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 macrophages 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. Digested footpad tissues were sorting and cytospin experiments, a FACSAria II (BD Biosciences) was used to sort single-cell suspensions from popliteal LNs draining 24-h Con then gently dissociated using the plunger of a 1-ml syringe (BD) through A–inflamed footpads. After sorting, cells were cytospun onto slides and a 200-mm pore wire mesh (custom made) and then further filtered through a 40-mm nylon mesh (Becton Dickinson [BD]). Popliteal LNs were iso- stained with a Romanowsky stain (Diff-Quick) according to the manu- lated and gently dissociated using small forceps. LN tissue was digested facturer’s instructions. (20 min, 37˚C) with collagenase D (2 mg/ml) in RPMI medium (Wisent). Quantitative PCR Digested LNs were then incubated on ice for 5 min in 10 mM EDTA, then filtered through a 40-mm nylon mesh (BD). Single-cell suspensions from Total RNA from footpads was isolated using the RNeasy Mini plus Isolation either tissue were centrifuged and washed twice with “flow buffer” con- Kit (Qiagen). Reverse transcription into single-stranded cDNA was per- sisting of HBSS with 2 mM EDTA and 2% FBS. formed using the high-capacity reverse transcription kit (Powerscript from Clontech, Palo Alto, CA), which contains random primers. PCR primers Immunostaining and flow cytometry were designed using the Primer Express software (Applied Biosystems). Single-cell suspensions were incubated (5 min) with rat and mouse Real-time PCRs were performed using LightCycler 480 detection system serum (The Jackson Laboratory) containing Ab to CD16/32 (2.4G2; BD (Roche), and amplified DNA was detected by incorporation of SYBR Green Pharmingen), then stained for cell-surface markers (30 min, on ice). Samples (Roche). A standard curve was generated for each primer pair using a mixture were washed, resuspended in flow buffer, and analyzed using an LSR II of various normal and inflamed tissues. The advanced relative quantification flow cytometer (BD Biosciences) and FlowJo software (Tree Star). Abs method (LightCycler 480 software) was used to determine the relative gene included biotin- or fluorochrome-conjugated PE, PE-cyanine 7, allophy- expression levels in footpads, and data were normalized to both hypo- cocyanin, allophycocyanin–cyanine 7, Alexa Fluor 647, Pacific Blue, xanthine phosphoribosyltransferase and CD31. The following nucleotide FITC, eFluor 605, and eFluor 650. Second-step reagents were streptavidin- sequences of quantitative PCR primers (forward and reverse, respectively) conjugated FITC, PE, or allophycocyanin (BD Pharmingen). Cell-surface were used: IFN-g:59-TTGCCAAGTTTGAGGTCAACAA-39,59-GCTT- markers used were the following (from Biolegend): CD3 (17A2), B220 CCTGAGGCTGGATTCC-39; IL-1-b:59-AGTTGACGGACCCCAAAA- (RA-6B2), Ly6C/Ly6G or GR-1 (RB6-8C5), CD11c (N418), Thy 1.2 (30- GA-39,59-TGCTGCTGCGAGATTTGAAG-39; IL-6: 59-CTCCCAACAG- H12), CD45 (30-F11), CD11b (M1/70; eBioscience), CD11c (HL3; BD ACCTGTCTATACCA-39,59-TGCCATTGCACAACTCTTTTCT-39; TNF- Pharmingen), and CD49a (Ha31/8 [BD Pharmingen] and CD49a/a-1 a:59-GTAGCCCACGTCGTAGCAAAC-39,59-GCACCACTAGTTGGT- integrin directly conjugated to FITC was a gift from D. Topham). TGTCTTTGA-39; IL-4: 59-ACAGGAGAAGGGACGCCAT-39,59-GAA- GCCCTACAGACGAGCTCA-39; hypoxanthine phosphoribosyltransferase: In vitro detachment and chemotactic migration assays 59-CAAGCTTGCTGGTGAAAAGGA-39,59-TGAAGTACTCATTATAG- TCAAGGGCATATC-39; CD31: 59-AGGGAGCACACCGAGAGCTA-39, 2 2 Itga1+/+ and Itga1 / macrophages were isolated from 4-d thioglycollate- 59-TGGATACGCCATGCACCTT-39. treated peritoneal cavities (2 ml, i.p.) and incubated ex vivo for 24 h with LPS (100 ng/ml in RPMI and 10% FBS) to induce a1 integrin expression. Alternatively, peritoneal exudates from 24-h Con A (100 mg in PBS, 2 ml, Results i.p.)–stimulated mice were used either as mixed populations or enriched Macrophages in the inflamed footpad express a1b1 integrin for macrophages by adherence to plastic. Macrophages were allowed to adhere to plastic for 30 min at 37˚C and were detached using 10 mM Our initial aim was to assess the expression of a1b1 integrin on a EDTA. In both the thioglycollate and enriched Con A methods, macro- heterogeneous population of leukocytes in a peripheral inflam- phage purity was $95%. Macrophages/exudates were washed three times matory lesion. We selected the mannan binding lectin Con A, and differentially labeled with CFSE (1 mM, 5 min, 37˚C), CMPTX which has previously been shown to induce an inflammatory lesion (Celltracker red, 10 mM, 25 min, 37˚C), or CMAC (Celltracker blue, 25 mM, 30 min, 37˚C; all from Invitrogen Molecular Probes). Cell concen- in the mouse footpads with a mixed population of leukocytes (18). trations were adjusted to 107 cells/ml for staining. Detachment assays were Footpad swelling was serially measured and reached a maximum performed at 37˚C using collagen IV–coated parallel plate flow chamber increase at 24 h (Fig. 1A) consistent with previous studies (19). At +/+ 2/2 apparatus (IBIDI). Differentially “tagged” Itga1 and Itga1 macro- 24 h, footpads contained a clearly visible cellular inflammatory phages were suspended in HBSS at 107 cells/ml with 2 mg/ml fatty acid– free BSA (Sigma) and 2 mM EDTA on ice after the method of Tcherny- infiltrate (Fig. 1B–E). Given that the maximal Con A inflammatory chev et al. (17). Macrophages were diluted to 106 cells/ml in HBSS with 2 response occurred at 24 h, we chose this time point for subsequent mg/ml fatty acid–free BSA at 37˚C. These cells were immediately intro- experiments. The Journal of Immunology 3

FIGURE 1. Macrophages express the highest levels of a1 integrin in the inflamed footpad. (A) Con A–induced swelling reaches a maximum at 24 h. Footpad thickness was measured with a Mitutoyo micrometer, and a time-dependent increase in percent swelling relative to the contralateral control footpad was observed (two independent experiments, n = 5, mean 6 SEM). (B–E) Con A induces a highly cellular inflammatory exudate in the mouse footpad. H&E-stained cross sections of the normal (B, C) and 24-h Con A–inflamed footpads (D, E). Dashed boxes in low-magnification (original magnification 34) views (B, D) indicate the location of the intermediate-magnification (original magnification 320) views (C, E). Representative images from two independent experiments with n = 4 mice). (F) Flow cytometry of pooled popliteal LNs draining 24-h Con A–inflamed footpads (three independent experiments, n =9, three LNs from three mice per experiment). Cytospins of sorted cells were based on the indicated gates, and percentages were calculated from the immediately preceding parent gates. Representative images of cytospins: macrophages (Macs), eosinophils (Eos), and neutrophils (Neuts). Scale bars, 20 mm. (G) Flow cytometry of digested 24-h Con A–inflamed footpads gated on total CD45+ cells showing a1b1 integrin expression (Figure legend continues) 4 a1b1 INTEGRIN-MEDIATED ADHESION

