The role of cytokines in the generation of inflammation and tissue damage in experimental gram-positive meningitis.

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.

Cloning and characterization of a cDNA for murine macrophage inflammatory protein (MIP), a novel monokine with inflammatory and chemokinetic properties.

In the course of studies on cachectin/TNF being conducted in our laboratory, a novel macrophage product has been detected and characterized. Termed macrophage inflammatory protein or MIP, this protein appears to be an endogenous mediator of the inflammatory events induced by endotoxin. A cDNA cloned probe for this protein has been isolated from a lambda gt10 phage library prepared from poly(A)+ RNA obtained of endotoxin-induced RAW264.7 cells. The sequence codes for a 92 amino acid-long polypeptide, of which 69 amino acids correspond to the mature product. The sequence predicts a molecular weight of 7,889 and structural analysis of the protein indicates a characteristic signal sequence alpha-helix and a hydrophobic core. Sequence data also confirm no sequence similarity to any other protein listed in the Dayhoff data base.

Identification of proliferating dendritic cell precursors in mouse blood.

While 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 10(6) 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 autoradiography 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]thymidine-labeled progeny exhibit many typical dendritic cell features, including abundant MHC class II and a cytoplasmic granular antigen identified by monoclonal 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.

Myelopoietic enhancing effects of murine macrophage inflammatory proteins 1 and 2 on colony formation in vitro by murine and human bone marrow granulocyte/macrophage progenitor cells.

Two recently identified and purified murine macrophage inflammatory proteins MIP-1 and MIP-2 were tested in vitro both alone, and in combination with purified recombinant (r) murine (mu) GM-CSF, natural (n)muCSF-1, or rhuman (hu)G-CSF, for effects on mouse marrow CFU-GM, in combination with erythropoietin for effects on mouse marrow BFU-E, and in combination with rhuGM-CSF or rhuG-CSF for effects on human marrow CFU-GM. MIP-1 and MIP-2 did not stimulate, but did enhance by up to threefold, colony formation of mouse CFU-GM co-stimulated by rmuGM-CSF and nmuCSF-1, but not by rhuG-CSF, in the absence or presence of serum. MIP-1 and MIP-2 were maximally active at concentrations greater than or equal to 100 ng/ml and the actions appeared to be initiated during the DNA synthetic portion of the cell cycle. Neither MIP-1 nor MIP-2 at up to 1 microgram/ml had any effect on mouse BFU-E, in the absence or presence of erythropoietin. Both MIP-1 and MIP-2 had direct acting effects on purified mouse CFU-GM. The cloning efficiency of 200 purified cells plated with 50 U muCSF-1 was 82% with and 43% without MIP; the cloning efficiency with 50 U rmuGM-CSF was 65% with and 35% without MIP. MIP effects were not mimicked by bacterial LPS, rhuIL-1 alpha, rhuIL-6, or rmuIL-4, and were neutralized by their respective specific antibodies. MIP-1 and MIP-2 also enhanced endogenously stimulated and rhuGM-CSF-, but not rhuG-CSF-, stimulated colony formation by human marrow CFU-GM. These results demonstrate a new role for MIP-1 and MIP-2 in vitro as myelopoietic enhancing activities for CFU-GM.

Resolution of the two components of macrophage inflammatory protein 1, and cloning and characterization of one of those components, macrophage inflammatory protein 1 beta.

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.

Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties.

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.

Macrophage inflammatory protein-1: a prostaglandin-independent endogenous pyrogen.

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.

Shock and tissue injury induced by recombinant human cachectin.

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.

Inhibition of acute myelogenous leukemia progenitor proliferation by macrophage inflammatory protein 1-alpha.

