Published online before print February 13, 2007
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* Department of Dermatology, Kanazawa University Graduate School of Medical Science, Ishikawa, Japan;
Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; and
Department of Dermatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
1 Correspondence: Department of Dermatology, Kanazawa University Graduate School of Medical Science, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan. E-mail: fujimoto-m{at}umin.ac.jp
ABSTRACT
Immune complex (IC)-induced inflammation is mediated by inflammatory cell infiltration, a process that is highly regulated by expression of multiple adhesion molecules. The roles and interactions of ICAM-1 and VCAM-1, the major regulators of leukocyte firm adhesion, were examined in the cutaneous reverse-passive Arthus reaction using ICAM-1-deficient (ICAM-1–/–) mice and blocking mAb against VCAM-1. Within 8 h, IC challenge of wild-type mice induced edema, hemorrhage, interstitial accumulation of neutrophils and mast cells, as well as production of TNF-
and IL-6. All of these inflammatory parameters were reduced significantly in ICAM-1–/– mice. The blockade of VCAM-1 in wild-type mice did not affect any inflammatory parameters. In contrast, ICAM-1–/– mice treated with anti-VCAM-1 mAb had significantly reduced edema, hemorrhage, and neutrophil infiltration. Furthermore, VCAM-1 blockade in ICAM-1–/– mice suppressed cutaneous TNF- and IL-6 production. Thus, VCAM-1 plays a complementary role to ICAM-1 in the cutaneous Arthus reaction by regulating leukocyte accumulation and proinflammatory cytokine production.
Key Words: cell adhesion molecule • VLA-4 • neutrophil • vasculitis
INTRODUCTION
The pathogenesis of autoimmune diseases frequently involves the formation of IgG-containing immune complexes (IC) inducing inflammatory responses with significant tissue injury, commonly referred to as Type III hypersensitivity reaction. This IC injury has been implicated in the pathogenesis of vasculitis syndrome, systemic lupus erythematosus, rheumatoid arthritis, and cryoglobulinemia [1
]. The mechanisms by which the immune system controls effector responses to IC are of central importance for developing therapeutic strategies. The standard animal model for the inflammatory response in these IC-mediated diseases is the Arthus reaction [2
]. Analyses using gene knockout mice have revealed that activation of the complement system, especially C5a and its interaction with the C5a receptor, and of Fc
Rs on inflammatory cells, particularly mast cells, is required to initiate the Arthus reaction [3
4
5
6
7
8
]. In addition, accumulation of neutrophils and mast cells is necessary for the progression of the IC-mediated vascular tissue damage, which results in edema and hemorrhage [3
4
5
6
7
8
].
Leukocyte recruitment from the circulation into an extravascular site is pivotal to the inflammatory response. Leukocytes first tether and roll on vascular endothelial cells before they are activated to adhere firmly and then crawl and transmigrate to the extravascular space. This multistep process is regulated by the cooperative action of adhesion molecules on the endothelial cell and the leukocyte [9
, 10
]. The selectins primarily mediate tethering and rolling of leukocytes, and Ig superfamily members, including ICAM-1 (CD54) and VCAM-1 (CD106), are critical for the firm adhesion through the interaction with their integrin ligands, LFA-1 (CD11a/CD18) and VLA-4 (CD49d/CD29), respectively [11
]. ICAM-1 is constitutively expressed at low levels on the surface of endothelial cells, but its expression is increased significantly upon endothelial cell activation with cytokines or endotoxin [12
]. These stimuli also induce the expression of VCAM-1, which interacts with most leukocytes, except neutrophils, which express VLA-4 [13
]. VCAM-1 plays essential roles in various aspects of biological responses, including the development of inflammation. Deletion of the VCAM-1 gene produces a phenotype similar to deletion of the 4 integrin gene, with embryonic lethality as a result of cardiac and placental developmental abnormalities, although a few VCAM-1-deficient (VCAM-1–/–) mice survive [14
, 15
].
We previously reported roles and interaction of the selectins, major regulators of the initial step of leukocyte migration, in the cutaneous Arthus reaction, which was reduced by deficiency of P-selectin (CD62P) or L-selectin (CD62L), and it was not inhibited by loss of E-selectin (CD62E) expression [6 , 16 ]. Furthermore, E- and P-selectin blockade resulted in more reduction relative to P-selectin deficiency alone, suggesting the cooperative roles of these selectins in the cutaneous Arthus reaction [16 ]. We also reported that ICAM-1 plays a critical role in the cutaneous Arthus reaction, as ICAM-1–/– mice exhibit reduced Arthus reaction, which is associated with decreased infiltration of neutrophils and mast cells [17 ]. However, the relative contribution and interaction of ICAM-1 and VCAM-1, both of which predominantly regulate the firm adhesion process of leukocyte migration, to the Arthus reaction remain unknown. In this study, to clarify the role of VCAM-1 and interaction of ICAM-1 and VCAM-1 in the Arthus reaction, we examined inflammation induced by IC in mice lacking ICAM-1 and wild-type mice with anti-VCAM-1 mAb.