Cell suspensions from digested footpads and draining popliteal than the footpad, and the LN consists of entirely CFSE2 cells. LNs were stained for multiple cell-surface markers and analyzed by Previous studies using s.c. injected lymphocytes have shown that flow cytometry. A combination of markers including the GR-1 Ab, beyond 16 h, cells can be found in the circulation (26). Thus, at 4 CD11b, and side-scatter signal allowed us to identify specific and 8 h, the CFSE+ leukocytes had migrated directly from the myeloid subsets (20) (Fig. 1B). Cells were sorted and cytospins inflamed footpad and reached the popliteal LN via afferent lym- performed to validate this gating strategy, and we could clearly phatics, but had not yet entered the circulation. Based on these identify macrophages, eosinophils, and neutrophils microscopically data and prior studies showing that in vivo CFSE stabilizes by 4 h (Fig. 1F). Analysis of CD45+ cells in footpad single-cell suspen- (28), we chose the 4-h time point for subsequent investigations. sions revealed that macrophages expressed the highest levels of Experiments with pertussis toxin inhibited the accumulation of a1b1 integrin (Fig. 1G). We also found that blood monocytes and CFSE+ leukocytes in draining popliteal LN and confirmed that macrophages in normal and inflamed LNs preferentially express CFSE dye does not diffuse out of the footpad and label cells in the a1b1 integrin (Supplemental Fig. 1). Prior studies established the node (Supplemental Fig. 2). In summary, we developed a physio- expression a1b1 integrin on T cells in chronic inflammatory logical bioassay in which ISPL fluorescently labels a “cohort” of lesions and in vitro on macrophages (13, 16). Our data establish endogenous leukocytes in the inflamed footpad. These CFSE+ that in vivo monocytes express a1b1 integrin in the blood, and cells progressively exit the footpad and accumulate in the draining macrophages express a1b1 integrin in an acute inflammatory le- LN where they can be isolated and analyzed by flow cytometry. sion and the draining LN. 2 2 Itga1 / macrophages display accelerated exit from the Exit of leukocytes from the inflamed footpad can be tracked by inflamed footpad ISPL using the fluorescent dye CFSE We investigated the effect of genetic deletion of a1b1 integrin and In large animals, it has been possible to assess the exit of endog- how this affected leukocyte migration from the inflamed footpad enous leukocytes from peripheral tissues by surgical cannulation of to the draining LN. Inflamed footpads were treated with ISPL at afferent lymphatics (21–23). In murine models, data on leukocyte 24 h, and 4 h later, draining popliteal LNs were isolated (Fig. 3A). exit from tissues have been generated by adoptive transfer ex- LNs were then analyzed for CFSE+ cells of each subtype, and data periments including s.c. injection of leukocytes into footpads or were normalized to wild type Itga1+/+ migration (Fig. 3B, 3C). intratracheal delivery into the lungs (24–27); studies transferring Both Itga1+/+ and Itga12/2 CFSE+ macrophages were found in leukocytes into mouse footpads have established the draining draining popliteal LNs. However, CFSE+ macrophages consis- popliteal LN as their first destination, where they accumulate over tently displayed more migration in Itga12/2 mice relative to time. Itga1+/+ mice (Fig. 3C). In contrast, the migration of other CFSE+ Analogous to these adoptive transfer experiments, we sought to leukocyte subsets was similar in the draining popliteal LNs (Fig. assay the exit of endogenous leukocytes from the inflamed mouse 3C). These data suggested the possibility that Itga12/2 macro- footpad to the draining popliteal LN. We developed an ISPL tech- phages exited more efficiently from the inflamed footpad and this nique to fluorescently label endogenous leukocytes exclusively in the could account for their greater percentage in the draining LN. We inflamed footpad with CFSE (Fig. 2A). This technique avoids hypothesized that a1b1 integrin–mediated adhesion was “an- spillover of dye and resultant “bystander” labeling of the draining choring” macrophages in the inflamed footpad, effectively inhib- LN by transient occlusion of the draining lymphatic vessels. Dye iting their exit. spillover and “bystander” labeling of the draining LN would have We also considered alternative mechanisms that could have made it impossible to determine from where CFSE+ cells originated. explained our results. Collagen IV is the principal ligand for a1 CFSE is highly labile in aqueous environments; thus, rapid deacti- integrin, and collagen IV is highly expressed in basement mem- vation occurs in aqueous solutions in vitro and in tissues in vivo (28, branes. To verify that these well-known expression patterns were 29). Within 5 min after footpad injection, CFSE is rendered inert and similar in both Itga12/2 and Itga1+/+ mice, we stained histological unable to effectively label cells. Over time, however, CFSE+ cells sections for basement membranes and collagen IV (Supplemental migrate via afferent lymphatics and accumulate in the draining LN. Fig. 3). Similar expression patterns of basement membrane, as We analyzed footpads and their draining popliteal LNs 15 min well as abundance of collagen IV, were seen in both Itga12/2 and after ISPL. Approximately 70% of leukocytes in the excised Itga1+/+ mice (Supplemental Fig. 3). Another consideration we footpad tissue were CFSE+, whereas the draining popliteal LN was had was that, in theory, a different collagen-binding integrin could devoid of CFSE+ cells (Fig. 2B). CFSE+ leukocytes could be potentially “compensate” for a1 integrin deficiency. The collagen detected4hafterISPLinthedrainingpopliteal LN, and the I–binding integrin a2 is one possibility because collagen I is percentage of CFSE+ cells decreases in the inflamed footpad from plentiful in extravascular tissues. We stained for