Macrophage inflammatory protein-alpha (MIP-1 alpha), an 8-kDa peptide produced by stimulated macrophages, has been recently sequenced and cloned. In addition to its inflammatory effects, MIP-1 alpha inhibits proliferation of immature hematopoietic progenitors both in vitro and in vivo. Because the gene coding for MIP-1 alpha is expressed in peripheral blood cells obtained from patients with acute myelogenous leukemia (AML), we sought to evaluate the effect of MIP-1 alpha on AML precursors. We studied bone marrow samples from 21 AML patients using both the AML blast colony assay and the delta suspension culture assay. We found that recombinant human (rh) MIP-1 alpha significantly inhibits early and mature AML progenitors with sample-to-sample variability, by up to 79% at concentrations ranging from 40 to 1600 ng/ml. These results were obtained in the presence of fetal calf serum either alone or with granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, or interleukin-3. In contrast, rhMIP-1 alpha (400 ng/ml) did not significantly affect normal colony-forming unit granulocyte-macrophage (CFU-GM), or burst-forming unit-erythroid (BFU-E) proliferation. These data prompted us to delineate the inhibitory mechanism of MIP-1 alpha. Consequently, we used the thymidine suicide technique to measure DNA synthesis in AML progenitors and the enzyme-linked immunosorbent assay to quantify intracellular levels of interleukin-1 beta in AML blasts following incubation with MIP-1 alpha. We found that whereas MIP-1 alpha prevented AML progenitors from entering the proliferative phase of the cell cycle, it had no effect on interleukin-1 beta levels. Taken together, our data suggest that MIP-1 alpha may have clinical benefits in therapy for AML and should be considered for evaluation in a clinical setting.

MIP-1 alpha is a regulator of mitotic and meiotic DNA synthesis during spermatogenesis.

To find out whether macrophage inflammatory protein-1 alpha (MIP-1 alpha) has a role in the regulation of germ cell development, we studied its effects on spermatogenic stage-specific DNA synthesis in vitro. MIP-1 alpha increased the DNA synthesis of primitive type A2-4 spermatogonia and of premeiotic cells, whereas the DNA synthesis of more differentiated intermediate and type B spermatogonia was inhibited when cultured in the presence of MIP-1 alpha. An antibody against MIP-1 alpha cross-reacted with a protein of 15 kDa from every spermatogenic stage of rat seminiferous epithelium. Immunohistochemical studies with the same antibody revealed a complex pattern of MIP-1 alpha localization both in primitive and advanced spermatogenic cells. These observations suggest that MIP-1 alpha is a local regulator of mitotic and meiotic DNA synthesis.

Inhibition of erythroid differentiation of mouse erythroleukemia cells by a macrophage product(s).

Conditioned media from established murine macrophage cell lines (RAW264.7, P388D1, and WEHI-3) incubated with endotoxin in a serum-free medium contain an erythroid inhibitory activity (EIA) that inhibited dimethylsulfoxide-induced erythroid differentiation of mouse Friend virus-transformed erythroleukemia cells. Endotoxin itself has no EIA activity. Partial purification of EIA demonstrated that it is distinct from other macrophage products such as IL-1, TGF beta, ECGF, FGF, G-CSF, hepatocyte stimulating factor, interferon, PDGF, and cachectin/TNF. These findings indicate that EIA is a macrophage product distinct from other monokines.

Macrophage inflammatory proteins 1 and 2: members of a novel superfamily of cytokines.

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.

Unresponsiveness of primitive chronic myeloid leukemia cells to macrophage inflammatory protein 1 alpha, an inhibitor of primitive normal hematopoietic cells.

