The murine IL-8 homologues KC, MIP-2, and LIX are found in endothelial cytoplasmic granules but not in Weibel-Palade bodies

Epub ahead of print December 9, 2009 - doi:10.1189/jlb.0809532 Brief Conclusive Report The murine IL-8 homologues KC, MIP-2, and LIX are found in endothelial cytoplasmic granules but not in Weibel-Palade bodies Johanna Hol,* Linn Wilhelmsen,* and Guttorm Haraldsen†,1 *Institute and †Division of Pathology, University of Oslo, Oslo University Hospital, Oslo, Norway RECEIVED AUGUST 6, 2009; REVISED OCTOBER 15, 2009; ACCEPTED OCTOBER 30, 2009. DOI: 10.1189/jlb.0809532 ABSTRACT Rapid translocation of P-selectin from WPB to the surface of endothelial cells is crucial for early neutrophil recruitment to acute inflammatory lesions. Likewise, the chemokine CXCL8/IL-8 is sorted to WPB in human endothelial cells, but little is known about its functional importance in lack of a suitable animal model. Here, we explored the distribution of the functional IL-8 homologues CXCL1/KC, CXCL2/MIP-2, and CXCL5-6/LIX in resting and inflamed murine vessels by confocal microscopy and paired immunostaining with markers of WPB, discovering that these chemokines did not localize to WPB but displayed a granular pattern in a subset in healthy skin compatible with sorting to the type 2 endothelial compartment for regulated secretion. Moreover, all chemokines colocalized with VWF and P-selectin in platelets, suggesting that their storage in platelet ␣-granules might represent an alternative source of rapidly available, neutrophil-recruiting chemokines. In conclusion, WPB appear not to be involved in regulated secretion of chemokines in the mouse, and instead, the possible existence of type 2 granules and the role of platelets in rapid leukocyte adhesion deserve further attention. J. Leukoc. Biol. 87: 000 – 000; 2010. Introduction The recruitment of neutrophils to sites of inflammation is crucial to immune defense and tissue repair, but it is also of therapeutic interest, as it complicates organ transplantation, myocardial infarction, cerebral stroke, and bowel surgery in a mechanism commonly referred to as IRI [1]. To migrate into tissues, leukocytes must first adhere to the endothelial surface in a sequential interaction between endothelial and leukocyte Abbreviations: DCIP⫽dendritic cell inflammatory protein, GCP-2⫽granulocyte chemotactic protein 2, GRO␣⫽growth-related oncogene ␣, IRI⫽ ischemia-reperfusion injury, KC⫽cytokine-induced neutrophil-attracting chemokine, KLH⫽keyhole limpet hemocyanin, LIX⫽LPS-induced chemokine, PAF⫽platelet-activating factor, PF4⫽platelet factor 4, VWF⫽von Willebrand factor, WPB⫽Weibel-Palade bodies The online version of this paper, found at www.jleukbio.org, includes supplemental information. 0741-5400/10/0087-0001 © Society for Leukocyte Biology adhesion molecules [2]. In the most acute sequence of events in this multistep adhesion cascade, prestored P-selectin from WPB of endothelial cells is translocated to the endothelial cell surface in response to secretagogues such as thrombin, histamine, or hypoxia, followed by the action of ligands binding to G protein-coupled receptors on the leukocyte surface [3, 4]. The involvement of such receptors was first shown for PAF, a rapidly synthesized lipid mediator that is presented on the surface of endothelial cell membranes [5]. Indeed, inhibitors of PAF reduce pathology effectively in intestinal and cerebral IRI [6, 7], but although PAF is a powerful recruiter of leukocytes, it lacks the selectivity offered by the characteristic chemokine receptor profiles on different subsets of leukocytes [4]. The discovery that the chemokines CXCL8/IL-8 and CCL26/ eotaxin-3 [8 –10] can be stored in WPB thus appeared to suggest the existence of an alternative, rapid adhesion cascade, offering higher specificity than that containing PAF, and formed the concept