a2integrinin 15 min to 4 h (Fig. 2B). This is because some of the CFSE+ cells the inflamed LN and found that expression levels were com- exit the footpad and migrate to the draining LN. Furthermore, parable in Itga1+/+ and Itga12/2 mice (Supplemental Fig. 4A). unlabeled (CFSE2) leukocytes are recruited from the blood to the This establishes that collagen IV expression is similar between the Con A–inflamed footpad during the experiment and over time, genotypes, as is expression of an alternate collagen receptor a2 these cells significantly outnumber the CFSE+ population that integrin. Thus, these possible alternative mechanisms cannot ex- remains in the footpad tissue. plain our results. A time course was performed to characterize the exit of leu- To rule out the possibility that there were differences in the in- kocytes from the inflamed footpad. This showed that maximal LN flammatory responses between Itga1+/+ and Itga12/2 mice, we accumulation of CF45+CFSE+ leukocytes occurred between 4 and analyzed several parameters of inflammation and at 24 h, the in- 8 h after ISPL (Fig. 2C, 2D). The percentage of CFSE+ cells in the flammatory responses of these two genotypes were similar. There LN is relatively small because the LN has vastly more leukocytes were no statistical differences in comparing the relative abundance

(white histograms). The negative control (gray) is based on footpad cells from Con A–inﬂamed Itga12/2 mice using the same staining protocol. Rep- resentative staining of digested footpads from four independent experiments with n = 4 mice. The Journal of Immunology 5

FIGURE 2. Leukocytes in the inflamed footpad can be tracked to the draining LN using ISPL. Flow cytometry analysis was performed using cells isolated from 24-h Con A–inflamed and CFSE pulse-labeled footpads and draining popliteal LNs. (A) Schematic illustration of ISPL wherein cells are labeled in the footpad, exit via afferent lymphatics, and accumulate in the draining LN over time. (B) The percent of CFSE+ leukocytes in the inflamed footpad (FP) and the corresponding popliteal LN at 15 min and 4 h after ISPL. Representative plots from two independent experiments with four mice. (C and D) CFSE+ cells in draining popliteal LNs after ISPL at indicated time points, showing accumulation of CFSE+ cells that migrated from the footpad to the LN. Representative flow cytometry plots and data (mean 6 SEM) from three independent experiments with six to nine mice per time point. of common proinflammatory cytokine mRNAs, the absolute num- Although consistent with our hypothesis, these results could ber of cells in the draining popliteal LNs, footpad swelling, or potentially be attributed to treatment effects on the draining histological appearance (Fig. 3D–G). Thus, we conclude that the popliteal LN. It is assumed that CFSE+ leukocyte accumulation in observed difference in macrophage migration is unlikely due to the popliteal LN is consistent across different treatments. How- differences in inflammatory responses between the two genotypes. ever, Ab injected into the footpad would reach the draining LN If Itga12/2 macrophages exited more efficiently from the in- and could potentially affect leukocyte accumulation. To address flamed footpad, this could influence the resolution of inflamma- this possibility, we injected Evans blue dye directly into the tion. Con A induces an acute inflammatory response and swelling in footpad lesion and compared this with a calf injection (distal calf). the footpad that mostly resolves by 48 h (Fig. 1A). Nevertheless, When injected into the calf, Evans blue dye could not be detected we compared the relative abundance of proinflammatory cytokine in the inflamed footpad lesion (Fig. 4D), but calf injections still mRNAs in Itga1+/+ and Itga12/2 mice at 48 h, in addition to our reach the draining popliteal LN (20). Therefore, we used ISPL and 24-h data (Supplemental Fig. 4B). In contrast with the 24-h time injected a1b1 neutralizing Ab into the calf, outside of the in- point (Fig. 3D), we found that IL-1b and TNF-a mRNA levels were flammatory lesion (Fig. 4C). Results show that calf injections of reduced in Itga12/2 mice relative to wild type mice. This is con- a1b1 integrin neutralizing Ab have no effect on macrophage sistent with the possibility that more efficient exit of macrophages accumulation in the draining popliteal LN (Fig. 4C). The Ab from the site of inflammation leads to reduced local proinflammatory blockade only enhances macrophage exit if delivered directly into cytokine production and accelerated resolution of inflammation. the inflamed footpad. Given the bivalent nature of IgG molecules, it was possible that a b Blockade of 1 1 integrin results in accelerated macrophage the blocking Ab was cross-linking a1b1 integrin, which could exit from the inflamed footpad potentially induce “outside-in” signaling. The increased accumu- If adhesion via a1b1 integrin was retaining macrophages in the lation of macrophages in the popliteal LNs of mice treated with inflamed footpad, then disrupting its adhesive function should neutralizing Ab could be attributed to integrin cross-linking and result in more macrophages exiting the footpad over time. Mouse signaling, rather than a blockade of adhesion. To control for the footpads were inflamed with Con A and 24 h later, neutralizing Ab possible effects of integrin cross-linking, we sought to perform to a1b1 integrin was injected directly into the footpad, contem- a monovalent blockade of a1b1 integrin. To this end, we sought poraneously with ISPL (Fig. 4A). We compared this with control out a small-molecule inhibitor of a1b1 integrin adhesion. Small- IgG injections using the same protocol (Fig. 4A), and performed molecule integrin inhibitors have been described and are known as flow cytometry on single-cell suspensions from the draining LNs disintegrins, some of which are derived from viper venom. (Fig. 4B). Over a 4-h period, draining popliteal LNs were enriched Proteins from viper venom have been shown to have a wide va- for CFSE+ macrophages by ∼2-fold when footpads were treated riety of biological effects, some of which are specific to integrins with a1b1 blocking Ab as compared with control IgG (Fig. 4B, (30). The disintegrin Obtustatin, from the blunt-nosed viper Vipera 4C). Blockade of a1b1 integrin had no effect on other CFSE+ lebetina obtusa, is one such molecule. Obtustatin is a 41-aa leukocyte subsets (Fig. 4C). monovalent disintegrin (31), and it has been shown to block the 6 a1b1 INTEGRIN-MEDIATED ADHESION