Most primitive hematopoietic cells appear to be normally quiescent in vivo, whereas their leukemic counterparts in patients with chronic myeloid leukemia (CML) are maintained in a state of rapid turnover. This difference is also seen in the long-term culture system, where control of primitive hematopoietic progenitor proliferation is mediated by interactions of these cells with marrow-derived mesenchymal cells of the fibroblast lineage. We now show that exogenous addition of macrophage inflammatory protein 1 alpha (MIP-1 alpha) to normal long-term cultures can reversibly and specifically block the activation of "primitive" (high proliferative potential), but not "mature" (lower proliferative potential), progenitors in the adherent layer of these cultures. Moreover, addition of MIP-1 beta after primitive-progenitor activation can prevent the subsequent return of these cells to a quiescent state a few days later as shown previously in similar experiments using antibodies to transforming growth factor beta. This suggests that the level of MIP-1 alpha (or a related MIP-1 alpha agonist) produced in LTCs, like the level of transforming growth factor beta, may be necessary, but is not on its own sufficient, to mediate the inhibitory activity of the regulatory cells in the adherent layer. Addition of MIP-1 alpha to similar long-term cultures containing normal marrow adherent layers but supporting exclusively neoplastic (CML) hematopoiesis did not block the cycling of primitive neoplastic progenitors. A defect in the responsiveness of CML cells to MIP-1 alpha (or a similarly acting chemokine) would explain their deregulated proliferative behavior in this model and, by extrapolation to the in vivo setting, suggests a molecular mechanism whereby the leukemic clone may become amplified at the stem-cell level. In addition, these findings suggest approaches to the therapy of CML, using inhibitors such as MIP-1 alpha for the protection of primitive normal cells.

Identification and characterization of macrophage inflammatory protein 2.

In response to endotoxin, macrophages secrete a protein with a molecular mass of approximately 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 of the 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 factor-treated cells.

Production of interleukin-1 but not tumor necrosis factor by human monocytes stimulated with pneumococcal cell surface components.

While there is considerable evidence that both interleukin-1 (IL-1) and tumor necrosis factor (TNF) are central mediators of inflammation caused by gram-negative bacteria and endotoxin, the roles of these two mediators in gram-positive infection are unknown. Pneumococcal infections are characterized by an intense inflammatory reaction in infected tissues. Current evidence suggests that the component of the pneumococcus which causes this inflammation in many body sites is the cell wall. We determined the ability of native pneumococcal cell wall, lipoteichoic acid, and cell wall subcomponents to stimulate secretion of IL-1 and TNF from human monocytes. Each pneumococcal cell surface component was found to have a different specific activity for induction of IL-1. Teichoication was an important determinant of this activity: teichoicated species were at least 10,000-fold more potent than endotoxin and 100-fold more potent than teichoic acid-free peptidoglycan. IL-1-inducing activity was greatly reduced by chemical alteration of the teichoic acid. In contrast to endotoxin, cell wall did not induce production of TNF. This dissociation of the production of IL-1 and TNF during the response of the human monocyte to pneumococcal surface components suggests that, in at least some circumstances, the mechanisms for generation of an inflammatory response to infection may be fundamentally different between gram-positive and gram-negative disease.

Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1 alpha on long-term reconstituting stem cells in vivo.

A macrophage-derived inhibitor of early hematopoietic progenitors (colony-forming unit-spleen, CFU-A) called stem cell inhibitor was found to be identical to macrophage inflammatory protein-1 alpha (MIP-1 alpha). We investigated the effect of MIP-1 alpha on the earliest stem cells that sustain long-term hematopoiesis in vivo in a competitive bone marrow repopulation assay. Because long-term reconstituting (LTR) stem cells are normally quiescent, an in vivo model was first developed in which they are triggered to cycle. A first 5-fluorouracil (5-FU) injection was used to eliminate later progenitors, causing the LTR stem cells, which are normally resistant to 5-FU, to enter the cell cycle and become sensitive to a second 5-FU injection administered 5 days later. Human MIP-1 alpha administered from day 0 to 7 was unable to prevent the depletion of the LTR stem cells by the second 5-FU treatment, as observed on day 7 in this model, suggesting that the LTR stem cells were not prevented from being triggered into cycle despite the MIP-1 alpha treatment. However, the MIP-1 alpha protocol used here did substantially decrease the number of more mature hematopoietic progenitors (granulocyte-macrophage colony-forming cells [CFC], burst-forming unit-erythroid, CFCmulti, and preCFCmulti) recovered in the bone marrow shortly after a single 5-FU injection. In vitro, MIP-1 alpha had no inhibitory effect on the ability of these progenitors to form colonies. This study confirms the in vivo inhibitory effect of MIP-1 alpha on subpopulations of hematopoietic progenitors that are activated in myelodepressed animals. However, MIP-1 alpha had no effect on the long-term reconstituting stem cells in vivo under conditions in which it effectively reduced all later progenitors.

