Several cytokines, in particular tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), have been shown to be responsible for pathological reactions which may lead to shock and death observed in infection with Gram-negative bacteria and in response to endotoxins (lipopolysaccharides, LPS). Priming of mice with the avirulent Bacille Calmette Guérin (BCG) vaccine strain of Mycobacterium bovis increases the sensitivity of mice to the lethal effect of LPS and results in an efficient priming for cytokine production. In response to low doses (1 microgram/mouse) of LPS, BCG-primed mice produce interleukin-12 (IL-12) which controls IFN-gamma production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to suppress IFN-gamma production. However, the concentration of the biologically active IL-12 p70 heterodimer is similar in the serum of both BCG-primed or unprimed mice, reaching levels of 1-3 ng/ml at 3-6 h after LPS injection, whereas IFN-gamma production was observed only in BCG-primed mice. The priming effect of BCG on IFN-gamma production appears to be mostly due to its ability to increase TNF-alpha production, which acts as cofactor with LPS-induced IL-12 in inducing IFN-gamma production, as shown by the ability of injection of TNF-alpha and LPS (1 microgram/mouse), but not LPS alone, to induce IFN-gamma production. However, in addition to TNF-alpha, other LPS-induced cofactor(s) are required in cooperation with IL-12 to induce optimal IFN-gamma production, because co-injection of TNF-alpha and IL-12, sufficient to induce serum concentrations of both cytokines higher and more persistent than those obtained by injection of LPS, was not sufficient to induce IFN-gamma production in vivo. Neutralizing anti-IL-12 antibodies, in addition to inhibiting the in vivo LPS-induced IFN-gamma production, also completely protect BCG-primed mice injected with up to 10 micrograms of LPS from shock-induced death. Thus, IL-12 is required for IFN-gamma production and lethality in an endotoxic shock model in mice.

SummaryThe Shwartzman reaction is elicited by two injections of lipopolysaccharide (LPS) in mice. The priming LPS injection is given in the footpad, whereas the lethal LPS challenge is given intravenously 24 h later. The injection of interferon 3, (IFN-'y) or interleukin 12 (IL-12) instead of the LPS priming injection induced the lethal reaction in mice further challenged with LPS. Antibodies against IFN-q/when given together with the priming agent, prevented the lethal reaction in mice primed with either LPS, IL-12, or IFN-3'. Antibodies against IL-12, when given together with the priming agent, prevented the lethal reaction in mice primed with either LPS or IL-12 but not with IFN-% These results strongly suggest that LPS induces the release of IL-12, that IL-12 induces the production of IFN-% and that IFN-7 is the cytokine that primes macrophages and other cell types. Upon LPS challenge, the lethal Shwartzman reaction is induced by a massive production of inflammatory cytokines that act on the target sites already sensitized by IFN-7. If mixtures of TNF and IL-1 or mixtures of TNF and IFN-3' are used to challenge mice previously primed with IFN-7 or ILo12, mortality is induced. In the same conditions, the individual cytokines or a mixture of IL-1 and IFN-3' do not replace the LPS challenge. When the mice are primed with LPS, the combination of TNF, IL-1, and IFN-7 induced only a partial mortality incidence suggesting that the involvement of other LPS-induced factors. Bacterial LPS is a cell wall component of Gram-negative bacteria that is responsible for most of the toxic manifestations associated with bacterial infections. In mice, a lethal shock syndrome, known as the generalized Shwartzman reaction, can be elicited by two consecutive injections of LPS (for review see 1). A priming dose of LPS, injected intradermall), in the footpad (f.p.)l, is followed after 24 h by an intravenous challenge injection of LPS. After this challenge injection, which is not lethal per se, the mice die within the following 48 h from disseminated intravascular coagulation, vascular occlusion, hemorrhage, perivascular accumulation of leukocytes, and necrosis (2). This hypersensitivity reaction occurs only if the time interval between these injections is crucially fixed at between 18 and 24 h. There is no response when the intravenous challenge of LPS is given either earlier or later, or when the order of priming and challenge 1 Abbreviations used in thispaper: f.p., footpad; h, human; m, mouse; MIF, macrophage inhibiting factor; R, receptor. injections is reversed. FinaUy, the reaction does not occur if both the LPS injections are intradermal or if the priming dose of LPS exceeds an optimum. The careful dosage and timing of the LPS injections and the need of specific routes of administration indicate that the Shwartzman reaction is elicited by induced endogenous factors acting in a precise time sequence. IFN-'y, TNF, and IL-1 are known to be involved in the pathogenesis of the generalized Shwartzman reaction. IFN-7 seems important ...

Two novel human β-chemokines, Ckβ-8 or myeloid progenitor inhibitory factor 1 (MPIF-1), and Ckβ-6 or MPIF-2, were discovered as part of a large scale cDNA sequencing effort. The MPIF-1 and MPIF-2 cDNAs were isolated from aortic endothelium and activated monocyte libraries, respectively. Both of the cDNAs were cloned into a baculovirus vector and expressed in insect cells. The mature recombinant MPIF-1 protein consists of 99 amino acids and is most homologous to macrophage inflammatory protein (MIP)-1α, showing 51% identity. It displays chemotactic activity on resting T lymphocytes and monocytes, a minimal but significant activity on neutrophils, and is negative on activated T lymphocytes. MPIF-1 is also a potent suppressor of bone marrow low proliferative potential colony-forming cells, a committed progenitor that gives rise to granulocyte and monocyte lineages. The mature recombinant MPIF-2 has 93 amino acid residues and shows 39 and 42% identity with monocyte chemoattractant protein (MCP)-3 and MIP-1α, respectively. It displays chemotactic activity on resting T lymphocytes, a minimal activity on neutrophils, and is negative on monocytes and activated T lymphocytes. On eosinophils, MPIF-2 produces a transient rise of cytosolic Ca2+ and uses the receptor for eotaxin and MCP-4. In hematopoietic assays, MPIF-2 strongly suppressed the colony formation by the high proliferative potential colony-forming cell (HPP-CFC), which represents a multipotential hematopoietic progenitor.