During the innate immune response to infection, monocyte-derived cytokines (monokines), stimulate natural killer (NK) cells to produce immunoregulatory cytokines that are important to the host's early defense. Human NK cell subsets can be distinguished by CD56 surface density expression (ie, CD56 bright and CD56 dim ). In this report, it is shown that CD56 bright NK cells produce significantly greater levels of interferon-␥, tumor necrosis factor-␤, granulocyte macrophage-colony-stimulating factor, IL-10, and IL-13 protein in response to monokine stimulation than do CD56 dim NK cells, which produce negligible amounts of these cytokines. Further, qualitative differences in CD56 bright NK-derived cytokines are shown to be dependent on the specific monokines present. For example, the monokine IL-15 appears to be required for type 2 cytokine produc- IntroductionNatural killer (NK) cells are innate immune effectors that produce immunoregulatory cytokines, such as interferon (IFN)-␥ and granulocyte macrophage-colony-stimulating factor GM-CSF, critical to early host defense against a variety of viral, bacterial, and parasitic pathogens. [1][2][3][4] Human NK cells comprise approximately 10% of all peripheral blood lymphocytes and are characterized phenotypically by the presence of CD56 and the lack of CD3. 1 There are 2 distinct subsets of human NK cells identified by cell surface density of CD56. The majority (approximately 90%) of human NK cells are CD56 dim and express high levels of Fc␥RIII (CD16), whereas a minority (approximately 10%) are CD56 bright and CD16 dim/neg . 5 CD56 bright NK cells constitutively express the high-and intermediate-affinity IL-2 receptors and expand in vitro and in vivo in response to low (picomolar) doses of IL-2. [6][7][8] These NK cells also express the c-kit receptor tyrosine kinase whose ligand enhances IL-2-induced proliferation. 9,10 In contrast, resting CD56 dim NK cells express only the intermediate affinity IL-2 receptor, are c-kit neg , and proliferate weakly in response to high doses of IL-2 (1 to 10 nM) in vitro, even after induction of the high-affinity IL-2 receptor. 6,7 Resting CD56 dim NK cells are more cytotoxic against NK-sensitive targets than CD56 bright NK cells. 11 However, after activation with IL-2 or IL-12, CD56 bright cells exhibit similar or enhanced cytotoxicity against NK targets compared to CD56 dim cells. [11][12][13] NK cell subsets have differential natural killer receptor (NKR) NK cells constitutively express receptors for monocyte-derived cytokines (monokines) and produce critical cytokines, such as IFN-␥, in response to monokine stimulation. [17][18][19][20] In the current study we examine CD56 bright and CD56 dim NK cell production of multiple cytokines-including IFN-␥, tumor necrosis factor (TNF)-␤, IL-10, IL-13, TNF-␣, and GM-CSF-in response to stimulation with monokines. We show that CD56 bright NK cells are the primary population responsible for NK cell cytokine production in response to monokines. These data support a model whereby CD56 bright a...

Interferon-gamma-inducing factor (IGIF, interleukin-18) is a recently described cytokine that shares structural features with the interleukin-1 (IL-1) family of proteins and functional properties with IL-12. Like IL-12, IGIF is a potent inducer of interferon (IFN)-gamma from T cells and natural killer cells. IGIF is synthesized as a biologically inactive precursor molecule (proIGIF). The cellular production of IL-1beta, a cytokine implicated in a variety of inflammatory diseases, requires cleavage of its precursor (proIL-1beta) at an Asp-X site by interleukin-1beta-converting enzyme (ICE, recently termed caspase-1). The Asp-X sequence at the putative processing site in proIGIF suggests that a protease such as caspase-1 might be involved in the maturation of IGIF. Here we demonstrate that caspase-1 processes proIGIF and proIL-1beta with equivalent efficiencies in vitro. A selective caspase-1 inhibitor blocks both lipopolysaccharide-induced IL-1beta and IFN-gamma production from human mononuclear cells. Furthermore, caspase-1-deficient mice are defective in lipopolysaccharide-induced IFN-gamma production. Our results thus implicate caspase-1 in the physiological production of IGIF and demonstrate that it plays a critical role in the regulation of multiple proinflammatory cytokines. Specific caspase-1 inhibitors would provide a new class of anti-inflammatory drugs with multipotent action.

