Jennifer Major

Hypoxic induction of interleukin-8 gene expression in human endothelial cells.

Because leukocyte-mediated tissue damage is an important component of the pathologic picture in ischemia/reperfusion, we have sought mechanisms by which PMNs are directed into hypoxic tissue. Incubation of human endothelial cells (ECs) in hypoxia, PO2 approximately 14-18 Torr, led to time-dependent release of IL-8 antigen into the conditioned medium; this was accompanied by increased chemotactic activity for PMNs, blocked by antibody to IL-8. Production of IL-8 by hypoxic ECs occurred concomitantly with both increased levels of IL-8 mRNA, based on polymerase chain reaction analysis, and increased IL-8 transcription, based on nuclear run-on assays. Northern analysis of mRNA from hypoxic ECs also demonstrated increased levels of mRNA for macrophage chemotactic protein-1, another member of the chemokine superfamily of proinflammatory cytokines. IL-8 gene induction was associated with the presence of increased binding activity in nuclear extracts from hypoxic ECs for the NF-kB site. Studies with human umbilical vein segments exposed to hypoxia also demonstrated increased elaboration of IL-8 antigen compared with normoxic controls. In mice exposed to hypoxia (PO2 approximately 30-40 Torr), there was increased pulmonary leukostasis, as evidenced by increased myeloperoxidase activity in tissue homogenates. In parallel, increased levels of transcripts for IP-10, a murine homologue in the chemokine family related to IL-8, were observed in hypoxic lung tissue. Taken together, these data suggest that hypoxia constitutes a stimulus for leukocyte chemotaxis and tissue leukostasis.

Neutrophil chemoattractant genes KC and MIP-2 are expressed in different cell populations at sites of surgical injury

KC and macrophage‐inflammatory protein‐2 (MIP‐2) are CXC chemokines that exhibit distinct temporal patterns of expression in the skin following surgical injury. In situ hybridization analysis demonstrates that these two chemokines are expressed by distinct cell types at different times following injury. Dermal fibroblasts and endothelial cells are primarily responsible for KC expression in the skin 6 h following surgery. In contrast, MIP‐2 production appears to be restricted to infiltrating inflammatory leukocytes including neutrophils and monocytes, which appear later in the response. This cell type‐specific pattern of chemokine expression is recapitulated in vitro using isolated primary‐ and long‐term‐cultured cell types. Primary dermal fibroblasts stimulated with interleukin‐1α express predominantly KC and very little MIP‐2, and peritoneal exudate neutrophils produce as much or more MIP‐2 as KC following stimulation in vitro. Although a collection of exogenous stimuli can induce expression of KC and MIP‐2, the quantitative ratio for expression reflects the cell type and not the stimulus. The selective expression of KC over MIP‐2 in endothelial cells results from markedly greater KC gene transcription and not from alterations in the rate of mRNA decay. These results demonstrate that distinct CXC chemokines show restricted expression in myeloid versus nonmyeloid cell types and that patterns of chemokine expression at sites of inflammation in vivo reflect the temporally ordered contribution of these distinct cell types.

The effects of oxidized low density lipoproteins on inducible mouse macrophage gene expression are gene and stimulus dependent.

Oxidized LDL has been previously reported to suppress the expression of genes induced in mononuclear phagocytes by inflammatory stimuli. In this study we extend these findings to demonstrate that the suppressive effects of oxidized LDL vary depending upon the gene being monitored and the stimulus being used to induce or enhance its expression. The expression of a selection of LPS-inducible genes exhibited differential sensitivity to pretreatment with oxidized LDL. Furthermore, the ability of oxidized LDL to suppress gene expression varied markedly with the inducing stimulus used. TNF alpha and IP-10 mRNA expression induced by IFN gamma and IL-2 was markedly more sensitive to suppression by oxidized LDL than that induced by LPS. The cooperative effects of IFN gamma and LPS on the expression of the inducible nitric oxide synthase gene were suppressed by oxidized LDL while the antagonistic effect of IFN gamma on LPS-induced expression of the TNF receptor type II mRNA was not altered. The suppressive activity of LDL was acquired only after extensive oxidation and was localized in the extractable lipid component. These results suggest a potent and direct connection between the oxidative modification of LDL and the chronic inflammation seen in atherogenic lesions. Furthermore, the appreciable selectivity of oxidized LDL in mediating secondary control of cytokine gene expression demonstrates that the active material(s) is targeted to disrupt specific intracellular signaling pathways.