MATERIALS AND METHODS
Mice
ICAM-1–/– mice [18
], expressing residual amounts of ICAM-1 splice variants in the thymus and spleen but not in other organs, including skin [19
], were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). All mice were healthy, fertile, and did not display evidence of infection or disease. All mice were backcrossed between five and 10 generations onto the C57BL/6 genetic background. Mice used for experiments were 12–16 weeks old. Age-matched, wild-type littermates and C57BL/6 mice (The Jackson Laboratory) were used as controls with equivalent results, so all control results were pooled. All mice were housed in a pathogen-free barrier facility and screened regularly for pathogens. The Committee on Animal Experimentation of Kanazawa University Graduate School of Medical Science (Ishikawa, Japan) approved all studies and procedures.
Reverse-passive Arthus reaction
For cutaneous reverse-passive Arthus reactions, mice anesthetized by inhalation of diethyl ether were shaved on their dorsal skin and wiped with 70% alcohol. Rabbit IgG anti-chicken egg albumin antibodies (60 µg/30 µl; Cappel, Durham, NC, USA) were injected intradermally with a 29-gauge needle, followed immediately by an i.v. injection of chicken egg albumin (20 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) [5
]. The intradermal injection of purified polyclonal rabbit IgG (60 µg/30 µl; Sigma-Aldrich) followed by i.v. installation of chicken egg albumin served as a control. The solution of chicken egg albumin contained 1% Evans blue dye (Sigma-Aldrich). For a blocking study using mAb to VCAM-1 (Clone 429 MVCAM.A, rat IgG2a, 30 µg per mouse; BD PharMingen, San Diego, CA, USA), mAb were injected i.v. 30 min before IC challenge. Irrelevant, isotype-matched, purified rat IgG1 mAb (R3-34) and rat IgG2a mAb (R35-95) served as controls (30 µg per mouse; BD PharMingen).
Quantitation of edema and hemorrhage
Edema was evaluated by measuring the vascular leak 4 h after IC challenge [5
]. Mice were killed, and the skin containing the injection site was removed at the level of fascia above skeletal muscle and was reversed. The diameter of extravascular Evans blue dye on the fascia side of the injection site was measured directly. Evans blue dye binds to serum proteins and thereby can be used to quantify alterations in vascular permeability. The diameter of the major and minor axes of the blue spot was averaged for analysis. The amount of hemorrhage was assessed 8 h after IC challenge by direct macroscopic measurement of the purpuric spot. The diameter of the major and minor axes of the purpuric spot was averaged for analysis.
Histological examination and immunohistochemical staining
Tissues were harvested 4 or 8 h after IC challenge using a disposable, sterile, 6-mm punch biopsy (Maruho, Osaka, Japan) and assessed for tissue damage and number of infiltrating neutrophils and mast cells. Tissues were cut into halves, fixed in 3.5% paraformaldehyde, and then embedded in paraffin. Sections (6 µm) were stained using H&E for neutrophil evaluation and toluidine blue for mast cell staining. Neutrophil and mast cell infiltration was evaluated by counting extravascular neutrophils and mast cells in the entire section and averaging the numbers present in 10 serial skin sections from the injection site. Each section was examined independently by three investigators in a blinded manner, and the mean was used for analysis.
For immunohistochemistry, tissue sections of skin biopsies were fixed with acetone and then incubated with 10% normal rabbit serum in PBS (10 min, 37°C) to block nonspecific staining. Sections were then stained with rat mAb specific for mouse VCAM-1 (429 MVCAM.A, BD PharMingen), as described previously [13 ]. Rat IgG2a (BD PharMingen) was used as a control for nonspecific staining. Sections were then incubated sequentially (20 min, 37°C) with biotinylated rabbit antirat IgG secondary antibodies (Vectastain avidin-biotin complex method, Vector Laboratories, Burlingame, CA, USA) and then HRP-conjugated, avidin-biotin complexes (Vectastain ABC method, Vector Laboratories). Sections were finally developed with 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide and counterstained with methyl green.