A Con A CFSE or Diluent LN isolation C 3 -24 h 0 h 4 h * Itga1+/+ Itga1-/- B Diluent CFSE 2 FIGURE 3. Macrophage exit is more efﬁcient 0.03 10.57 1 from the footpads of Itga12/2 relative to Itga1+/+ Macs mice, although the Con A inﬂammatory response relative to wild type

Fold change in migration 0 at 24 h is similar in both genotypes. (A) Timeline MacsEos Neuts CD11cT cells B cells of footpad injections and isolation of draining 0.00 2.39 +/+ 2/2 D 1000 Itga1+/+ popliteal LNs in Itga1 and Itga1 mice. (B) Eos Itga1-/- Flow cytometry CFSE gating strategy for popli- 100 teal LN leukocyte subtypes is illustrated. In each 10

independent experiment, inﬂamed footpads were expression 0.00 7.20 ND

Relative mRNA 1 injected with diluent or CFSE, and the CFSE gate Neuts IFN-γ IL-1β IL-6 TNFα IL-4 was set based on diluent-injected footpads. (C) + +/+ Recovery of CFSE cells in draining popliteal E 6 Itga1 Itga1-/- ) ) 5 ) 6 6 LNs. Flow cytometry data are presented as the 0.48 15.09 4 5 fold change relative to the mean of the Itga1+/+ 4 4 4 group (three independent experiments with n =6 CD11c 3 3 3 or 7 mice, mean 6 SD, *p # 0.05, Student t test). 2 2 2 1 1 1 LN cells (x 10 LN cells (x 10 D–G LN cells (x 10 ( ) Comparison of inflammatory responses 0.07 1.20 0 0 0 24 h after Con A injection reveal no statistical 2 2 Eos differences between Itga1 / and Itga1+/+ mice. T cells Total Macs Neuts CD11c T cells B cells (D) Relative mRNA abundance of selected pro- F 60 +/+ inflammatory cytokines from footpads. Data Itga1 Itga1-/- were normalized to contralateral noninflamed 0.08 0.82 40 footpads (three independent experiments with n = B cells 20 6 E 3 or 4 mice, mean SEM). ( ) Absolute num- Increase in thickness (%) bers of cells in draining popliteal LNs (three in- FSC 0 dependent experiments with n = 6 or 7 mice, CFSE Footpads (24 h) mean 6 SD). (F) Percent increase in footpad G Itga1+/+ Itga1-/- swelling (two independent experiments with n = 4 or 5 mice, mean 6 SEM). (G) Comparable histological features in footpads (representative images from two independent experiments with n = 4 mice). Scale bars, 20 mm. ND, Not detected.

adhesion of a1b1 integrin to collagen IV with high specificity. and T cells from Itga12/2 mice exhibit reduced adhesion to col- Because Obtustatin interacts with a1b1 integrin monovalently, we lagen IV (13, 14, 31). Macrophages in the inflamed footpad ex- could perform a functional blockade without cross-linking the press relatively high levels of a1b1 integrin compared with other integrin (31). cell types (Fig. 1C), but whether macrophages also use a1b1 We initiated a blockade of a1b1 integrin in the inflamed mouse integrin to adhere to collagen IV is unknown. We therefore per- footpads using Obtustatin or its inactivated form ethylpyridylated- formed in vitro detachment assays to competitively assess the Obtustatin (EP-Obtustatin) as a control (31). Similar to the Ab relative strength of adherence between Itga1+/+ and Itga12/2 blockade, Obtustatin treatment resulted in a modest but repro- macrophages. ducible enrichment of CFSE+ macrophages in the draining LN Macrophages were harvested from inflamed peritoneal cavities when compared with controls (Fig. 4E). Consistent with the Ab of Itga1+/+ and Itga12/2 mice, differentially labeled in vitro with treatments, increased migration was only seen in the macrophage either CFSE or CMPTX (Cell tracker red), then admixed in ap- population (Fig. 4E). Given these results and the monovalent na- proximately equal numbers (Fig. 5A). These macrophages were ture of Obtustatin, it is unlikely that the altered macrophage mi- introduced into a parallel plate flow chamber system coated with gration was due to “outside-in” signaling triggered by integrin collagen IV and allowed to adhere (Fig. 5A). After exposure to cross-linking caused by Ab treatment. These data reinforce the a detaching shear force of 10 dynes/cm2,anaverageof∼56% hypothesis that a1b1 integrin specifically mediates macrophage more Itga1+/+ macrophages remained adherent to the collagen adhesion and retention in the inflamed footpad, inhibiting mac- IV–coated plates compared with Itga12/2 macrophages (Fig. 5B). rophage exit. Pretreatment of macrophages with either a1b1 integrin blocking Ab or Obtustatin eliminated this difference (Fig. 5B). These Adhesion to collagen IV reduces macrophage chemotactic results provide direct evidence that Itga12/2 macrophages have a migration efficiency relative deficiency in adhesion to collagen IV and that a1b1integrin We next tested the adhesive function of a1b1 integrin in vitro. mediates this adhesion. Previous studies have shown that collagen IV is a relatively spe- To investigate the role of a1b1 integrin–mediated adhesion cific ECM ligand for a1b1 integrin, and that embryonic fibroblasts in the context of migration, we performed chemotaxis assays. The Journal of Immunology 7

FIGURE 4. Blockade of a1b1 integrin results in more efficient macrophage exit from inflamed footpads. (A) Experimental timeline showing footpad treatments and isolation of the draining popliteal LN. (B) Flow cytometry gating strategy for CFSE+ LN cells at 4 h after ISPL. In each independent experiment, the CFSE gate was set based on diluent-injected inflamed footpads. The CD11c gate was set based on the fluorescence minus one. (C) Leukocyte exit efficiency relative to the mean of the IgG control treatment group. Function-blocking Ab to a1 integrin (Ha31/8) was injected directly into the footpad or into the calf. Control footpads were injected with IgG. Three independent experiments with n = 6–11 mice (mean 6 SEM). Only the macrophage population shows statistical significance (*p # 0.05 by ANOVAwith Bonferroni post test). (D) The distribution of Evans blue dye 30 min after injection into the footpad (left) or the distal calf (right). The right footpad of both mice was inflamed for 24 h with Con A. Blue dye is not detectable in the footpad of the calf-injected mouse. (E) Leukocyte exit efficiency using direct footpad injections of either Obtustatin, a specific a1 integrin blocking protein (disintegrin), or inactivated Obtustatin (EP-Obtustatin). Data are presented as the fold change relative to the mean of the EP-Obtustatin treatment group. Three independent experiments with n = 6–11 mice (mean 6 SEM). Only the macrophage population shows statistical significance (*p # 0.05, Student t test).

