MIP-1␣[CCL3] acting on the CCR1 receptor mediates neutrophil migration in immune inflammation via sequential ␣ release of TNF- and LTB4 Cleber D. L. Ramos,* Claudio Canetti,*,† Janeusa T. Souto,‡,§ Joa˜ o S. Silva,‡ Cory M. Hogaboam,¶ Sergio H. Ferreira,* and Fernando Q. Cunha*,1 Departments of *Pharmacology and ‡Biochemistry and Immunology, School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Brazil; §Department of Microbiology and Parasitology, Federal University of Rio Grande do Norte, Natal, RN, Brazil; and †Division of Pulmonary & Critical Care Medicine and ¶Department of Pathology, University of Michigan, Ann Arbor Abstract: In the present study, we investigated nists might have a therapeutic potential. J. Leukoc. the involvement of macrophage-inflammatory pro- Biol. 78: 167–177; 2005. tein-1␣ (MIP-1␣)[CC chemokine ligand 3 (CCL3)], MIP-1␤[CCL4], regulated on activation, normal Key Words: chemokines ⅐ chemokine receptors ⅐ chemotaxis T expressed and secreted (RANTES)[CCL5], and CC chemokine receptors (CCRs) on neutrophil mi- gration in murine immune inflammation. Previ- INTRODUCTION ously, we showed that ovalbumin (OVA)-triggered neutrophil migration in immunized mice depends on the sequential release of tumor necrosis factor Neutrophil migration is a complex process, which results ␣ ␣ mainly from the release of neutrophil chemotactic factors by (TNF- ) and leukotriene B4 (LTB4). Herein, we show increased mRNA expression for MIP- resident cells, inducing rolling and adhesion of neutrophils on 1␣[CCL3], MIP-1␤[CCL4], RANTES[CCL5], and endothelial cells, followed by their transmigration to the ex- travascular space [1, 2]. Apart from its importance in host CCR1 in peritoneal cells harvested from OVA-chal- defense, the migration of neutrophils to the inflammatory site lenged, immunized mice, as well as MIP-1␣[CCL3] is, at least in part, responsible for tissue damage observed in and RANTES[CCL5] but not MIP-1␤[CCL4] proteins several inflammatory diseases such as rheumatoid arthritis, in the peritoneal exudates. OVA-induced neutrophil glomerulonephritis, autoimmune vasculitis, and inflammatory migration response was muted in immunized MIP- -؊ ؊ bowel disease [3–5], highlighting the importance of under 1␣[CCL3] / mice, but it was not inhibited by treat- standing the mechanisms involved in leukocyte migration. ment with antibodies against RANTES[CCL5] or ␤ ␤ ␣ ␣ ␤ ␣ Interleukin-1 (IL-1 ), tumor necrosis factor (TNF- ), com- MIP-1 [CCL4]. MIP-1 [CCL3] mediated neutro- plement fragment C5a, lipid mediators, such as platelet-acti- phil migration in immunized mice through induc- ␣ vating factor 4 and leukotriene B4 (LTB4), and more recently, tion of TNF- and LTB4 synthesis, as these medi- chemokines are the main chemotactic mediators involved in ators were detected in the exudates harvested from neutrophil recruitment to sites of inflammation [6]. OVA-challenged immunized wild-type but not MIP- Chemokines are important mediators involved in the migra- ؊/؊ ␣ 1 [CCL3] mice; administration of MIP- tion and activation of the various subsets of leukocytes [7]. ␣ 1 [CCL3] induced a dose-dependent neutrophil Chemokines are divided into four subfamilies, based on the migration, which was inhibited by treatment with position of one or two cysteine residues located near the N ؊/؊ ␣ an anti-TNF- antibody in TNF receptor 1 (p55 )- terminus of the protein: C (lymphotactin[XCL1]), CC {i.e., deficient mice or by MK 886 (a 5-lipoxygenase in- monocyte chemoattractant protein-1 (MCP-1)[CC chemokine ␣ hibitor); and MIP-1 [CCL3] failed to induce LTB4 ligand 2 (CCL2)], macrophage-inflammatory protein-1␣ (MIP- ␣ ؊/؊ production in p55 mice. MIP-1 [CCL3] used 1␣)[CCL3], MIP-1␤[CCL4], regulated on activation, normal T CCR1 to promote neutrophil recruitment, as OVA or expressed and secreted (RANTES)[CCL5], eotaxin[CCL11], MIP-1␣[CCL3] failed to induce neutrophil migration pulmonary and activation-regulated chemokine/dendritic cells ؊/؊ ؊/؊ in CCR1 mice, in contrast to CCR5 mice. In (DC)-chemokine 1[CCL18])}, CXC {i.e., growth-related onco- summary, we have demonstrated that neutrophil mi- gration observed in this model of immune inflamma- tion is mediated by MIP-1␣[CCL3], which via CCR1, induces the sequential release of TNF-␣ and LTB . 1 Correspondence: Departamento de Farmacologia, Faculdade de Medicina 4 de Ribeira˜o Preto, USP, Avenida Bandeirantes, 3900 Ribeira˜o Preto, Sa˜o Therefore, whether a similar pathway mediates neu- Paulo, 14049-900 (Brazil). E-mail: [email protected] trophil migration in human immune-inflammatory Received April 13, 2004; revised March 8, 2005; accepted March 13, 2005; diseases, the development of specific CCR1 antago- doi: 10.1189/jlb.0404237. 0741-5400/05/0078-167 © Society for Leukocyte Biology Journal of Leukocyte Biology Volume 78, July 2005 167 ␣ ␣ gene- (GRO- )[CXC chemokine ligand 1 (CXCL1)], IL-8- induces neutrophil migration via a LTB4-dependent mecha- [CXCL8], monokine induced by interferon-␥ (IFN-␥)[CXCL9], nism [38]. IFN-inducible protein 10 (IP-10)[CXCL10])}, and CX3C In the present study, we investigated the role of MIP- (fraktalkine[CX3CL1]). The CXC subfamily is subdivided ac- 1␣[CCL3], MIP-1␤[CCL4], and RANTES[CCL5] during OVA- cording to the presence or the absence of the Glu-Leu-Arg induced neutrophil migration. We demonstrated that OVA- (ELR) motif positioned in the NH2-terminal region before the induced neutrophil migration in immunized mice was mediated first cysteine residue. ELRϩCXC chemokines cause, preferen- by MIP-1␣[CCL3] but not by RANTES[CCL5] or MIP- – ␤ ␣ tially, the recruitment of neutrophils, and ELR CXC chemo- 1 [CCL4] via the release of TNF- and LTB4. In addition, we kines are more specific for lymphocytes [8]. The release of showed that MIP-1␣[CCL3] activated CCR1 but not CCR5 to ELRϩCXC chemokines such as MIP-2[CXCL2], keratinocyte- promote neutrophil migration. derived chemokine[CXCL1], GRO-␣[CXCL1], epithelial-de- rived neutrophil-activating factor-78[CXCL5], or IL-8[CXCL8] correlates with neutrophil infiltration observed in experimental models of inflammation and in human inflammatory diseases, MATERIALS AND METHODS such as rheumatoid arthritis, inflammatory bowel disease, sar- coidosis, and psoriasis [9–12]. Furthermore, inhibition of these Animals chemokines with antibodies or the use of knockout mice for these chemokines or their receptors reduces neutrophil migra- Male BALB/c and C57Bl/6 (WT) mice and mice with a targeted disruption of the MIP-1␣ (C57Bl/6 MIP-1␣[CCL3]Ϫ/Ϫ), TNF-␣ receptor type I (C57Bl/6 tion in different models of inflammation [13–16]. In human Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ p55 / ), CCR1, or CCR5 (BALB/c CCR1 / and C57Bl6 CCR5 / , respec- models, the chemokines induce neutrophil migration mainly tively), weighing 18–22 g, were housed