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Inflammatory Lesion Macrophage Exit from a Peripheral 1 Integrin

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Inflammatory Lesion Macrophage Exit from a Peripheral 1 Integrin Published March 18, 2013, doi:10.4049/jimmunol.1202097 The Journal of Immunology a1b1 Integrin-Mediated Adhesion Inhibits Macrophage Exit from a Peripheral Inflammatory Lesion Henry M. Becker,*,†,‡ Jacob Rullo,*,† Mian Chen,* Magar Ghazarian,* Sungho Bak,* Haiyan Xiao,* John B. Hay,‡ and Myron I. Cybulsky*,† Integrins are adhesion molecules critical for the recruitment of leukocytes from blood into peripheral tissues. However, whether integrins are also involved in leukocyte exit from peripheral tissues via afferent lymphatics to the draining lymph node remains poorly understood. In this article, we show that adhesion by the collagen IV–binding integrin a1b1 unexpectedly inhibited macrophage exit from inflamed skin. We monitored macrophages exiting mouse footpads using a newly developed in situ pulse labeling technique. Blockade of a1b1 integrin or genetic deletion (Itga12/2) increased macrophage exit efficiency. Chemotaxis assays through collagen IV showed more efficient migration of Itga12/2 macrophages relative to wild type. Given that macro- phages are key orchestrators of inflammation, a1b1 integrin adhesion may represent a mechanism for regulating inflammatory responses by controlling macrophage exit or persistence in inflamed tissues. The Journal of Immunology, 2013, 190: 000–000. he extent and duration of leukocyte accumulation at the The family of integrins associated with the b1 subunit (CD29) site of tissue inflammation can have a profound effect on includes several that bind ECM molecules, and some of these are T the course of inflammation, as well as on tissue injury. found on leukocytes, including macrophages (7). This b1 subset of Leukocyte accumulation in tissues is dependent on multiple factors integrins was first identified on T cells but appeared only after days including the rate of recruitment from the blood, active retention, or weeks of in vitro culture; hence they were named very late Ags apoptosis, and egress from tissues, resulting in migration to re- 1–6, and since that time, additional a subunits have been found that gional lymph nodes (LNs) via afferent lymphatics. Tissue entry of can associate with the b1 chain (7–10). Very late Ag 1 (a1b1 leukocyte subsets requires adhesion to the vascular endothelium integrin or CD49a/CD29) on T cells was the first of the b1 group to and is controlled by a well-described combinatorial cascade of be characterized, and it was found to bind ECM, in particular, events involving several families of molecules including integrins collagen IV, but not collagen I (11). Later studies further charac- (1, 2). Classic experiments have shown that the absence or terized a1b1 integrin expression on T cells, as well as confirming blockade of leukocyte integrins inhibits blood to tissue migration its ability to adhere to collagen IV (12–14). The a1b1 integrin was in both homeostatic and inflammatory conditions (1, 3, 4). Al- also shown to be important in inflammatory responses, required for though the importance of leukocyte integrins for blood-to-tissue T cell–mediated inflammation including delayed-type hypersensi- migration has been firmly established, their potential role in ex- tivity and resistance to viral infection (13, 15). More recently, data travascular tissues is still poorly understood. Some studies using have emerged demonstrating a critical role for a1b1 integrin in inflamed lymphatic endothelium in vitro have suggested that there macrophage activation showing that the binding of Semaphorin7A are integrin-dependent steps during T cell entry into afferent (Sema7A) expressed on T cells to a1b1 integrin expressed on mac- lymphatics (5). Other studies in mouse ear explants have found rophages is required for effective macrophage inflammatory cyto- integrins to be dispensable for dendritic cell migration and entry kine secretion (16). into afferent lymphatics (6). If integrins are important in extra- Current models hold that when leukocyte integrins bind their vascular leukocyte migration, then integrins that can bind extra- ligands, this facilitates cell migration. Therefore, integrins are cellular matrix (ECM) constituents would be a logical subset to generally viewed as adhesion molecules with a promigratory investigate. function. Whether integrin-mediated adhesion can also inhibit leukocyte migration is less clear. Intriguingly, data generated from integrin-deficient dendritic cells demonstrated normal migration *Toronto General Research Institute, University Health Network, Toronto, Ontario through noninflamed extravascular tissues (6). This suggests the M5G 2C4, Canada; †Department of Laboratory Medicine and Pathobiology, Univer- sity of Toronto, Toronto, Ontario M5G 1L7, Canada; and ‡Department of Immu- possibility that in the appropriate biological context, integrins nology, University of Toronto, Toronto, Ontario M5G 1L7, Canada might play an alternate adhesive role as “anchoring” molecules, Received for publication July 30, 2012. Accepted for publication