Transwell microporous membranes were coated with either con- CCR7 and sphingosine-1 phosphate (S1P) receptor, the S1P re- trol BSA or different ECM molecules: collagen IV, collagen I, ceptor having originally been discovered as crucial for lymphocyte laminin, or an ECM gel containing multiple ECM molecules in- exit from secondary lymphoid organs (24, 26, 27, 33). In contrast, cluding 70% collagen IV. Peritoneal macrophages from Itga1+/+ leukocyte entry into tissues from the blood is governed by a multi- and Itga12/2 mice were differentially labeled, admixed, and step cascade that is mediated by a series of adhesion molecules placed in the upper chamber; the lower chamber contained the including integrins (2, 34). The adhesive role of integrins in the chemokine CCL21, creating a chemotactic gradient to drive mi- blood to tissue leukocyte migration is well established. The ob- gration (Fig. 5C). jective of this study was to investigate the potential role of integrin- Consistent with our prediction, when membranes were coated mediated adhesive mechanisms in controlling leukocyte exit from with collagen IV–containing substrates, Itga1+/+ macrophage a peripheral inflammatory lesion. migration was less efficient compared with Itga12/2 macrophages Our data demonstrate a role for integrin-mediated adhesion of (Fig. 5C). However, there was no difference in macrophage mi- macrophages in inflamed tissues, specifically the collagen IV– gration efficiency across substrates that lacked collagen IV such binding integrin a1b1. To our surprise, rather than functioning in as BSA, collagen I, or laminin (Fig. 5C). These data support the a promigratory capacity, a1b1 integrin inhibited macrophage exit hypothesis that macrophages expressing a1b1 integrin adhere to from a peripheral inflammatory lesion. Staining of the inflamed collagen IV and this decreases their chemotactic migration effi- mouse footpad cells revealed that macrophages expressed the ciency. highest levels of a1b1 integrin (Fig. 1G). Consistent with this staining pattern, a1b1 integrin blockade or genetic deletion af- Discussion fected the exit of macrophages from the inflammatory lesion, but Leukocytes at inflammatory lesions constitute an important com- not other leukocytes. We postulate that a1b1 integrin “anchors” ponent of the immune response. The accumulation and persistence macrophages in the inflamed footpad, inhibiting their exit. To our of leukocytes in inflamed tissues is regulated by multiple mech- knowledge, this is the first study to directly demonstrate the anisms including the balance between leukocyte entry from the migration-inhibiting function of a leukocyte integrin from a periph- blood and leukocyte exit to afferent lymphatics and the draining eral inflammatory lesion. LN (32). Leukocyte exit from peripheral tissues relies on the We present a novel experimental technique that we have called expression of G protein–coupled chemokine receptors such as ISPL. Using the fluorescent dye CFSE, ISPL exclusively labels 8 a1b1 INTEGRIN-MEDIATED ADHESION

ligands induced neutrophil-dominated responses, consistent with previously published data (38–40). This led us to select the mannan binding lectin Con A. Con A–induced robust inflammation and lesions contained a heterogeneous population of inflammatory cells including a broad range of different leukocyte subsets (18, 19) (Figs. 1B–G, 3G). Moreover, there were no significant differences in the inflammatory responses between Itga12/2 and Itga1+/+ mice, suggesting the effect on macrophages was not due to differences in inflammation (Fig. 3D–G). Thus, the Con A inflammatory reaction allowed us to analyze, compare, and contrast a mixed population of inflammatory leukocytes exiting the footpad. We specifically tested macrophage adhesion and migration to collagen IV substrates in vitro. Collagen IV is a specific ligand for a1b1 integrin (11). Wild-type macrophage adhesion to collagen IV was strengthened via a1b1 integrin relative to Itga12/2 macrophages; blocking of a1b1 integrin abolished this adhesive advantage (Fig. 5A, 5B). The function of a1b1 integrin was also tested in transwell chemotaxis experiments. Macrophages migrated to the CCR7 ligand CCL21, but transwell membranes coated with substrates containing collagen IV inhibited wild type macrophage migration (Fig. 5C). Wild-type macrophage migra- FIGURE 5. Itga12/2 macrophages display an adhesion deficit to col- tion efficiency could be reduced to as low as ∼25% relative to 2 2 lagen IV and migrate across collagen IV–coated membranes with higher Itga1 / macrophages, and this was achieved by coating transwell efficiency relative to Itga1+/+ macrophages. (A) Representative images of membranes with ECM gel (∼70% collagen IV; Fig. 5C). The adherent macrophages. Original magnification 320. (B) Data from de- ECM gel was visibly thicker than other coatings, and we speculate +/+ 2/2 tachment assays. Itga1 and Itga1 macrophages were labeled with that this provided more ligand binding sites and thus a greater either CFSE (green) or CMPTX (red), incubated with BSA, Ha31/8, or opportunity for adhesion via a1b1 integrin. These data show that Obtustatin, and infused onto collagen IV–coated parallel plate flow macrophage chemotactic migration can be inhibited by a1b1 chambers. After 5 min (preshear), macrophages were exposed to fluid shear stress (10 dynes/cm2, 1 min), and the percentage of macrophages integrin binding to collagen IV. remaining adherent (postshear) was calculated for each genotype. Three The a1b1 integrin is important