The caspase family represents a new class of intracellular cysteine proteases with known or suspected roles in cytokine maturation and apoptosis. These enzymes display a preference for Asp in the P1 position of substrates. To clarify differences in the biological roles of the interleukin-1␤ converting enzyme (ICE) family proteases, we have examined in detail the specificities beyond the P1 position of caspase-1, -2, -3, -4, -6, and -7 toward minimal length peptide substrates in vitro. We find differences and similarities between the enzymes that suggest a functional subgrouping of the family different from that based on overall sequence alignment. The primary specificities of ICE homologs explain many observed enzyme preferences for macromolecular substrates and can be used to support predictions of their natural function(s). The results also suggest the design of optimal peptidic substrates and inhibitors.A growing body of evidence supports important roles for the interleukin-1␤ converting enzyme (ICE) 1 (1, 2) and its homologs (recently renamed caspases (3)) in cytokine maturation and apoptosis. The caspase gene family, defined by protein sequence homology but also characterized by conservation of key catalytic and substrate-recognition amino acids, includes caspase-2 (4), caspase-3 (5-7), caspase-4 (8 -10), caspase-5 (10), caspase-6 (11), caspase-7 (12-14), caspase-8 (15-17), caspase-9 (18, 19), and caspase-10 (17). Each is an intracellular cysteine protease that shares with the serine protease granzyme B specificity for Asp in the P1 position of substrates. The specific biological roles and interrelationships of these enzymes are for the most part unknown and are areas of active investigation in many laboratories.A role for caspase-1 in inflammation is supported by several lines of evidence. Caspase-1-deficient mice, and cells derived from those animals, are deficient in IL-1␤ maturation and are resistant to endotoxic shock (20,21). Peptidic inhibitors of caspase-1 can be effective in blocking maturation and release of IL-1␤ by cultured cells (1) and in whole animals (22, 23) and of inflammation in animal models (24,25). The selectivity of the inhibitors employed in these studies among the caspases has not been demonstrated, and so the precise role of each caspase in inflammation is uncertain. Nevertheless the results uphold the promise of caspase-1 and/or its homologs as targets for anti-inflammatory drug discovery.Caspases play important roles in apoptosis signaling and effector mechanisms. Sequence alignments reveal homology with Ced-3 (26), a nematode cysteine protease (27, 28) that is required for cell death. The viral proteins CrmA and p35 are antiapoptotic and act by inhibition of caspases (29,30). A bacterial invasin induces apoptosis by binding to and activating caspase-1 specifically (31). Caspase-3 is necessary and sufficient for apoptosis in one acellular model (6); however, in mice the essential function of this enzyme is limited to apoptosis in the brain (32). A hallmark of apoptosis is the pr...

These studies demonstrate that treatment of human U‐937 cells with ionizing radiation (IR) is associated with activation of a cytoplasmic myelin basic protein (MBP) kinase. Characterization of the kinase by gel filtration and in‐gel kinase assays support activation of a 40 kDa protein. Substrate and inhibitor studies further support the induction of protein kinase C (PKC)‐like activity. The results of N‐terminal amino acid sequencing of the purified protein demonstrate identity of the kinase with an internal region of PKC delta. Immunoblot analysis was used to confirm proteolytic cleavage of intact 78 kDa PKC delta in control cells to the 40 kDa C‐terminal fragment after IR exposure. The finding that both IR‐induced proteolytic activation of PKC delta and endonucleolytic DNA fragmentation are blocked by Bcl‐2 and Bcl‐xL supports an association with physiological cell death (PCD). Moreover, cleavage of PKC delta occurs adjacent to aspartic acid at a site (QDN) similar to that involved in proteolytic activation of interleukin‐1 beta converting enzyme (ICE). The specific tetrapeptide ICE inhibitor (YVAD) blocked both proteolytic activation of PKC delta and internucleosomal DNA fragmentation in IR‐treated cells. These findings demonstrate that PCD is associated with proteolytic activation of PKC delta by an ICE‐like protease.

Recent studies have shown that protein kinase C (PKC) δ is proteolytically activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. However, the relationship of PKCδ cleavage to induction of apoptosis is unknown. The present studies demonstrate that full-length PKCδ is cleaved at DMQD330N to a catalytically active fragment by the cysteine protease CPP32. The results also demonstrate that overexpression of the catalytic kinase fragment in cells is associated with chromatin condensation, nuclear fragmentation, induction of sub-G1 phase DNA and lethality. By contrast, overexpression of full-length PKCδ or a kinase inactive PKCδ fragment had no detectable effect. The findings suggest that proteolytic activation of PKCδ by a CPP32-like protease contributes to phenotypic changes associated with apoptosis.

The baculovirus antiapoptotic protein p35 inhibited the proteolytic activity of human interleukin-1 beta converting enzyme (ICE) and three of its homologs in enzymatic assays. Coexpression of p35 prevented the autoproteolytic activation of ICE from its precursor form and blocked ICE-induced apoptosis. Inhibition of enzymatic activity correlated with the cleavage of p35 and the formation of a stable ICE-p35 complex. The ability of p35 to block apoptosis in different pathways and in distantly related organisms suggests a central and conserved role for ICE-like proteases in the induction of apoptosis.

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For complex diseases in which multiple mediators contribute to overall disease pathogenesis by distinct or redundant mechanisms, simultaneous blockade of multiple targets may yield better therapeutic efficacy than inhibition of a single target. However, developing two separate monoclonal antibodies for clinical use as combination therapy is impractical, owing to regulatory hurdles and cost. Multi-specific, antibody-based molecules have been investigated; however, their therapeutic use has been hampered by poor pharmacokinetics, stability and manufacturing feasibility. Here, we describe a generally applicable model of a dual-specific, tetravalent immunoglobulin G (IgG)-like molecule--termed dual-variable-domain immunoglobulin (DVD-Ig)--that can be engineered from any two monoclonal antibodies while preserving activities of the parental antibodies. This molecule can be efficiently produced from mammalian cells and exhibits good physicochemical and pharmacokinetic properties. Preclinical studies of a DVD-Ig protein in an animal disease model demonstrate its potential for therapeutic application in human diseases.