Fever and feeding in the rat: actions of intrahypothalamic interleukin-6 compared to macrophage inflammatory protein-1 beta (MIP-1 beta).

The chemokines, macrophage inflammatory protein-1 (MIP-1) and its subunit MIP-1 beta, induce an intense fever in the rat when they are injected directly into the anterior hypothalamic, pre-optic area (AH/POA), a region containing thermosensitive neurons. The purpose of this study was to compare the central action on body temperature (Tb) of MIP-1 beta with that of interleukin-6 (IL-6), which also has been implicated in the cerebral mechanism underlying the pathogenesis of fever. Following the stereotaxic implantation in the AH/POA of guide cannulae for repeated micro-injections, radio transmitters which monitor Tb continuously were inserted intraperitoneally in each of 15 male Sprague-Dawley rats. Each micro-injection was made in a site in the AH/POA in a volume of 1.0 microliter of pyrogen-free artificial CSF, recombinant murine MIP-1 beta, or recombinant human IL-6. MIP-1 beta in a dose of 25 pg evoked an intense fever characterized by a short latency, a mean maximum rise in Tb of 2.4 +/- 0.21 degrees C reached by 3.7 +/- 0.42 hr, and a duration exceeding 6.5 hr. Injected into homologous sites in the AH/POA, IL-6 induced a dose dependent fever of similar latency and a mean maximal increase in Tb of 1.2 +/- 0.25 degrees C, 1.8 +/- 0.15 degrees C, and 2.1 +/- 0.22 degrees C and duration of 6.2 +/- 1.28 hr, 6.7 +/- 0.49 hr, and 6.8 +/- 0.65 hr when given in doses of 25, 50, and 100 ng, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta 1, macrophage inflammatory protein-1 alpha, and interleukin-8 gene expression is lower in stimulated human neonatal compared with adult mononuclear cells.

Hematopoiesis is developmentally immature in the newborn compared with the adult. Diminished gene expression of several positive hematopoietic regulators has been observed in activated cord compared with adult peripheral blood mononuclear cells (MNC; Cairo et al. Pediatr Res, 30:362, 1991 and Cairo et al, Pediatr Res, 31:574, 1992). However, altered expression of negative hematopoietic regulators during states of increased demand may also contribute to the pathogenesis of newborn dyshematopoiesis. To test this hypothesis, we measured protein levels of transforming growth factor-beta 1 (TGF-beta 1) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) in the conditioned media of human umbilical cord and adult MNC using specific enzyme-linked immunosorbent assays. There was significantly less TGF-beta 1 in culture supernatants of cord versus adult MNC after 24, 72, and 120 hours of stimulation (P < .05), and significantly less MIP-1 alpha in cord versus adult supernatants after 72 hours and 120 hours of stimulation (P < .01). We then examined the mRNA expression of the negative regulators TGF-beta 1, MIP-1 alpha, and interleukin-8 (IL-8) in cord and adult MNC using Northern blot hybridization followed by quantitative densitometry. Cord MNC expressed significantly less TGF-beta 1 mRNA than adult MNC 6 hours and 72 hours after stimulation (P < .001). Cord MNC expressed significantly less MIP-1 alpha mRNA than adult MNC 6 hours (P < .01), 24 hours (P < .001), and 72 hours after stimulation (P < .001). Cord MNC also expressed significantly less IL-8 mRNA than adult MNC 6 hours after stimulation (P < .001). Therefore, decreased mRNA accumulation appears to coincide with reduced cytokine expression in the activated cord MNC. There were no significant differences in the transcription rates determined by nuclear run-on assay of either the TGF-beta 1 or MIP-1 alpha gene in cord versus adult MNC after 6 hours of stimulation, suggesting that the reduced TGF-beta 1 and MIP-1 alpha mRNA in activated cord MNC may be secondary to alteration in posttranscriptional regulation. The present results, together with those of our previous studies, suggest that the altered expression of both positive and negative hematopoietic regulators may be involved in the immaturity of host defense in human neonates.