for initiating inflammation, as independent experiments with n = 11 (mean 6 SEM, *p # 0.05, Student t well as the maintenance of chronic inflammatory responses. Mice test). (C) Chemotactic migration of differentially labeled and admixed genetically deficient in a1b1 integrin or treated with systemic Itga1+/+ and Itga12/2 macrophages. Transwell membranes were coated a1b1 integrin neutralizing Ab show decreased inflammation in the with the indicated substrates (ECM = ECM gel). The lower chamber effector phase of T cell–mediated responses such as delayed-type contained CCL21 (10 nM). Macrophages that migrated from the upper hypersensitivity, decreased chronic inflammation, and decreased into the lower chamber over 18 h were collected and analyzed by flow resistance to viral infection (12, 13, 15, 35, 37). Intriguingly, re- cytometry. Data are presented as percent of input cells, and all groups were 2 2 duced numbers of T cells were found at inflammatory lesions in normalized to the mean of the Itga1 / group (3 independent experiments 2 2 Itga1 / mice or mice treated with a1b1 integrin neutralizing Ab, with n = 6–12, mean 6 SEM, *p # 0.05, Student t test). and it was postulated that a1b1 integrin was “retaining” T cells at these inflammatory sites (12, 15, 35). This attractive hypothesis is a cohort of endogenous leukocytes in the inflamed footpad, but the consistent with our results showing that a1b1 integrin inhibits draining LN remains unlabeled (Fig. 2B). Over time, leukocytes macrophage exit from the inflamed footpad. Subsequent studies exit the inflamed footpad via afferent lymphatics and accumulate showed that T cell–dependent inflammatory responses required in the popliteal LN. Accumulated CFSE+ cells in the LN can then T cell–macrophage interaction for effective macrophage produc- be isolated and identified by flow cytometry. The major advantage tion of proinflammatory cytokines (16). This novel macrophage of our approach is the labeling of endogenous leukocyte pop- activation pathway depends on the direct binding of Sema7A on ulations in situ in mice. This is a significant advance because Tcellstoa1b1 integrin on macrophages. Notably, both Sema7A2/2 leukocytes in inflamed tissues may represent unique populations and Itga12/2 mice are deficient in the effector phase of T cell– with specific localizations in the tissue microenvironment. In ad- mediated inflammatory responses establishing an in vivo role for dition, the endogenous distribution of leukocytes may be impor- these molecules (13, 16). Macrophages are critical for peripheral tant for their access to adhesive and chemotactic ligands. It is our inflammatory responses, and their presence or absence can have hope that ISPL will complement other approaches and help ex- dramatic effects (41, 42). Given that the persistence of macrophages pand the repertoire of useful in vivo methods. in tissues and that the cytokines they secrete are important in the During the process of choosing an inflammatory stimulus, we maintenance of inflammatory responses, we speculate that more considered several important factors including effective recruit- rapid macrophage exit could lead to decreased inflammation. This ment of a heterogeneous population of leukocytes and induction of implies that control of macrophage exit from tissues could represent 2 2 comparable acute inflammatory responses in both Itga1 / and an additional regulatory mechanism for inflammatory processes. Itga1+/+ mice to enable comparisons between these genotypes. However, whether a1b1 integrin can modulate these responses via 2 2 Because Itga1 / mice or mice systemically perfused with a1b1 macrophage persistence or exit from inflamed tissues remains to be integrin blocking Ab have reduced T cell–mediated immune elucidated. responses (15, 16, 35–37), we considered T cell–independent in- Integrins involved in blood-to-tissue migration, such as a4b1or flammatory stimuli. Initially, we used TLR ligands such as LPS aLb2 (LFA-1), are constitutively expressed on particular leuko- and polyinosinic-polycytidylic acid. However, we found that TLR cyte subsets and, in fact, require a chemokine receptor–mediated The Journal of Immunology 9 activation step for effective adhesion (7, 34). In contrast, in T cells Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 453: 51–55. a1b1 integrin generally requires an inflammatory stimulus to be 7. Hynes, R. O. 2002. Integrins: bidirectional, allosteric signaling machines. Cell expressed and to date has been found predominantly in extra- 110: 673–687. vascular tissues (15, 43). Moreover, unlike a4b1oraLb2, genetic 8. Hemler, M. E. 1990. VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu. Rev. Immunol. 8: 365–400. deletion or blockade of a1b1 integrin has no effect on blood-to- 9. Hemler, M. E., C. Huang, and L. Schwarz. 1987. The VLA protein family. tissue leukocyte recruitment (12,15).Inextravasculartissues, Characterization of five distinct cell surface heterodimers each with a common leukocyte exit is mediated by chemokine receptors such as CCR7 130,000 molecular weight beta subunit. J. Biol. Chem. 262: 3300–3309. 10. Takada, Y., C. Huang, and M. E. Hemler. 1987. Fibronectin receptor structures in (24, 26), as well as S1P receptor (27). Leukocytes are chemo- the VLA family of heterodimers. Nature 326: 607–609. tactically attracted to afferent lymphatics that express the CCR7 11. Vandenberg, P., A. Kern, A. Ries, L. Luckenbill-Edds, K. Mann, and K. Ku¨hn. 1991. Characterization of a type IV collagen major cell binding site with affinity ligand CCL21 (44). CCR7-dependent tissue exit has been ob- to the alpha 1 beta 1 and the alpha 2 beta 1 integrins. J. Cell Biol. 113: 1475– served in both homeostatic and inflammatory conditions (24, 26). 