of the WPB as a “Swiss army knife” of leukocyte adhesion. IL-8 is the best-characterized member of a group of chemokines characterized by a Glu-Leu-Arg motif preceding the conserved CXC motif of two cysteines separated by a single aa (ELR⫹ CXC chemokines) [4]. These chemokines induce recruitment and activation of neutrophils by binding CXCR1 (IL-8 and CXCL6/GCP-2) and CXCR2 (all ELR⫹CXC chemokines) expressed on the neutrophil surface [4]. By contrast, rodents lack a direct homologue of IL-8, but the chemokines CXCL1/KC, CXCL2/MIP-2, and CXCL5-6/LIX are regarded as functional homologues of IL-8 and have been found to contribute to the pathology of a number of neutrophil-dependent animal models of disease, including IRI [11–14]. Although neither KC, MIP-2, nor LIX are direct homologues of IL-8, each belongs to the same major cluster of chemokines (human chromosome 4q13.3; mouse chromosome 5qE2), which represent closely related chemokines involved in neutrophil recruitment [15]. Chemokines differentiate via gene-duplication events, some of which are so recent that there is not always a clear correlation between human and mouse chemo1. Correspondence: Division of Pathology, Oslo University Hospital, Sognsvannsveien 20, 0027 Oslo, Norway. E-mail: guttorm.haraldsen@rrresearch.no Volume 87, March 2010 Journal of Leukocyte Biology Copyright 2009 by The Society for Leukocyte Biology. 1 kines. One example of this is LIX, which is most similar to human CXCL5/epithelial cell-derived neutrophil-activating peptide-78 and CXCL6/GCP-2 but a direct homologue of neither [16, 17]. Moreover, LIX could be considered the most likely candidate for WPB residency, as it, like IL-8, is capable of binding CXCR1 and CXCR2 [18]. The group of ELR⫹CXC murine chemokines also includes CXCL3/DCIP, CXCL7, and CXCL15/lungkine [16]. Little is known about these latter three, their endothelial production has not been described in the mouse, and antibodies are unavailable (DCIP) or have not worked convincingly in our hands (CXCL7 and lungkine). This study has therefore focused on KC, MIP-2, and LIX. The importance of endothelial WPB release in IRI has been demonstrated in laboratory animals by inhibition of WPB exocytosis [19] or lack of WPB biogenesis (based on VWF deficiency [20]), resulting in reduced IRI pathology. Moreover, vascular P-selectin levels increased rapidly after reperfusion and several hours before mRNA levels rose, also consistent with WPB exocytosis [21]. Accordingly, blocking of P-selectin inhibited rolling and neutrophil recruitment after IRI, LPS, or chemokine exposure [6, 22, 23]. Finally, mice deficient in Pselectin were relatively protected against the effects of IRI, indicating that transcriptionally regulated E-selectin cannot fully compensate for the lack of P-selectin, perhaps exactly because it appears later on the vascular surface [24]. By contrast, although IL-8 is known to play an important role in the pathology of IRI [11], and the in vitro and in vivo storage of IL-8 in WPB is well-documented [8, 9], the functional relevance of WPB storage of chemokines has not been evaluated. Although KC is sometimes claimed to be stored in murine WPB, presumably based on the assumption of IL-8 homology, no experimental evidence supports this claim. The aim of this study was therefore to determine whether KC, MIP-2, or LIX localizes to murine WPB. For this purpose, we evaluated noninflamed and LPS-injected skin samples from C57Bl6 mice by double-immunofluorescent staining and confocal microscopy. Although almost perfect colocalization of P-selectin and VWF was observed in postcapillary venules and capillaries, the distribution of KC, MIP-2, and LIX was incompatible with WPB storage. On the other hand, weak but consistent signals for KC, LIX, and to a lesser degree, MIP-2 were observed within a subset of vessels of healthy skin, pointing