RNA isolation and real-time PCR
Tissues were harvested 4 or 8 h after IC challenge using a disposable, sterile, 6-mm punch biopsy (Maruho) and cut into halves. All skin samples were snap-frozen in liquid nitrogen and stored at –80°C before use. Total RNA was isolated from frozen tissue with Qiagen RNeasy spin columns (Qiagen Ltd., Crawley, UK) and then was reversely transcribed into cDNA according to the protocol for the RT System (Promega, Madison, WI, USA). The expressions of VCAM-1, IL-6, and TNF- mRNA were analyzed using a real-time PCR quantification method according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). Sequence-specific primers and probes were designed by Pre-Developed TaqMan® assay reagents (Applied Biosystems). Real-time PCR (one cycle at 50°C for 2 min, one at 95°C for 10 min; 40 cycles at 92°C for 15 s and at 60°C for 60 s) was performed on an ABI Prism 7000 sequence detector (Applied Biosystems). GAPDH was used to normalize the mRNA. To compare target gene and housekeeping GAPDH gene mRNA expression, the relative expression of real-time PCR products was determined using the 
comparative threshold cycle (Ct) method [20
]. The fold induction = 2–[Ct], where Ct = the threshold cycle, and Ct = [Ct gene interest (unknown sample)-Ct GAPDH (unknown sample)] – [Ct gene interest (calibrator sample)-Ct GAPDH (calibrator sample)]. One of the control samples was chosen as a calibrator sample. Each sample was examined in duplicate, and the mean Ct was used in the equation.
Statistical analysis
The Mann-Whitney U-test was used to determine the level of significance of differences between the sample means, and Bonferroni’s test was used for multiple comparisons. A P value of less than 0.05 was considered statistically significant. All values are shown as the mean ± SEM.
RESULTS
VCAM-1 expression in ICAM-1–/– mice in the cutaneous reverse-passive Arthus reaction
VCAM-1 expression on activated endothelial cells of the lung, heart, liver, kidney, and skin is induced by stimulation with proinflammatory cytokines [21
]. First, VCAM-1 expression during the cutaneous Arthus reaction was examined immunohistochemically in ICAM-1–/– mice. In normal skin from ICAM-1–/– mice, VCAM-1 was scarcely detected on endothelial cells (Fig. 1A
). In contrast, 4 h after IC induction, there was a substantial increase in VCAM-1 expression on the endothelium of blood vessels (Fig. 1B)
. This up-regulation remained observed 8 h after IC induction (Fig. 1C)
. This expression pattern was also similarly observed in wild-type mice (data not shown). Regional VCAM-1 mRNA levels were comparable between wild-type mice and ICAM-1–/– mice at 4 and 8 h (Fig. 1D)
. Therefore, VCAM-1 expression may play a role in the cutaneous reverse-passive Arthus reaction.
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Figure 1. VCAM-1 expression in the skin from ICAM-1–/– mice during a cutaneous-passive Arthus reaction. VCAM-1 expression in the skin before IC challenge (A) and in inflamed skin after 4 h (B) and 8 h (C) of IC challenge was assessed by immunohistochemistry using anti-VCAM-1 antibody. Sections were counterstained with methyl green. Original magnification, x100. (D) VCAM-1 mRNA levels in the skin from wild-type mice or ICAM-1–/– mice during a cutaneous-passive Arthus reaction before IC challenge (0 h) and after 4 h and 8 h. Transcript levels were quantified by real-time RT-PCR analysis and were normalized relative to endogenous GAPDH levels. Values represent mean (±SEM) relative to transcript levels of wild-type mice at 4 h from four to five mice in each group.
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Figure 2. The effect of loss or blockade of ICAM-1 and VCAM-1 on edema and hemorrhage in the cutaneous reverse-passive Arthus reaction. Mice were injected intradermally with rabbit IgG anti-chicken egg albumin antibody, followed by systemic chicken egg albumin and 1% Evans blue dye. After 4 or 8 h, dorsal skins were assessed from ICAM-1–/– mice treated with anti-VCAM-1 mAb or control antibody and from wild-type mice treated with anti-VCAM-1 mAb or control antibody. (A) Edema was evaluated as the diameter of extravasated Evans blue spot. Wild-type mice, which received an intradermal injection of polyclonal rabbit IgG, followed by i.v. installation of chicken egg albumin, served as control. (B) Hemorrhage after 8 h was assessed as the diameter of the purpuric spot. Edema and hemorrhage were inhibited significantly in ICAM-1–/– mice treated with anti-VCAM-1 mAb or control antibody compared with wild-type mice treated with anti-VCAM-1 mAb or control antibody for both panels (P<0.05). Horizontal bars indicate mean values for each group of mice. N.S., Not significant.