1483. In contrast, we demonstrate that a1b1 integrin adhesion inhibits 12. Andreasen, S. O., A. R. Thomsen, V. E. Koteliansky, T. I. Novobrantseva, A. G. Sprague, A. R. de Fougerolles, and J. P. Christensen. 2003. Expression and macrophage exit from a peripheral inflammatory lesion (Figs. 3C, functional importance of collagen-binding integrins, alpha 1 beta 1 and alpha 2 4C, 4E). We speculate that there could be an antagonistic rela- beta 1, on virus-activated T cells. J. Immunol. 171: 2804–2811. tionship between a1b1 integrin and chemokine receptors such as 13. de Fougerolles, A. R., A. G. Sprague, C. L. Nickerson-Nutter, G. Chi-Rosso, P. D. Rennert, H. Gardner, P. J. Gotwals, R. R. Lobb, and V. E. Koteliansky. CCR7. This implies that balancing these opposing forces would 2000. Regulation of inflammation by collagen-binding integrins alpha1beta1 and be important in determining the extent of exit or persistence of alpha2beta1 in models of hypersensitivity and arthritis. J. Clin. Invest. 105: 721– 729. macrophages in tissues; however, this remains to be investigated. 14. Gardner, H., J. Kreidberg, V. Koteliansky, and R. Jaenisch. 1996. Deletion of Based on our studies, we speculate that other integrins might integrin alpha 1 by homologous recombination permits normal murine devel- function to inhibit exit of leukocytes from the periphery in a manner opment but gives rise to a specific deficit in cell adhesion. Dev. Biol. 175: 301– 313. similar to a1b1 integrin. ECM binding integrins could be con- 15. Ray, S. J., S. N. Franki, R. H. Pierce, S. Dimitrova, V. Koteliansky, sidered prime candidates and include a2b1, a5b1 a6b1, and A. G. Sprague, P. C. Doherty, A. R. de Fougerolles, and D. J. Topham. 2004. The avb3 (7). The inflammatory state of the tissue milieu is also likely collagen binding alpha1beta1 integrin VLA-1 regulates CD8 T cell-mediated immune protection against heterologous influenza infection. Immunity 20: to be important and could affect both the expression patterns and 167–179. function of leukocyte integrins. 16. Suzuki, K., T. Okuno, M. Yamamoto, R. J. Pasterkamp, N. Takegahara, H. Takamatsu, T. Kitao, J. Takagi, P. D. Rennert, A. L. Kolodkin, et al. 2007. In conclusion, we have demonstrated a new role for leukocyte Semaphorin 7A initiates T-cell-mediated inflammatory responses through integrins in inflamed extravascular tissues. In addition to the well- alpha1beta1 integrin. Nature 446: 680–684. described promigratory role of integrins in the blood-to-tissue 17. Tchernychev, B., B. Furie, and B. C. Furie. 2003. Peritoneal macrophages express both P-selectin and PSGL-1. J. Cell Biol. 163: 1145–1155. paradigm, we show that a1b1 integrin adhesion specifically in- 18. Kulidjian, A. A., A. C. Issekutz, and T. B. Issekutz. 2002. Differential role of E- hibits macrophage exit from a peripheral inflammatory lesion. selectin and P-selectin in T lymphocyte migration to cutaneous inflammatory Given the important functions of macrophages as multifunctional reactions induced by cytokines. Int. Immunol. 14: 751–760. 19. Shier, W. T., J. T. Trotter, III, and C. L. Reading. 1974. Inflammation induced by phagocytes involved in the initiation, maintenance, and resolution concanavalin A and other lectins. Proc. Soc. Exp. Biol. Med. 146: 590–593. of inflammation (41, 42, 45–47), the migration-inhibiting function 20. Iannacone, M., E. A. Moseman, E. Tonti, L. Bosurgi, T. Junt, S. E. Henrickson, S. P. Whelan, L. G. Guidotti, and U. H. von Andrian. 2010. Subcapsular sinus of a1b1 integrin may be considered significant. Our data identify macrophages prevent CNS invasion on peripheral infection with a neurotropic a1b1 integrin as a possible target for regulating macrophage mi- virus. Nature 465: 1079–1083. gration at an inflammatory site, the broader implication being the 21. Issekutz, T. B., W. Chin, and J. B. Hay. 1980. Lymphocyte traffic through granulomas: differences in the recovery of indium-111-labeled lymphocytes in potential to modulate local inflammation and immune responses. afferent and efferent lymph. Cell. Immunol. 54: 79–86. 22. Lascelles, A. K., and B. Morris. 1961. Surgical techniques for the collection of Acknowledgments lymph from unanaesthetized sheep. Q. J. Exp. Physiol. Cogn. Med. Sci. 46: 199– 2/2 205. We thank A. Pozzi (Vanderbilt University, Nashville, TN) for Itga1 23. Young, A. J., J. B. Hay, and C. R. Mackay. 1993. Lymphocyte recirculation and mice, C. Marcinkiewicz (Temple University, Philadelphia, PA) for Obtus- life span in vivo. Curr. Top. Microbiol. Immunol. 184: 161–173. tatin and EP-Obtustatin, D. Topham (University of Rochester, Rochester, 24. Bromley, S. K., S. Y. Thomas, and A. D. Luster. 2005. Chemokine receptor NY) for directly conjugated Ha31/8 Ab, C. Guidos and S. Zhao (University CCR7 guides T cell exit from peripheral tissues and entry into afferent lymphatics. Nat. Immunol. 6: 895–901. of Toronto) for advice on flow cytometry, and S. Winer (University of 25. Cavanagh, L. L., R. Bonasio, I. B. Mazo, C. Halin, G. Cheng, A. W. van der Toronto) for comments on the manuscript. Velden, A. Cariappa, C. Chase, P. Russell, M. N. Starnbach, et al. 2005. Acti- vation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells. Nat. Immunol. 6: 1029–1037. Disclosures 26. Debes, G. F., C. N. Arnold, A. J. Young, S. Krautwald, M. Lipp, J. B. Hay, and The authors have no financial conflicts of interest. E. C. Butcher. 