A cDNA clone of murine macrophage inflammatory protein 2 (MIP-2) has been isolated from a library prepared from lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and the nucleotide sequence determined. This cDNA was used to clone cDNAs for human homologues of MIP-2 from a library prepared from phorbol myristate acetate-treated and LPS-stimulated U937 cells. Two homologues were isolated and sequenced. Human MIP-2 alpha and MIP-2 beta are highly homologous to each other and to a previously isolated gene, human gro/melanoma growth-stimulating activity (MGSA). These three human genes, MIP-2 alpha, MIP-2 beta, and gro/MGSA, constitute a sub-family within the cytokine family represented by platelet factor 4 and interleukin 8.

Macrophages secrete a wide variety of proteins that mediate many aspects of acute and chronic inflammation (for review see reference 1). While some of these factors have been well characterized (e.g., IL-1 [2] and cachectin/TNF [3]), others remain poorly defined . Recently, we described the purification and characterization ofa new monokine found in the culture medium of an LPS-stimulated mouse macrophage tumor cell line (RAW264 .7) (4). This protein, termed macrophage inflammatory protein or MIP, has several properties indicative ofan endogenous mediator ofinflammation (e.g ., neutrophil attraction and activation) . Since MIP represents an important new addition to the family of activated macrophage products, it is important to investigate its structure and regulation on the molecular level. Here we describe the cloning and sequencing of the cDNA for murine MIP. Materials and MethodsConstruction ofthe cDNA Library. RAW264.7 cells were obtained from American Type Culture Collection (Rockville, MD) and grown in RPMI 1640 (Gibco Laboratories, Grand Island, NY) supplemented with 20 mM Hepes and 10% FCS (HyClone Laboratories, Logan, UT) until they reached confluency. The cells were then washed five times in HBSS (Gibco Laboratories) and the medium was replaced with serum-free RPMI supplemented with 1 Rg/ml of LPS W (Escherichia coli 0127:B8, Difco Laboratories, Detroit, MI). The cells were incubated at 37°C for 2 h and total RNA was extracted by the addition of6 M guanidinium thiocyanate (5) . Poly(A)' RNA was then isolated by two cycles of oligo-dT-cellulose chromatography, essentially as described by Maniatis et al. (6) . Double-stranded cDNA was prepared from the poly(A)' selected RNA as described by Gubler and Hoffman (7) . After methylation of the internal Eco RI sites and addition of Eco RI linkers, the cDNA was inserted into the Eco RI sites of the bacteriophage %gtl0 (8) .Construction of the Probe Pools . Oligonucleotide probe pools were synthesized as described by Warner et al. (9) against amino acids 22-30 of a partial NH2-terminal sequence . This portion ofthe polypeptide was selected because ofits lower degeneracy in the codon dictionary when compared with the remainder of the sequence . The resulting probe pools are two 512-fold degenerate pools of 26 nucleotides in length.

A number of macrophage-derived mediators have been implicated in the vascular changes of inflammation. We recently reported the isolation of a novel monokine, macrophage inflammatory protein 1 (MIP-1), which causes local inflammatory responses in vivo, and induces superoxide production by neutrophils in vitro. Purified native MIP-1 comprises two peptides with very similar physical characteristics. We report here the resolution of MIP-1 into component peptides by SDS-hydroxylapatite chromatography, and compare the NH2-terminal sequences of the two peptides, now referred to as MIP-1 alpha and MIP-1 beta. A synthetic oligonucleotide probe pool corresponding to the NH2-terminal amino acid sequence of MIP-1 beta was used to isolate a cDNA clone containing its coding sequence. The sequence codes for a 109 amino acid-long polypeptide, of which 69 amino acids correspond to the mature product. Comparison of this MIP-1 beta cDNA with our previously cloned MIP-1 alpha sequence reveals that the MIP-1 peptides, members of a growing family of potential inflammatory mediators, are distinct but highly homologous (58.9% sequence identity) products of different genes.

To determine the regions of interleukin-8 (IL-8) that allow high affinity and interleukin-8 receptor type 1 (IL8R1)-specific binding of chemokines, we produced chimeric proteins containing structural domains from IL-8, which binds to both IL8R1 and interleukin-8 receptor type 2 (IL8R2) with high affinity, and from GRO␥, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity.

We have previously shown that treatment of endothelial cells with minimally modified LDL (MM-LDL) induces the binding of monocytes to unknown endothelial receptor molecules. We now report that a member of the GRO family of chemokines plays a role in MM-LDL-induced monocyte binding. A cDNA library made from rabbit aortic endothelial cells (RAEC) treated with MM-LDL was expression screened for molecules inducing binding of a human monocyte cell line (THP-1). A cDNA was isolated with 75% homology to GRO. GRO mRNA levels were significantly elevated after exposure of RAEC or human aortic endothelial cells (HAEC) to MM-LDL. HAEC treated with MM-LDL displayed an increase in a surface-associated protein that bound to antibody against GRO despite low levels of GRO in the medium. Antibody to GRO significantly inhibited the binding of monocytes to MM-LDL-treated RAEC and HAEC. The increase in GRO expression and monocyte binding were reduced by incubating MM-LDL-treated endothelial cells with heparin (in a method that releases heparan sulfate bound molecules from the cell surface). These results suggest that GRO related chemokines are bound to the surface of MM-LDL-treated endothelial cells and may contribute to the monocyte adhesion induced by MM-LDL. (J. Clin.

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IL-8 is a potent proinflammatory cytokine that has a key role in the recruitment and activation of neutrophils during inflammation. IL-8 reacts with neutrophils via two distinct types of IL-8-R. Receptor-specific Abs were raised against peptides derived from the first extracellular domain of each IL-8-R. Anti-IL-8-R1 and anti-IL-8-R2 selectively block 125I-IL-8 binding to rIL-8-R type 1 or 2, respectively. The anti-peptide Abs were used to assess the role of each receptor in the chemotactic response of neutrophils to GRO alpha and to IL-8. Anti-IL-8-R2 blocks GRO alpha-induced chemotaxis of neutrophils. Chemotaxis to GRO alpha is not inhibited by anti-IL-8-R1. Thus GRO alpha stimulates chemotaxis exclusively through IL-8-R2 and independently of IL-8-R1. Surprisingly, anti-IL-8-R1 inhibits the majority (78 +/- 3%) of IL-8-induced neutrophil chemotaxis. Only a minor proportion of IL-8-induced chemotaxis (29 +/- 5%) is inhibited by anti-IL-8-R2. These findings indicate that chemotaxis to IL-8 is mediated predominantly by type 1 IL-8-Rs and suggest that IL-8-R1 is an appropriate target for therapeutic strategies to limit neutrophil influx in diseases where neutrophils contribute to pathophysiology.