mature mast cells exist exclusively within tissues, al- phase of immune responses could not be fully excluded since these though mast cell numbers increase at sites of inflammation (46). experimental systems required systemic Ag sensitization before Since mature mast cells from the blood accumulate in the CNS Ag challenge. Catalina et al. (23) have shown that effector func- within 1Ð2 h in response to altered physiological conditions (47), tions, including leukocyte entry into inflammatory sites of the skin, it is possible that mature mast cells also migrate from the circu- are intact in L-selectinϪ/Ϫ mice, while the major defect in respon- lation into inflamed sites. Consistent with this, peritoneal mast siveness is due to a lack of T cell sensitization in draining lymph cells expressed significant levels of both L-selectin and CD18 (Fig. nodes. In studies by Xu et al. (22), leukocyte recruitment into the 6). Furthermore, Arthus reaction-induced mast cell accumulation skin was reduced within the first 4 days of sensitization in L-se- in both skin and the peritoneum was substantially reduced in mice lectinϪ/Ϫ mice, but not after 9 days of sensitization. Based on this, lacking L-selectin, ICAM-1, or both adhesion molecules (Fig. 2). Xu et al. (22) concluded that the impaired migration of naive T It is unlikely that mast cells migrated from surrounding tissues into cells into the draining lymph nodes impaired Ag sensitization in sites of inflammation since ICAM-1 expression was only detected L-selectinϪ/Ϫ mice, rather than the subsequent impairment of leu- on the endothelium during IC-induced inflammation (Fig. 7), and kocyte recruitment into the skin. However, the finding that L-se- mast cell numbers increased rapidly in the peritoneal cavity of

FIGURE 7. ICAM-1 expression in skin from wild-type mice during a cutaneous passive Arthus reaction. ICAM-1 expression in normal skin (A) and in inflamed skin after4h(B) and8h(C) of IC challenge was as- sessed by immunohistochemistry us- ing anti-ICAM-1 Abs. Sections were counterstained with methyl green. Original magnification, ϫ100. The Journal of Immunology 2977 wild-type mice following inflammation (Fig. 2B). Although the References exact routes of mast cell migration into inflamed foci were not 1. Kohl, J., and J. E. Gessner. 1999. On the role of complement and Fc ␥-receptors determined in this study, the results suggest that L-selectin and in the Arthus reaction. Mol. Immunol. 36:893. 2. Arthus, M. 1903. Injections re«pete«es de serum de cheval chez le lapin. V. R. Soc. ICAM-1 regulate mast cell recruitment from the circulation. Biol. 55:817. Mature peritoneal mast cells expressed CD18 and L-selectin 3. Sylvestre, D. L., and J. V. Ravetch. 1994. Fc receptors initiate the Arthus reac- tion: redefining the inflammatory cascade. Science 265:1095. (Fig. 6). Consistent with this finding, Mac-1 (CD11b/CD18) is 4. Sylvestre, D., R. Clynes, M. Ma, H. Warren, M. C. Carroll, and J. V. Ravetch. expressed by immature mast cells derived from mouse bone mar- 1996. Immunoglobulin G-mediated inflammatory responses develop normally in row and mature peritoneal mast cells (41). CD18 expression is also complement-deficient mice. J. Exp. Med. 184:2385. 5. Baumann, U., J. Kohl, T. Tschernig, K. Schwerter-Strumpf, J. S. Verbeek, detected on mast cells in normal human skin, and human mast cell R. E. Schmidt, and J. E. Gessner. 2000. A codominant role of Fc␥RI/III and C5aR lines express CD18 (48). With regard to L-selectin expression, a in the reverse Arthus reaction. J. Immunol. 164:1065. 6. Hazenbos, W. L., J. E. Gessner, F. M. Hofhuis, H. Kuipers, D. Meyer, previous study has shown that immature mast cells derived from I. A. Heijnen, R. E. Schmidt, M. Sandor, P. J. Capel, M. Daeron, et al. 1996. mouse bone marrow do not express L-selectin (49). A second Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc␥RIII (CD16) deficient mice. Immunity 5:181. study has shown that L-selectin is not expressed on mature primary 7. Zhang, Y., B. F. Ramos, and B. A. Jakschik. 