2005. Chemokine receptor CCR7 required for T lymphocyte exit from peripheral tissues. Nat. Immunol. 6: 889–894. 27. Ledgerwood, L. G., G. Lal, N. Zhang, A. Garin, S. J. Esses, F. Ginhoux, M. Merad, H. Peche, S. A. Lira, Y. Ding, et al. 2008. The sphingosine 1-phos- References phate receptor 1 causes tissue retention by inhibiting the entry of peripheral 1. Butcher, E. C., and L. J. Picker. 1996. Lymphocyte homing and homeostasis. tissue T lymphocytes into afferent lymphatics. Nat. Immunol. 9: 42–53. Science 272: 60–66. 28. Becker, H. M., M. Chen, J. B. Hay, and M. I. Cybulsky. 2004. Tracking of 2. Ley, K., C. Laudanna, M. I. Cybulsky, and S. Nourshargh. 2007. Getting to the leukocyte recruitment into tissues of mice by in situ labeling of blood cells with site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immu- the fluorescent dye CFDA SE. J. Immunol. Methods 286: 69–78. nol. 7: 678–689. 29. Quah, B. J., H. S. Warren, and C. R. Parish. 2007. Monitoring lymphocyte 3. Berlin, C., R. F. Bargatze, J. J. Campbell, U. H. von Andrian, M. C. Szabo, proliferation in vitro and in vivo with the intracellular fluorescent dye carbox- S. R. Hasslen, R. D. Nelson, E. L. Berg, S. L. Erlandsen, and E. C. Butcher. yfluorescein diacetate succinimidyl ester. Nat. Protoc. 2: 2049–2056. 1995. alpha 4 integrins mediate lymphocyte attachment and rolling under 30. Gould, R. J., M. A. Polokoff, P. A. Friedman, T. F. Huang, J. C. Holt, J. J. Cook, physiologic flow. Cell 80: 413–422. and S. Niewiarowski. 1990. Disintegrins: a family of integrin inhibitory proteins 4. Jalkanen, S., A. C. Steere, R. I. Fox, and E. C. Butcher. 1986. A distinct en- from viper venoms. Proc. Soc. Exp. Biol. Med. 195: 168–171. dothelial cell recognition system that controls lymphocyte traffic into inflamed 31. Marcinkiewicz, C., P. H. Weinreb, J. J. Calvete, D. G. Kisiel, S. A. Mousa, synovium. Science 233: 556–558. G. P. Tuszynski, and R. R. Lobb. 2003. Obtustatin: a potent selective inhibitor of 5. Johnson, L. A., S. Clasper, A. P. Holt, P. F. Lalor, D. Baban, and D. G. Jackson. alpha1beta1 integrin in vitro and angiogenesis in vivo. Cancer Res. 63: 2020– 2006. An inflammation-induced mechanism for leukocyte transmigration across 2023. lymphatic vessel endothelium. J. Exp. Med. 203: 2763–2777. 32. Issekutz, T. B., G. W. Chin, and J. B. Hay. 1981. Lymphocyte traffic through 6. La¨mmermann, T., B. L. Bader, S. J. Monkley, T. Worbs, R. Wedlich-So¨ldner, chronic inflammatory lesions: differential migration versus differential retention. K. Hirsch, M. Keller, R. Fo¨rster, D. R. Critchley, R. Fa¨ssler, and M. Sixt. 2008. Clin. Exp. Immunol. 45: 604–614. 10 a1b1 INTEGRIN-MEDIATED ADHESION

33. Matloubian, M., C. G. Lo, G. Cinamon, M. J. Lesneski, Y. Xu, V. Brinkmann, 40. Medzhitov, R. 2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol. M. L. Allende, R. L. Proia, and J. G. Cyster. 2004. Lymphocyte egress from 1: 135–145. thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 41. Thepen, T., A. J. van Vuuren, R. C. Kiekens, C. A. Damen, W. C. Vooijs, and 427: 355–360. J. G. van De Winkel. 2000. Resolution of cutaneous inflammation after local 34. Butcher, E. C. 1991. Leukocyte-endothelial cell recognition: three (or more) elimination of macrophages. Nat. Biotechnol. 18: 48–51. steps to specificity and diversity. Cell 67: 1033–1036. 42. Wang, H., T. Peters, D. Kess, A. Sindrilaru, T. Oreshkova, N. Van Rooijen, 35. Conrad, C., O. Boyman, G. Tonel, A. Tun-Kyi, U. Laggner, A. de Fougerolles, A. Stratis, A. C. Renkl, C. Sunderko¨tter, M. Wlaschek, et al. 2006. Activated V. Kotelianski, H. Gardner, and F. O. Nestle. 2007. Alpha1beta1 integrin is macrophages are essential in a murine model for T cell-mediated chronic crucial for accumulation of epidermal T cells and the development of psoriasis. psoriasiform skin inflammation. J. Clin. Invest. 116: 2105–2114. Nat. Med. 13: 836–842. 43. Gebhardt,T.,L.M.Wakim,L.Eidsmo,P.C.Reading,W.R.Heath,andF.R.Carbone. 36. Fiorucci, S., A. Mencarelli, B. Palazzetti, A. G. Sprague, E. Distrutti, A. Morelli, 2009. Memory T cells in nonlymphoid tissue that provide enhanced local immunity T. I. Novobrantseva, G. Cirino, V. E. Koteliansky, and A. R. de Fougerolles. during infection with herpes simplex virus. Nat. Immunol. 10: 524–530. 2002. Importance of innate immunity and collagen binding integrin alpha1beta1 44. Gunn, M. D., K. Tangemann, C. Tam, J. G. Cyster, S. D. Rosen, and in TNBS-induced colitis. Immunity 17: 769–780. L. T. Williams. 1998. A chemokine expressed in lymphoid high endothelial 37. Krieglstein, C. F., W. H. Cerwinka, A. G. Sprague, F. S. Laroux, M. B. Grisham, venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc. V. E. Koteliansky, N. Senninger, D. N. Granger, and A. R. de Fougerolles. 2002. Natl. Acad. Sci. USA 95: 258–263. Collagen-binding integrin alpha1beta1 regulates intestinal inflammation in ex- 45. Mosser, D. M., and J. P. Edwards. 2008. Exploring the full spectrum of mac- perimental colitis. J. Clin. Invest. 110: 1773–1782. rophage activation. Nat. Rev. Immunol. 8: 958–969. 38. Iwasaki, A., and R. Medzhitov. 2004. Toll-like receptor control of the adaptive 46. Zhang, X., and D. M. Mosser. 2008. Macrophage activation by endogenous immune responses. Nat. Immunol. 5: 987–995. danger signals. J. Pathol. 214: 161–178. 39. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu. 47. Gordon, S., and P. R. Taylor. 2005. Monocyte and macrophage heterogeneity. Rev. Immunol. 20: 197–216. Nat. Rev. Immunol. 5: 953–964.