in temperature-controlled rooms (22– via the activation of the CXC chemokine receptor 1 (CXCR1) 25°C) and received water and food ad libitum in the animal facility of the and CXCR2 [17, 18]. In this context, substances that inhibit Department of Pharmacology or Immunology, School of Medicine of Ribeira˜o these chemokines receptors, such as repertaxin, have been Preto, University of Sa˜o Paulo (Brazil). ␣ Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ tested to inhibit human neutrophil migration [19, 20]. Breeding pairs of MIP-1 [CCL3] , p55 , and CCR5 were pur- chased from the Jackson Laboratories (Bar Harbor, MA). Breeding stocks The CC chemokines have been described as preferential backcrossed to C57Bl/6 were obtained and housed in a sterile laminar flow chemoattractants and activators of mononuclear cells and eo- cabinet until experiments were conducted. The breeding pairs of CCR1Ϫ/Ϫ sinophils [21–23]. However, recent studies demonstrate that mice were a gift of Prof. Craig Gerard (Children’s Hospital, Harvard Medical this chemokine subtype may also be involved in neutrophil School, Boston, MA). migration in murine models [24, 25]. MIP-1␣[CCL3] is pro- duced by a variety of cells, including lymphocytes, monocytes/ Active sensitization macrophages, mast cells, basophils, epithelial cells, and fibro- BALB/c, C57Bl/6, MIP-1␣[CCL3]Ϫ/Ϫ, CCR1Ϫ/Ϫ, and CCR5Ϫ/Ϫ mice were blasts, and it binds to CC chemokine receptor 1 (CCR1) and immunized as described previously [34]. Briefly, on day 0, the animals re- CCR5 with high affinity to exert its biological effects [26–30]. ceived a single subcutaneous (s.c.) injection of OVA (100 ␮g) in 0.2 mL of an MIP-1␣[CCL3] also induces calcium influx in a dose-depen- emulsion containing 0.1 mL phosphate-buffered saline (PBS) and 0.1 mL dent manner and the chemotaxis of murine neutrophils in vitro complete Freund’s adjuvant (CFA; Sigma Chemical Co., St. Louis, MO). The animals were given booster injections of OVA dissolved in incomplete [31]. Its administration in mice induces neutrophil influx and Freund’s adjuvant (Sigma Chemical Co.) on days 7 and 14. Control mice were protected mice against Cryptococcus neoformans infection [32]. injected s.c. with 0.2 mL of an emulsion containing equal volumes of PBS and Furthermore, in 1999, Das et al. [33] demonstrated that neu- CFA, followed by boosters containing PBS and incomplete Freund’s adjuvant. trophil recruitment observed in a model of immune inflamma- Twenty-one days after the initial injection, the immunized and control animals ␮ tion was inhibited partially by anti-MIP-1␣[CCL3] treatment. were challenged by intraperitoneal (i.p.) injection with OVA (10 g/cavity) dissolved in 0.2 mL PBS or PBS alone, and the neutrophil migration was However, the CCR subtype(s) that this chemokine uses as well determined 2, 4, and 8 h after, as described below. In addition, the peritoneal as the mechanisms by which MIP-1␣[CCL3] induces neutro- exudates were collected 1.5 h after PBS or OVA injection to measure the phil recruitment have not been characterized. It is important to TNF-␣, MIP-1␣[CCL3], MIP-1␤[CCL4], and RANTES[CCL5] by enzyme- mention that although the MIP-1␣[CCL3] production is also linked immunosorbent assay (ELISA; see below). The MIP-1␣[CCL3] concen- increased in patients with rheumatoid arthritis and sarcoidosis tration was also measured 30 min, 2 h, 4 h, and 8 h after OVA injection.