1991. Augmentation of reverse mast cells isolated from human lung and uterus (50). However, Arthus reaction by mast cells in mice. J. Clin. Invest. 88:841. tissue fragments were first treated with collagenase in the second 8. Zhang, Y., B. F. Ramos, and B. A. Jakschik. 1992. Neutrophil recruitment by tumor necrosis factor from mast cells in immune complex peritonitis. Science study and the isolated mast cells were then cultured for at least 258:1957. 24 h. L-selectin is rapidly lost from the cell surface of leukocytes 9. Sylvestre, D. L., and J. V. Ravetch. 1996. A dominant role for mast cell Fc receptors in the Arthus reaction. Immunity 5:387. following cellular activation (15). Furthermore, incubating lym- 10. Hopken, U. E., B. Lu, N. P. Gerard, and C. Gerard. 1997. Impaired inflammatory Downloaded from phocytes overnight at 4¡C can also result in the complete loss of responses in the reverse Arthus reaction through genetic deletion of the C5a L-selectin from the cell surface (51). Therefore, it is likely that receptor. J. Exp. Med. 186:749. 11. Butcher, E. C. 1991. Leukocyte-endothelial cell recognition: three (or more) steps L-selectin expression was endoproteolytically released from the to specificity and diversity. Cell 67:1033. cell surface during mature mast cell isolation. Nonetheless, the 12. Ley, K., and T. F. Tedder. 1995. Leukocyte interactions with vascular endothe- lium: new insights into selectin-mediated attachment and rolling. J. Immunol. present study reveals that mature mast cells freshly isolated from 155:525.

the peritoneal cavity express significant levels of L-selectin. 13. Springer, T. A. 1995. Traffic signals on endothelium for lymphocyte recirculation http://www.jimmunol.org/ and leukocyte emigration. Annu. Rev. Physiol. 57:827. ICAM-1 is expressed on many types of cells, with its expression 14. Tedder, T. F., D. A. Steeber, A. Chen, and P. Engel. 1995. The selectins: vascular up-regulated by proinflammatory cytokines in vitro (26). Aug- adhesion molecules. FASEB J. 9:866. mented ICAM-1 expression is also observed on keratinocytes, fi- 15. Tedder, T. F., X. Li, and D. A. Steeber. 1999. The selectins and their ligands: adhesion molecules of the vasculature. Adv. Mol. Cell Biol. 28:65. broblasts, and infiltrating leukocytes in the inflamed skin in vivo 16. Spertini, O., F. W. Luscinskas, M. A. Gimbrone Jr., and T. F. Tedder. 1992. (52, 53). Furthermore, ICAM-1 expression by fibroblasts may me- Monocyte attachment to activated human vascular endothelium in vitro is medi- ated by leukocyte adhesion molecule-1 (L-selectin) under non-static conditions. diate neutrophil migration through fibroblast layers within tissues J. Exp. Med. 175:1789. (54, 55), and ICAM-1 expressed on lung epithelial cells supports 17. Spertini, O., F. W. Luscinskas, G. S. Kansas, J. M. Munro, J. D. Griffin, the adhesion and retention of neutrophils (56). Therefore, it is pos- M. A. Gimbrone, Jr., and T. F. Tedder. 1991. Leukocyte adhesion molecule-1

(LAM-1, L-selectin) interacts with an inducible endothelial cell ligand to support by guest on September 27, 2021 sible that ICAM-1 expression on cells other than endothelial cells leukocyte adhesion. J. Immunol. 147:2565. might be involved in the migration or retention of leukocytes 18. Brady, H. R., O. Spertini, W. Jimenez, B. M. Brenner, P. A. Marsden, and T. F. Tedder. 1992. Neutrophils, monocytes and lymphocytes bind to cytokine- within the perivascular area of inflamed skin during the Arthus activated kidney glomerular endothelial cells through L-selectin (LAM-1) in reaction. However, our finding that ICAM-1 was expressed exclu- vitro. J. Immunol. 149:2437. 19. Arbones, M. L., D. C. Ord, K. Ley, H. Radich, C. Maynard-Curry, D. J. Capon, sively by skin endothelium in the Arthus reaction (Fig. 7) excludes and T. F. Tedder. 