to the possibility that another storage granule for chemokines, resembling the endothelial type 2 granule in humans [10], may be of functional importance. We discovered this compartment when observing that the chemokines CXCL1/GRO␣ and CCL2/MCP-1 were subject to regulated secretion from human endothelial cells but were not present in WPB [10]. In addition, all chemokines colocalized with VWF and P-selectin in platelets, suggesting their storage in platelet ␣-granules and possibly representing an alternative, rapidly available source of chemokines. MATERIALS AND METHODS Reagents LPS (Escherichia coli, serotype 026:B6) was purchased from Sigma-Aldrich (St. Louis, MO, USA), histamine (Solu Prick SQ kit) from ALK-Abello A/S (Horsholm, Denmark), and tyramide signal amplification reagents from Molecular Probes, Invitrogen (Paisley, UK). Table 1 specifies sources of antibodies. Animal models Skin samples for immunofluorescent staining were obtained from adult C57BL6 mice of both sexes (Taconic, Hudson, NY, USA). Samples of healthy skin (n⫽9) and skin injected with 5 ␮g LPS at 2 (n⫽2), 4 (n⫽2), 8 (n⫽2), 24 (n⫽5), and 48 (n⫽2) h before sacrifice to produce inflammation known to up-regulate neutrophil-attracting chemokines were fixed in 10% formalin for 24 h before paraffin-embedding. All animal experiments were performed according to national legislation and local guidelines. Immunofluorescence analysis Staining by conventional methods has, in our hands, not proven sensitive enough to detect reliably more than the brightest endothelial signal, a problem that we have resolved successfully by using tyramide signal amplification. Formalin-fixed, paraffin-embedded specimens were cut in 4 ␮m sections and placed on polysine-coated microscope slides (LSL, Rochdale, UK). The sections were de-waxed by incubation with xylene for 2 ⫻ 5 min, rehydrated in a series of alcohol, washed in PBS, and subjected to antigen retrieval in Tris-HCl buffer, pH 9.0, at 100°C on a water-bath for 20 min. The slides were left to cool in the buffer for 20 –30 min and incubated for 10 min with 3% H2O2 to block endogenous peroxidase with 1% tyramide sig- TABLE 1. Antibodies Used for Immunofluorescent Stainings Epitope Antibody Conjugate Working concentration Murine KC Murine MIP-2 Murine LIX Murine P-selectin Human VWF rabbit polyclonal rabbit polyclonal rabbit polyclonal goat polyclonal rabbit polyclonal – – – – – 2 ␮g/ml 2 ␮g/ml 1 ␮g/ml 3.3 ␮g/ml 1/2000 Human VWF Murine Ly-6G KLH goat polyclonal rat monoclonal rabbit polyclonal – – – 1 ␮g/ml 1/400 2 ␮g/ml Rabbit IgG Rabbit IgG Goat IgG donkey polyclonal donkey polyclonal donkey polyclonal HRP Alexa Fluor® 488 Cy3 2 Journal of Leukocyte Biology Volume 87, March 2010 1/100 1/600 1/600 Producer (product number) PeproTech (Rocky Hill, NJ, USA) (500-P115) Peprotech (500-P130) Peprotech (500-P146) R&D Systems (Minneapolis, MN, USA) (AF737) Dako (Denmark) (A0082) Santa Cruz Biotechnology (Santa Cruz, CA, USA) (PA1-74050) BD PharMingen (San Jose, CA, USA) (551459) Sigma-Aldrich (H0892) Jackson ImmunoResearch Labs (West Grove, PA, USA) (711-035-152) Molecular Probes, Invitrogen (A-11034) Jackson ImmunoResearch Labs (711-165-152) www.jleukbio.org Hol et al. KC, MIP-2, and LIX in murine endothelium nal amplification blocking reagent (Molecular Probes, Invitrogen) for 60 min, with affinity-purified primary antibodies to Ly-6G, KC, MIP-2, LIX, P-selectin, and/or VWF for 20 h at 4°C, with secondary antibodies for 105 min, with Alexa Fluor威 488 tyramide (Molecular Probes, Invitrogen) for 10 min, and with Hoechst nuclear dye for 5 min, followed by air-drying and mounting in polyvinyl alcohol. Slides were washed in PBS between incubations and dipped in distilled H2O before drying. A full overview of antibodies and working concentrations are provided in Table 1. An affinity-purified, concentration-matched antibody