February 14, 2013. inhibiting rather than facilitating leukocyte migration. We inves- This work was supported by the Canadian Institutes of Health Research (Operating tigated the function of a1b1 integrin in an inflammatory model Grant MOP-89740 to M.I.C.; Canadian Graduate Scholarship Doctoral Award to J.R.) using a newly developed in situ pulse labeling technique. Our and the Heart and Stroke Foundation of Ontario (Career Investigator Award to M.I.C.). results show that the collagen IV–binding integrin a1b1 inhibits Address correspondence and reprint requests to Dr. Henry M. Becker and Dr. Myron macrophage exit from a peripheral inflammatory lesion. Cybulsky, University of Toronto, 101 College Street, TMDT 3-303, Toronto, ON M5G 1L7, Canada,. E-mail addresses: [email protected] (H.M.B.) and [email protected] (M.C.) Materials and Methods The online version of this article contains supplemental material. Animals Abbreviations used in this article: ECM, extracellular matrix; EP, ethylpyridylated; 2/2 ISPL, in situ pulse labeling; LN, lymph node; Sema7A, Semaphorin7A; S1P, a1 integrin knockout mice (Itga1 ) bred onto a C57BL/6 background sphingosine-1 phosphate. for at least 12 generations were a gift from A. Pozzi. For experiments, C57BL/6 (Jackson Laboratories) and Itga12/2 mice were between 8 and Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 12 wk old. All animal experiments were approved by the University Health www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202097 2 a1b1 INTEGRIN-MEDIATED ADHESION Network Animal Care Committee and performed according to institutional duced into the flow chamber and allowed to adhere for 5 min. Images of and federal guidelines. four high-powered fields were acquired by fluorescence microscopy; then ashearforceof10dynes/cm2 was applied for 1 min to induce cell de- Footpad injections and in situ pulse labeling tachment. After flow was stopped, images of four additional high- powered fields were obtained, and macrophages that remained adherent Con A (20 mgin20ml PBS; Sigma) was injected into mouse footpads using a 50-ml Hamilton syringe. The fluorescent dye CFSE (Invitrogen before and after shear were counted. Chemotactic migration assays were m Molecular Probes) was prepared according to the manufacturer’s instruc- performed in Corning Costar Transwell chambers with 5- Mmicropo- tions and further diluted in PBS to obtain a final concentration of 1.44 mM. rous membrane filters separating the upper and lower chambers. Mem- m Mice were lightly anesthetized using isoflurane via nosecone, and a tour- branes were coated overnight (4˚C) with collagen IV (2.5 g/ml), collagen I (1 mg/ml), laminin (10 mg/ml), or fatty acid–free BSA (10 mg/ niquet was applied at the distal calf. CFSE (10 ml) was injected into a Con ml; Sigma), then rinsed with PBS. ECM gel (Sigma; 10 mlusedundi- A–inflamed footpad using a 10-ml Hamilton syringe fitted with a custom- made 33-gauge needle. The tourniquet was removed 15 min after the luted, “neat”) was applied to membranes for 10 min (22˚C) and allowed CFSE injection, and mice were returned to cages. In some experiments, to polymerize (as per manufacturer’s instructions). Transwell experi- inflamed footpads were first injected with Ab (10 mg Ha31/8 or control Ab, ments were carried out at 37˚C in DMEM with 2 mg/ml fatty acid–free BSA and 1:100 Penicillin V/Streptomycin (Wisent). Two-hundred micro- Armenian Hamster IgG2, l1, both NA/LE; BD Pharmingen), Obtustatin 2 2 Itga1+/+ Itga1 / (10 mg; a gift from C. Marcinkiewicz), or pertussis toxin (List Bio- liters total of differentially labeled and macrophages or Con A exudates in approximately equal proportions (5 3 106 cells/ml) chemicals). After 3 min, a tourniquet was applied and in situ pulse labeling were introduced into the upper chamber. The lower chamber contained (ISPL) was performed. CCL21 (10 nM; PeproTech). Macrophages were allowed to chemotac- Cell isolation tically migrate through the coated membranes for 18 h, at which point the cells that had migrated to the lower chamber were isolated, immu- Single-cell suspensions were prepared from mouse footpads and popliteal nostained as required, and analyzed by flow cytometry. Data from each LNs. In both cases and regardless of treatment, cells excluded propidium in vitro assay were normalized to the ratio of Itga1+/+ and Itga12/2 input iodide and there was no difference between Itga1+/+ and Itga12/2 staining. macrophages. For isolation of cells from the footpad, mice were euthanized by carbon dioxide or isofluorane overdose and perfused with ice-cold PBS via the left Histology, cytospins, and cell sorting ventricle. Footpad tissue (∼8 by 4 mm) was excised from the plantar surface, cut into small squares (,1 mm), and digested (1 h, 37˚C) with Footpads were excised and fixed for at least 24 h in 10% buffered formalin. Liberase CI (250 mg/ml; Roche) and DNase I (50 mg/ml; Roche) in In some experiments, footpads were decalcified using HCl/EDTA before m DMEM (Sigma). EDTA (10 mM final concentration) was then added and embedding in paraffin, sectioning (5 or 2 m), and staining with H&E. For samples were incubated on ice for 5 min.
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