1994. Lymphocyte homing and leukocyte rolling and migration this possibility. Moreover, this finding suggests that endothelial are impaired in L-selectin (CD62L) deficient mice. Immunity 1:247. ICAM-1 expression primarily mediates leukocyte accumulation 20. Tang, M. L. K., L. P. Hale, D. A. Steeber, and T. F. Tedder. 1997. L-selectin is involved in lymphocyte migration to sites of inflammation in the skin: delayed during the Arthus reaction. rejection of allografts in L-selectin-deficient mice. J. Immunol. 158:5191. Two dominant pathways contribute to initiation of the cutaneous 21. Tedder, T. F., D. A. Steeber, and P. Pizcueta. 1995. L-selectin deficient mice have ␥ impaired leukocyte recruitment into inflammatory sites. J. Exp. Med. 181:2259. Arthus reaction: a Fc RIII-dependent pathway and a complement- 22. Xu, J., I. S. Grewal, G. P. Geba, and R. A. Flavell. 1996. Impaired primary T cell dependent pathway using the C5aR (5, 6, 9). The loss of Fc␥RIII responses in L-selectin-deficient mice. J. Exp. Med. 183:589. results in a 60% reduction in edema formation and neutrophil re- 23. Catalina, M. D., M. C. Carroll, H. Arizpe, A. Takashima, P. Estess, and M. H. Siegelman. 1996. The route of antigen entry determines the requirement for cruitment compared with wild-type mice, whereas C5aR defi- L-selectin during immune responses. J. Exp. Med. 184:2341. ciency results in a 30Ð50% reduction (5, 10). The present study 24. Kunkel, E. J., and K. Ley. 1996. Distinct phenotype of E-selectin-deficient mice. E-selectin is required for slow leukocyte rolling in vivo. Circ. Res. 79:1196. demonstrates that edema, hemorrhage, and neutrophil accumula- 25. Ley, K. E., D. Bullard, M. L. Arbones, R. Bosse, D. Vestweber, T. F. Tedder, and tion are inhibited by 30Ð40% with L-selectin deficiency, 40Ð50% A. L. Beaudet. 1995. Sequential contribution of L- and P-selectin to leukocyte rolling in vivo. J. Exp. Med. 181:669. with ICAM-1 deficiency, and 50Ð60% with the combined loss of 26. Dustin, M. L., R. Rothlein, A. K. Bhan, C. A. Dinarello, and T. A. Springer. 1986. L-selectin and ICAM-1 (Figs. 1 and 2A). Thus, blocking the func- Induction by IL-1 and interferon-␥: tissue distribution, biochemistry, and function tion of L-selectin, ICAM-1, or both adhesion molecules inhibits of a natural adherence molecule (ICAM-1). J. Immunol. 137:245. 27. Steeber, D. A., M. A. Campbell, A. Basit, K. Ley, and T. F. Tedder. 1998. the Arthus reaction to a similar extent as blocking either the C5aR Optimal selectin-mediated rolling of leukocytes during inflammation in vivo re- or Fc␥RIII pathways. Thus, cell adhesion molecules, including quires intercellular adhesion molecule-1 expression. Proc. Natl. Acad. Sci. USA 95:7562. L-selectin and ICAM-1, may play a critical role in Arthus reaction 28. Sligh Jr., J. E., C. M. Ballantyne, S. S. Rich, H. K. Hawkins, C. W. Smith, initiation, in addition to its progression. This suggests that L-se- A. Bradley, and A. L. Beaudet. 1993. Inflammatory and immune responses are lectin and ICAM-1 are potential therapeutic targets for human IC- impaired in mice deficient in intercellular adhesion molecule 1. Proc. Natl. Acad. Sci. USA 90:8529. mediated diseases such as vasculitis syndrome and some collagen 29. Xu, H., J. A. Gonzalo, Y. St. Pierre, I. R. Williams, T. S. Kupper, R. S. Cotran, diseases. However, it should be noted that genetic deficiency of T. A. Springer, and J.-C. Guiterrez-Ramos. 1994. Leukocytosis and resistance to septic shock in intercellular adhesion molecule 1-deficient mice. J. Exp. Med. L-selectin or ICAM-1 in the development of Arthus reaction was 180:95. a fundamentally different situation than the selective inhibition of 30. Nagaoka, T., Y. Kaburagi, Y. Hamaguchi, M. Hasegawa, K. Takehara, D. A. Steeber, T. F. Tedder, and S. Sato. 2000. Delayed wound healing in the the function of these adhesion molecules during the course of an absence of intercellular adhesion molecule-1 or L-selectin expression. IC-mediated disease. Am. J. Pathol. 157:237. 2978 ICAM-1 AND L-SELECTIN IN THE ARTHUS REACTION

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