Cachectin (tumor necrosis factor), a protein produced in large quantities by endotoxin-activated macrophages, has been implicated as an important mediator of the lethal effect of endotoxin. Recombinant human cachectin was infused into rats in an effort to determine whether cachectin, by itself, can elicit the derangements of host physiology caused by administration of endotoxin. When administered in quantities similar to those produced endogenously in response to endotoxin, cachectin causes hypotension, metabolic acidosis, hemoconcentration, and death within minutes to hours, as a result of respiratory arrest. Hyperglycemia and hyperkalemia were also observed after infusion. At necropsy, diffuse pulmonary inflammation and hemorrhage were apparent on gross and histopathologic examination, along with ischemic and hemorrhagic lesions of the gastrointestinal tract, and acute renal tubular necrosis. Thus, it appears that a single protein mediator (cachectin) is capable of inducing many of the deleterious effects of endotoxin.

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.

SummaryWhile it has been known that dendritic cells arise from proliferating precursors in situ, it has been difficult to identify progenitors in culture. We find that aggregates of growing dendritic cells develop in cultures of mouse blood that are supplemented with granulocyte/macrophage colony-stimulating factor (GM-CSF) but not other CSFs. The dendritic cell precursor derives from the Ia-negative and nonadherent fraction. The aggregates of developing dendritic cells appear at about 1 wk of culture, with 100 or more such clusters being formed per 106 blood leukocytes. The aggregates can be dislodged and subcultured as expanding clusters that are covered with cells having the motile sheet-like processes ("veils") of dendritic cells. By about 2 wk, large numbers of single, major histocompatibility complex (MHC) class II-rich dendritic cells begin to be released into the medium. Combined immunoperoxidase and [3H]thymidine autoradiograph}, show that the cells that proliferate within the aggregate lack certain antigenic markers that are found on mature dendritic cells. However, in pulse-chase protocols, the [3H]thymidinelabeled progeny exhibit many typical dendritic cell features, including abundant MHC class II and a cytoplasmic granular antigen identified by monodonal antibody 2A1. The progeny dendritic cells are potent stimulators of the mixed leukocyte reaction and can home to the T-dependent areas of lymph node after injection into the footpads. We conclude that mouse blood contains GM-CSF-dependent, proliferating progenitors that give rise to large numbers of dendritic cells with characteristic morphology, mobility, phenotype, and strong T cell stimulatory function.

Cytokines mediate many host responses to bacterial infections. We determined the inflammatory activities of five cytokines in the central nervous system: TNF-alpha, IL-1 alpha, IL-1 beta, macrophage inflammatory protein 1 (MIP-1), and macrophage inflammatory protein 2 (MIP-2). Using a rabbit model of meningeal inflammation, each cytokine (except IL-1 beta) induced enhanced blood brain barrier permeability, leukocytosis in cerebrospinal fluid, and brain edema. Homologous antibodies to each mediator inhibited leukocytosis and brain edema, and moderately decreased blood brain barrier permeability. In rabbits treated with anti-CD-18 antibody to render neutrophils dysfunctional for adhesion, each cytokine studied lost the ability to cause leukocytosis and brain edema. After intracisternal challenge with pneumococci, antibodies to TNF or IL-1 prevented inflammation, while anti-MIP-1 or anti-MIP-2 caused only a 2-h delay in the onset of inflammation. We suggest these cytokines have multiple inflammatory activities in the central nervous system and contribute to tissue damage during pneumococcal meningitis.

A number of studies of inflammation and of cell growth and transformation have recently converged by defining two related families of cytokines. The first, represented by macrophage inflammatory protein 1, is composed of several gene products that have been identified in activated T cells, macrophages, and fibroblasts. The biological activities of this family are still being characterized but so far include effects on neutrophils, monocytes, and hematopoietic cells. The second, represented by macrophage inflammatory protein 2, includes platelet products such as platelet factor 4 and beta-thromboglobulin as well as several other recently described gene products that have effects on a number of cell types including neutrophils, fibroblasts, hematopoietic cells, and melanoma cells. The two families are structurally related and may have evolved from a common ancestral gene that duplicated and then diverged. Their differential control and expression in a wide variety of cell types suggests that they may have multiple functions in regulating inflammation and cell growth.

The haemopoietic system has three main compartments: multi-potential stem cells, intermediate stage progenitor cells and mature cells. The availability of simple reproducible culture systems has made possible the characterization and purification of regulators of the progenitor cells, including colony-stimulating factors and interleukins. In contrast, our knowledge of the regulators involved in the control of stem cell proliferation is limited. The steady-state quiescent status of the haemopoietic stem cell compartment is thought to be controlled by locally acting regulatory elements present in the stromal microenvironment, but their purification has been hampered by the lack of suitable culture systems. We have recently developed a novel in vitro colony assay that detects a primitive cell (CFU-A) which has similar proliferative characteristics, in normal and regenerating bone marrow, to the CFU-S (haemopoietic stem cells, as defined by the spleen colony assay) and which responds to CFU-S-specific proliferation regulators. We have now used this assay to purify to homogeneity a macrophage-derived reversible inhibitor of haemopoietic stem cell proliferation (stem cell inhibitor, SCI). Antibody inhibition and sequence data indicate that SCI is identical to a previously described cytokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and that SCI/MIP-1 alpha is functionally and antigenically identical to the CFU-S inhibitory activity obtained from primary cultures of normal bone marrow cells. The biological activities of SCI/MIP-1 alpha suggest that it is a primary negative regulator of stem cell proliferation and that it has important therapeutic applications in protecting haemopoietic stem cells from damage during cytotoxic therapies for cancer.

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.