to KLH was used as an irrelevant control for the antichemokine reagents. Tissue sections were analyzed with a Nikon Ellipse E800 microscope with dry lenses or with a Nikon Ellipse 80i with Nikon plan fluor oil objectives 20⫻/0.75, 40⫻/1.30, and 100⫻/ 1.30. Images were obtained using Dlympus AnalySIS and Olympus Cell ˆR image acquisition image analysis software. A B RESULTS AND DISCUSSION KC, MIP-2, and LIX associate with a subset of vessels in resting skin but show a distribution different from that of P-selectin Sections of healthy mouse skin were immunostained for the chemokines with P-selectin and VWF as vessel markers. Vascular staining was evaluated as absent, cytoplasmic, membranous (Fig. 1), or granular (Fig. 2, and see Fig. 4). In addition to a uniform, strong signal in epithelial cells (data not shown), all chemokines were associated with vessels of healthy skin (Figs. 2 and 3) but showed different distributions. Staining for the WPB constituent P-selectin resulted in a strong granular signal in postcapillary venules and some capillaries but a weak or absent signal in arterioles (Fig. 2). This distribution has been reported previously [25] and is consistent with postcapillary venules being the main site of leukocyte extravasation. In contrast to P-selectin, the chemokines KC, MIP-2, and LIX were also found in endothelial cells of arterioles and capillaries (Fig. 2). Staining for KC revealed signals in a high proportion of endothelial cells and was mainly cytoplasmic (Fig. 3A), sometimes membranous (Fig. 2B), and in occasional vessels, granule-like in appearance (Figs. 2A and 4A). The sparse number of intravascular platelets observed were also positive for KC, as were a number of perivascular and other tissue-resident cells, some cells associated with lymphatic vessels and scattered lymphatic endothelial cells. MIP-2 was seen mainly as a strong signal in platelets (Fig. 3B) but was also found as granule-like cytoplasmic structures in occasional vascular endothelial cells (Figs. 2 and 4B). Compared with KC, much smaller amounts of MIP-2 were found in endothelial cells of healthy skin. However, adipose cells showed a marked granular MIP-2 staining pattern, and some periarteriolar muscle cells stained positive for the chemokine (Fig. 2A, arrow). LIX was seen in endothelial cells as a cytoplasmic signal, which was coarsely granular or cytoplasmic in appearance (Figs. 2 and 3C), and was also associated with numerous perivascular cells, amongst them, muscle cells surrounding arterioles (Fig. 2A, arrow). Like KC and MIP-2, the LIX signal was also seen in platelets. Furthermore, a number of other tissue-resident cells were positive for LIX. www.jleukbio.org C D Figure 1. Patterns of vascular signal. Signal in vessels was evaluated to be absent (A), mainly cytoplasmic (B), membranous (C), or strong membranous and cytoplasmic (D). Example micrographs are from sections of control skin (A), skin 24 h after LPS (B and D), and skin 48 h after LPS (C), all stained with antibodies to KC, as detailed in Materials and Methods. Original scale bars, 10 ␮m. Endothelial KC, MIP-2, and LIX signals are upregulated after LPS stimulation of skin and show a shift toward membranous localization IL-8 is found in WPB of vessels in healthy human skin and intestine [8], leading us to propose that this may be driven by the continuous exposure to exogenous antigen [8]. This hypothesis is supported by the observation that endothelial cells derived from the umbilical cord require proinflammatory activation before IL-8 is synthesized and subject to WPB storage [8, 9]. We therefore speculated that the commensal environment of laboratory animals raised in minimal disease units may not sufficiently challenge chemokine synthesis and sorting. Based on this assumption, we also analyzed skin samples Volume 87, March 2010 Journal of Leukocyte Biology 