In response to endotoxin, macrophages secrete a protein with a molecular mass of %6000 Da and with an affinity for heparin. This protein, which we term "macrophage inflammatory protein 2," is a potent chemotactic agent for human polymorphonuclear leukocytes. In addition, subcutaneous administration of the monokine causes a localized inflammatory reaction. Partial N-terminal sequence data reveal similarity to a family of proteins, the archetype of which is platelet factor 4. Although macrophage inflammatory protein 2 is a distinct member ofthe platelet factor 4 family, its sequence is most closely related to that of the gro/KC gene product, which is expressed in transformed or platelet-derived growth factortreated cells.One hallmark of the acute inflammatory state is the recruitment and activation of polymorphonuclear leukocytes (PMNs). Numerous mediators have been shown to be involved in this process, including leukotrienes (1, 2); complement components (3, 4); cachectin/tumor necrosis factor (TNF) (5-7); bacterial products (4,8); neutrophil-activating protein 1 (NAP-1), a recently described monokine with sequence relatedness to the platelet factor 4 (PF4) family (9)(10)(11)(12)(13)(14)(15); and another monokine, macrophage inflammatory protein 1 (MIP-1) (16).We have previously shown that endotoxin-stimulated macrophages secrete two proteins that bind to heparin-Sepharose and elute only with high-salt buffer (16 Research, Leuven, Belgium), and purified human NAP-1 protein was given by T. Yoshimura and E. Leonard (National Cancer Institute, Bethesda, MD). All other reagents were obtained from Sigma.Cell Culture. The mouse macrophage cell line RAW 264.7 was obtained from American Type Culture Collection. The cells were maintained in culture and stimulated with endotoxin to produce conditioned medium as previously described (16).Purification of MIP-2. MIP-2 was purified by using methodology previously described for MIP-1 (16). The degree of purification was followed by SDS/PAGE with silver staining. In brief, 2 liters of conditioned supernatant from endotoxinstimulated RAW 264.7 cells were concentrated and diafiltrated against 20 mM Tris HCl (pH 8.0) and applied to a Mono Q 10/10 (anion-exchange) column (Pharmacia LKB Biotechnology, Rahway, NJ). Greater than 90% of the MIP-2 was observed not to bind to the column and was recovered in the effluent.The peak MIP-2-containing fractions were applied to a heparin-conjugated Sepharose (Pharmacia LKB Biotechnology) column equilibrated with 20 mM Tris HCl (pH 8.0) and eluted with a 0-2 M NaCl linear gradient in the same buffer. MIP-2 eluted at -0.75 M NaCl. Peak fractions were concentrated in a Centricon ultrafiltration device with a molecular weight cutoff of 3000 (Amicon, Danvers, MA) and applied to a Superose 12 (gel-filtration; Pharmacia LKB Biotechnology) column equilibrated with 100 mM ammonium acetate. From 2 liters of RAW 264.7 conditioned medium (which equaled -100 mg total protein), we generally isolated 0.5 mg of MIP-2 as assessed by the Bradford protein...
We report the identification and purification of a new inflammatory monokine synthesized by the macrophage tumor cell line RAW 264.7 in response to endotoxin. This monokine, which we term "macrophage inflammatory protein" (MIP), is a doublet with an apparent molecular mass of approximately 8,000 daltons on SDS-PAGE but forms aggregates of greater than 2 x 10(6) daltons as assessed by gel filtration. Partial NH2-terminal amino acid sequence data reveal no significant homology with any previously described protein. Although the monokine is anionic under physiological conditions, it is one of two major macrophage-secreted proteins that bind to heparin at high salt concentrations. At 100 ng/ml or greater, MIP is chemokinetic for human polymorphonuclear cells and triggers hydrogen peroxide production. Subcutaneous injection of 10 ng or greater of MIP into footpads of C3H/HeJ mice elicits an inflammatory response, characterized by neutrophil infiltration. These findings suggest that MIP is an endogenous mediator that may play a role in the host responses that occur during endotoxemia and other inflammatory events.
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.
Macrophage inflammatory protein-1 (MIP-1) produced a monophasic fever of rapid onset whose magnitude was equal to or greater than that of fevers produced with either recombinant human cachectin (or tumor necrosis factor) or recombinant human interleukin-1. However, in contrast to these two endogenous pyrogens, the fever induced by MIP-1 was not inhibited by the cyclooxygenase inhibitor ibuprofen. Thus, MIP-1 may participate in the febrile response that is not mediated through prostaglandin synthesis and clinically cannot be ablated by cyclooxygenase inhibitors.
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