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Figure 3. Arthus reaction-induced recruitment of neutrophils and mast cells in the skin from ICAM-1–/– mice treated with anti-VCAM-1 mAb or control antibody and from wild-type mice treated with anti-VCAM-1 mAb or control antibody at 4 and 8 h after IC challenge. Numbers of neutrophils and mast cells per skin section were determined by counting in H&E- and toluidine blue-stained skin sections, respectively. All values represent the mean ± SEM of results obtained from five to 10 mice in each group. *, P < 0.05; **, P < 0.01, versus wild-type mice with control Ig. , P < 0.05; , P < 0.01, versus ICAM-1–/– mice with control Ig.
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Figure 4. Histological tissue sections showing neutrophil infiltration in the skin of ICAM-1–/– mice treated with anti-VCAM-1 mAb or control antibody and of wild-type mice treated with anti-VCAM-1 mAb or control antibody at 4 h (A) and 8 h (B) after IC challenge. Neutrophils were revealed by H&E staining. Original magnification, x100.
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Figure 5. Histological tissue sections showing mast cell accumulation in the skin of ICAM-1–/– mice treated with anti-VCAM-1 mAb or control antibody and of wild-type mice treated with anti-VCAM-1 mAb or control antibody at 4 h (A) and 8 h (B) after IC challenge. Mast cells (arrows) were detected as cells with metachromatic staining of granules in toluidine blue-stained sections. Original magnification, x100.
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mRNA levels were reduced in ICAM-1–/– mice (41%, P<0.005) relative to wild-type mice and in ICAM-1–/– mice with VCAM-1 blockade (15%, P<0.05) compared with ICAM-1–/– mice (Fig. 6B)
. However, IL-6 and TNF- mRNA were not decreased in wild-type mice treated with ant-VCAM-1 mAb compared with wild-type mice treated with control antibody. Thus, the reduced cutaneous inflammatory responses by blockade of adhesion molecules were generally associated with the reduced release of IL-6 and TNF-.
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Figure 6. TNF- and IL-6 mRNA expression in the skin of ICAM-1–/– mice treated with anti-VCAM-1 mAb or control antibody and of wild-type mice treated with anti-VCAM-1 mAb or control antibody at 4 h after IC challenge. Relative mRNA expression was quantified by real-time RT-PCR. All values represent the mean ± SEM of results obtained from five mice in each group.
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In the present study, IC-induced edema and hemorrhage were inhibited significantly in ICAM-1–/– mice treated with mAb to VCAM-1 compared with ICAM-1 deficiency alone, whereas they were not inhibited by blockade of VCAM-1 alone in wild-type mice (Fig. 2) . The results indicate that ICAM-1 and VCAM-1 contribute cooperatively to the development of the cutaneous-passive Arthus reaction. ICAM-1 plays a dominant role, as VCAM-1 blockade did not alter the reaction in wild-type mice but reduced it in the absence of ICAM-1 expression. Furthermore, although the reduced Arthus reaction by loss of ICAM-1 expression correlated with neutrophil and mast cell accumulation in the skin, VCAM-1 blockade in combination with ICAM-1 deficiency decreased neutrophil accumulation but did not affect mast cell infiltration. Therefore, VCAM-1 expression mainly regulates the infiltration of neutrophils but not mast cells in the cutaneous-passive Arthus reaction. In contrast to the current study, a significant effect of VCAM-1 blockade has been observed in the reverse-passive Arthus reaction in cremaster muscle [22 ]. Antibodies to VCAM-1 alone reduced adhesion and emigration to control levels. Therefore, relative contribution of VCAM-1 may be different between tissues.
Mast cells play an important role in the passive Arthus reaction [8 ]. Although the exact routes of mast cells into inflammatory sites have not been determined yet, immature mast cells derived from bone marrow progenitors are thought to migrate through the circulation into tissues and subsequently differentiate into mature mast cells [23 ]. In this study, IC challenge induced rapid mast cell accumulation, which was decreased significantly in ICAM-1–/– mice, and no further inhibition was observed with a blockade of VCAM-1 in ICAM-1–/– mice. There are two distinct populations of mast cells based on phenotype, histochemistry, and proteoglycan content: mucosal mast cells, found in the mucosa of intestines and the lungs, and connective tissue mast cells located in the skin, peritoneal cavity, and other nonmucosal sites [24 ]. Connective tissue mast cells express substantially less VLA-4 [25 ]. Therefore, it is possible that mast cell accumulation in the cutaneous Arthus reaction is independent of the interaction between VCAM-1 and VLA-4.