3 B A P-sel KC A c c V V V V MIP-2 20µm 20µm 50µm 50µm Figure 2. Vascular distribution of KC, MIP-2, and LIX in healthy skin. Signal for KC, MIP-2, and LIX was found in arterioles (A, labeled A), venules (A, labeled V), and capillaries (B, labeled c) of healthy skin, and P-selectin (P-sel) was restricted to venules and some capillaries (A and B). MIP-2 and LIX signal was also seen in periarteriolar muscle cells (A, arrows). Original scale bars, 50 ␮M (A) and 20 ␮M (B). MIP-2 P-sel c V V V V c 20µm 20µm 50µm 50µm A P-sel A A LIX P-sel KC A P-sel P-sel LIX A c V c V 20µm 50µm control A P-sel A c V 20µm 50µm in a time course after intradermal injection of the TLR2 ligand LPS. To facilitate the identification of neutrophils as a functional readout for inflammation, we also stained for the granulocyte marker Ly-6G (Supplemental Fig. 1), demonstrating granulocyte recruitment within 2 h of LPS injection. Costaining for the chemokines KC, MIP-2, or LIX and Pselectin or VWF revealed that all chemokines were up-regulated in the endothelium within 2 h of LPS exposure (Supplemental Fig. 2). The KC signal was increased dramatically compared with healthy skin. Bright membranous staining was seen on most vessels (Fig. 3E), and a strong signal was seen in intraluminal and infiltrating granulocytes. Lymphatic endothelial cells also stained positive for the chemokine in the perinuclear region as well as on the cell membrane. Moreover, increased signal was observed on perivascular cells, partly including periarteriolar muscle cells and other tissue resident cells. The staining intensity of KC on endothelial cells was maximal at 2 and 4 h after LPS injection, judged by comparison with the signal on intraluminal leukocytes (Supplemental Fig. 2). At 24 and 48 h, the endothelial signal became increasingly cytoplasmic, although strong membranous staining was still present. LPS stimulation also increased the MIP-2 signal in the skin. To the largest extent, it was associated with increased numbers 4 Journal of Leukocyte Biology P-sel control c V 20µm 50µm Volume 87, March 2010 20µm 50µm of platelets on the endothelial surface and within clusters of granulocytes, but there was also staining consistent with localization to the endothelial Golgi apparatus and the surface membrane (Fig. 3F). There was furthermore a strong increase in staining of periarteriolar muscle cells and increased granular staining within endothelial and adipose cells. LIX signal after LPS stimulation was found as membranous and granular endothelial staining (Fig. 3G). Compared with KC and MIP-2, the difference in LIX signal observed between healthy and LPS-exposed skin was less prominent. In some vessels, staining of the Golgi was apparent. In contrast to healthy skin, the membranous endothelial staining was stronger than that of the perivascular cells at 2 and 4 h after LPS exposure. This relation was reverted at later time-points, and the endothelial staining again appeared more cytoplasmic. Some perinuclear signal was also observed in lymphatic endothelial cells. Endothelial KC, MIP-2, and LIX are not stored in WPB but show a granular signal compatible with sorting to the type II compartment for regulated secretion High magnification microscopy confirmed that P-selectin and VWF colocalized within endothelial cells of healthy and LPS-ex- www.jleukbio.org Hol et al. KC, MIP-2, and LIX in murine endothelium A E KC healthy B P-sel KC 24 h LPS F MIP-2 healthy C P-sel MIP-2 24 h LPS G P-sel 24 h LPS H VWF 24 h LPS posed skin, consistent with their storage in WPB (Fig. 4). However, neither marker of WPB colocalized with KC, MIP-2, or LIX in endothelial cells of healthy or inflamed skin (Fig. 4, A–C; data for inflamed skin are not shown). By contrast, we observed a granular signal for all chemokines in scattered vessels (Fig. 4, A–C, arrowheads), compatible with sorting to the type 2 compartment for regulated secretion. We therefore considered our recent discovery that IL-8 targeting to WPB depends on an exposed aspartic acid in loop 2 (the loop between ␤-sheet 2 and 3) [26], which is absent in KC, MIP-2, and LIX. Instead, they all have loop 2 sequences similar to CXCL1/GRO␣ and CXCL4/PF4 — human chemokines that are targeted to endothelial type 2 compartments [10, 27] and platelet ␣-granules [28], respectively. Based on loop 2 sequences, it is therefore not surprising that the murine IL-8 homologues failed to target WPB. On the other hand, eotaxin-3, another chemokine targeted to WPB, does not have an aspartic acid in this loop and must therefore be targeted www.jleukbio.org P-sel Figure 3. KC, MIP-2, and LIX are found in association with some vessels of resting skin but are strongly up-regulated after LPS exposure. Formalin-fixed, paraffinembedded samples of healthy skin (n⫽9) and skin 24 h after LPS injection (n⫽5) from C57Bl6 mice were immunostained with antibodies to KC (A and E), MIP-2 (B and F), LIX (C and G), KLH as an irrelevant control (H), P-selectin (A–H), and VWF (D). The green (upper) and red (lower) channels are shown as smaller panels to the right of each merged image, except in D, where green channels are shown in the lower right panel and red channels in the upper right panel. Original scale bars, 50 ␮m. The green channel images have been obtained with different exposure times to reduce overexposure in sections with strong signal: C, E, and G, 50 ms; A, D, and F, 100 ms; and B and H, 200 ms. control P-sel healthy P-sel LIX LIX healthy D P-sel P-sel by unknown mechanisms [10]. Before we know more about the exact mechanisms underlying targeting of chemokines to compartments for regulated secretion, it is difficult to predict their subcellular localization on the basis of sequence homology only. The lack of CXCR2-activating chemokines in murine WPB means that they are subject to regulated secretion from other endothelial compartments or not subject to such regulation. Unfortunately, endothelial type 2 granules are defined currently based on their content of chemokines (GRO␣ and CCL2/ MCP-1) and their secretagogue responsiveness in vitro [10], whereas additional markers have not been identified. Thus, at this point, we are restricted in our ability to determine whether the granular staining that we observed is related to this compartment. Alternatively, it is possible that the granules observed belong to the constitutive secretory pathway and that our observation reflects a low level of constitutive chemokine secretion. Yet, Volume 87, March 2010 Journal of Leukocyte Biology 5 A KC B VWF Figure 4. KC, MIP-2, and LIX do not colocalize with P-selectin and VWF within endothelial cells but are seen as a granular signal. Formalin-fixed, paraffinembedded samples of healthy skin from C57Bl6 mice were immunostained with antibodies to KC (A), MIP-2 (B), LIX (C), KLH as an irrelevant control (E), VWF (A–D), and P-selectin (D). Insets in main photos show higher magnification of selected areas. The green (upper) and red (lower) channels are shown as smaller panels to the right of each merged image, except in D, where green channels are shown in the lower right panel and red channels in the upper right panel. Original scale bars, 20 ␮m. Arrowheads point to chemokine-containing granules. C LIX D VWF E MIP-2 VWF P-sel VWF control VWF another possibility would be that they reflect the transcytosis known to mediate vascular presentation of chemokines generated in the perivascular area [29, 30]. However, although both of these mechanisms are in play during inflammation, they would appear to disturb the gatekeeper function of resting vessels and are therefore unlikely to explain our observation. In platelets, KC, MIP-2, and LIX colocalize with P-selectin and VWF, consistent with storage