Neutrophils are another critical effector cell in the cutaneous Arthus reaction. In contrast to mast cell accumulation, the current study has demonstrated that VCAM-1 had a complementary role to ICAM-1 in neutrophil recruitment. Although neutrophils have generally been considered to lack VLA-4 expression [26
], several reports have provided evidence for the presence of VLA-4 expression on human and rodent neutrophils [27
28
29
]. We previously reported positive expression of VLA-4 in peritoneal neutrophils from wild-type mice and ICAM-1–/– mice [17
]. Furthermore, studies have demonstrated that VCAM-1 and ICAM-1 play important roles in neutrophil emigration in vivo [21
]. In a CD18 null mouse model of myocardial ischemia and reperfusion injury, neutrophil emigration on cardiac endothelial monolayer efficiency was reduced further (90%) after VCAM-1 antibody blockade [30
]. Moreover, in a murine model of endotoxic shock, increased expression of VCAM-1 was observed in the liver. Pretreatment with anti-VCAM-1 antibody decreased neutrophil transmigration into the liver parenchyma and attenuated liver tissue damage [13
]. Constitutive VLA-4 expression on resting rat neutrophils is also shown to mediate neutrophil accumulation in the inflamed joints of rats with adjuvant-induced arthritis as well as in dermal inflammation sites. Blocking VLA-4 together with LFA-1 inhibited neutrophil migration to dermal inflammatory reactions by 30–70%. Thus, although VLA-4 by itself had no effect on neutrophil accumulation, it strongly inhibits neutrophil accumulation when combined with anti-LFA-1 in cutaneous inflammation [29
]. In the current study, after 4 h of IC challenge, neutrophil numbers were reduced significantly in ICAM-1–/– mice compared with wild-type mice, but moreover, the combined loss or blockade of ICAM-1 and VCAM-1 led to greater reductions than the loss of ICAM-1 alone by 
40% (Fig. 3A)
. Taken together, the results suggest that VCAM-1 plays a pivotal role in neutrophil accumulation in the passive Arthus reaction.
The current study is consistent with our previous observations that the loss or blockade of L-, E-, and P-selectins and ICAM-1 completely abrogated mast cell infiltration but not neutrophil infiltration, indicating that there exists an adhesion pathway independent of all selectins and ICAM-1 for neutrophil recruitment [17 ]. In addition, a recent study has shown that VLA-4 is the predominant E- and P-selectin-independent mechanism for leukocyte migration to skin in response to various stimuli [31 ]. Collectively, during the process of the cutaneous Arthus reaction, which selectins, ICAM-1, and VCAM-1 regulate cooperatively, VCAM-1 expression is important for neutrophil infiltration.
Proinflammatory cytokines, including TNF- and IL-6, are produced and released from mast cells, neutrophils, and monocytes [32
, 33
]. TNF- and IL-6 levels, after 4 h within skin at the site of IC deposition, were decreased significantly by the loss of ICAM-1 in comparison with wild-type mice. VCAM-1 blockade had an additive effect in ICAM-1–/– mice but not in wild-type mice, which is likely to result from the decreased neutrophil infiltration (Fig. 3A)
. Thus, deficiency or blocking of adhesion molecules regulates leukocyte trafficking, which in turn influences the survival and activation by their role in outside-in signaling [34
]. Therefore, these results suggest that ICAM-1 and VCAM-1 control neutrophil accumulation cooperatively or possibly their function after IC challenge and thereby influence the release of proinflammatory cytokines from these leukocytes, leading to the regulation of the Arthus reaction, which is a useful, simplified model to evaluate direct roles of adhesion molecules in the effector phase of inflammation.
The current study, together with the results from our previous studies, demonstrates the relative contribution of these molecules: Edema and hemorrhage are inhibited by 30–40% with L-selectin deficiency, by 45% with ICAM-1 deficiency, by 40–60% with P-selectin deficiency or blockade, by 50% with L-selectin and ICAM-1 deficiency, by 75% with E- and P-selectin blockade, by 100% with E- and P-selectin blockade in L-selectin and ICAM-1 deficiency, and by 80% with VCAM-1 blockade in ICAM-1 deficiency [6
, 17
]. In this study, although VCAM-1 alone plays a minor role, orchestrated regulation with ICAM-1 takes a fundamental part in the cutaneous-passive Arthus reaction.
ACKNOWLEDGEMENTS
This work is supported by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan. We thank Ms. M. Matsubara and Y. Yamada for technical assistance.
Received October 10, 2006; revised January 3, 2007; accepted January 15, 2007.
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