in platelet ␣-granules During inflammation, another possible source of chemokines on the vascular surface may be blood-borne platelets. Human platelets are known to store several chemokines as well as Pselectin and VWF in ␣-granules [28, 31, 32], and in fact, activated platelets can deposit chemokines on the surface of endothelial cells [33, 34]. Within the vessel lumen and on the endothelial surface, we observed that P-selectin and VWF colocalized in coalescing round to irregular signal patches of a size comparable with platelets (Fig. 5). Many aggregates of leu6 Journal of Leukocyte Biology Volume 87, March 2010 kocytes and platelets were seen in LPS-exposed skin in this study, and increased numbers of adherent platelets were seen in inflamed areas. We concluded that they were platelets and observed signals for KC, MIP-2, and LIX that colocalized with P-selectin (Fig. 5, A–C) and VWF (data not shown). MIP-2 gave the strongest signal in this location, followed closely by KC, and LIX provided a slightly weaker signal. We interpret our stainings to suggest strongly that KC, MIP-2, and LIX are stored in murine platelet ␣-granules, representing a rapidly available source of the neutrophil-recruiting chemokine. Supporting this assumption, the loop 2 sequences of the chemokines closely resemble that of loop 2 of PF4, which has been shown to be critical for the targeting of PF4 to ␣-granules [28]. We conclude that functional homologues of IL-8 in the mouse (KC, MIP-2, and LIX) were not stored in endothelial WPB, although KC and LIX (and to a lesser degree, MIP-2) were associated with a subset of vessels in healthy tissue. All three chemokines were markedly up-regulated in the endothe- www.jleukbio.org Hol et al. KC, MIP-2, and LIX in murine endothelium A P-sel KC Merge B P-sel MIP-2 Merge C P-sel LIX Merge D VWF P-sel Merge E P-sel Control Merge lium after LPS exposure, but a colocalization with WPB remained undetectable. In contrast, KC and MIP-2 (and to a lesser degree, LIX) colocalized with P-selectin and VWF in platelets, suggesting that ␣-granules could represent a rapidly available pool of neutrophil-recruiting chemokines. Furthermore, it is possible that the granular chemokine signal within endothelial cells of healthy tissues may represent a murine equivalent of the endothelial type 2 compartment found in humans. Future studies should use mice doubledeficient in CXCR2 and the recently characterized murine homologue of CXCR1 and ask to what extent chemokines binding these receptors are involved in the rapid recruitment of neutrophils. www.jleukbio.org Figure 5. KC, MIP-2, and LIX colocalize with P-selectin and VWF in blood platelets. Formalin-fixed, paraffin-embedded samples of LPS-exposed skin from C57Bl6 mice (n⫽5) were immunostained with antibodies to KC (A), MIP-2 (B), LIX (C), P-selectin (all), VWF (D), and KLH as an irrelevant control (E). The three panels to the right of the main panels show a larger magnification of red, green, and merged channels from selected squares. Original scale bars, 10 ␮m. AUTHORSHIP J. H. and G. H. designed the study, and J. H. and L. W. performed the experiments. J. H. and G. H. performed microscopy and evaluation of images. ACKNOWLEDGMENTS The authors thank the Research Council of Norway and Health Region South of Norway for financial support; Aaste Aursjø, Vigdis Wendel, and Linda Manley for technical assistance; and Per Brandtzaeg for critical reading of the manuscript. Volume 87, March 2010 Journal of Leukocyte Biology 7 REFERENCES 1. Litt, M. R., Jeremy, R. W., Weisman, H. F., Winkelstein, J. A., Becker, L. C. (1989) Neutrophil depletion limited to reperfusion reduces myocardial infarct size after 90 minutes of ischemia. Evidence for neutrophilmediated reperfusion injury. Circulation 80, 1816 –1827. 2. Springer, T. A. 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