IL-4 Pretreatment Selectively Enhances Cytokine and Chemokine Production in Lipopolysaccharide-Stimulated Mouse Peritoneal Macrophages

Although well recognized for its anti-inflammatory effect on gene expression in stimulated monocytes and macrophages, IL-4 is a pleiotropic cytokine that has also been shown to enhance TNF-α and IL-12 production in response to stimulation with LPS. In the present study we expand these prior studies in three areas. First, the potentiating effect of IL-4 pretreatment is both stimulus and gene selective. Pretreatment of mouse macrophages with IL-4 for a minimum of 6 h produces a 2- to 4-fold enhancement of LPS-induced expression of several cytokines and chemokines, including TNF-α, IL-1α, macrophage-inflammatory protein-2, and KC, but inhibits the production of IL-12p40. In addition, the production of TNF-α by macrophages stimulated with IFN-γ and IL-2 is inhibited by IL-4 pretreatment, while responses to both LPS and dsRNA are enhanced. Second, the ability of IL-4 to potentiate LPS-stimulated cytokine production appears to require new IL-4-stimulated gene expression, because it is time dependent, requires the activation of STAT6, and is blocked by the reversible protein synthesis inhibitor cycloheximide during the IL-4 pretreatment period. Finally, IL-4-mediated potentiation of TNF-α production involves specific enhancement of mRNA translation. Although TNF-α protein is increased in IL-4-pretreated cells, the level of mRNA remains unchanged. Furthermore, LPS-stimulated TNF-α mRNA is selectively enriched in actively translating large polyribosomes in IL-4-pretreated cells compared with cells stimulated with LPS alone.

Chemokine expression in trinitrochlorobenzene-mediated contact hypersensitivity

The expression of the murine IP‐10 and MCP‐1 genes has been examined in the skin of mice during contact hypersensitivity reactions to the hapten trinitrochlorobenezene (TNCB). In both naive and passively sensitized animals, challenge with TNCB resulted in elevated expression of both genes as early as 4 h as detected by Northern hybridization analysis. Twenty‐four hours after challenge, expression was markedly reduced in naive animals but remained elevated in sensitized animals. This prolonged expression of chemokine gene products correlates with the tissue swelling response generally used as a measure of delayed‐type hypersensitivity (DTH) in this model and suggests that the continued expression of these genes results from the stimulation of hapten‐specific T helper cells. Examination of cell type expression patterns by in situ hybridization using 3H‐radiolabeled riboprobes confirmed the results of Northern hybridization experiments. Both genes were expressed predominantly in cells exhibiting the morphology of connective tissue fibroblasts, although the distribution of cells positive for IP‐10 mRNA expression differed from that of cells expressing MCP‐1 mRNA. IP‐10 expression was localized almost exclusively to a population of connective tissue cells surrounding the fur follicle. MCP‐1 expression was rarely found associated with fur follicles but instead was distributed throughout the dermis in cells embedded in the collagenous extracellular matrix. Surprisingly, neither endothelial cells lining the small vessels located deep within the dermis nor epidermal keratinocytes were positive under any of the conditions utilized in the present study. Expression of both IP‐10 and MCP‐1 has been previously reported in a variety of distinct cell types in vitro. The present results indicate that only a subset of the cell types with such potential are stimulated to express these chemokine genes in vivo during hapten‐mediated DTH responses, implying the presence of subtle cell type‐ and tissue‐specific control mechanisms. J. Leukoc. Biol. 55: 452–460; 1994.

The Structural Basis of Force Generation by the Mitotic Motor Kinesin-5.

Background: Kinesin-5 motors are important for formation and maintenance of the bipolar mitotic spindle. Results: ATP binding triggers coupled conformational changes of kinesin-5 specific structural elements in the microtubule-bound motor domain. Conclusion: Kinesin-5 mechanochemistry is tuned to its cellular functions. Significance: Subnanometer resolution structure determination of microtubule-bound kinesin-5s and kinetics experiments reveal the molecular basis of their motor properties and of drug inhibition. Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement. These studies reveal coupled, nucleotide-dependent conformational changes that explain many of this motors properties. We find that ATP binding induces a ratchet-like docking of the neck linker and simultaneous, parallel docking of the N-terminal cover strand. Loop L5, the binding site for allosteric inhibitors of kinesin-5, also undergoes a dramatic reorientation when ATP binds, suggesting that it is directly involved in controlling nucleotide binding. Our structures indicate that allosteric inhibitors of human kinesin-5, which are being developed as anti-cancer therapeutics, bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Together, our data reveal the precision with which the molecular mechanism of kinesin-5 motors has evolved for force generation.

A lipopolysaccharide-inducible macrophage gene (D3) is a new member of an interferon-inducible gene cluster and is selectively expressed in mononuclear phagocytes

We previously reported the isolation and characterization of cDNA clones encoding novel lipopolysac‐ charide (LPS)‐inducible mRNAs from murine peritoneal macrophages. We now present the complete coding sequence of a cDNA previously termed D3. Analysis of multiple clones from a murine macrophage cDNA library provided a complete cDNA sequence of approximately 1.6 kb. The corresponding RNA contains a single open reading frame encoding a hydrophilic protein composed of 425 amino acids and is characterized by a region including three perfect and two imperfect repeats of a seven‐ amino‐acid sequence. Based on nucleotide and deduced amino acid sequence, this mRNA is a new member of a previously described multigene cluster of interferon‐ inducible genes termed the Mouse 200 series genes. This new sequence most closely resembles gene 204 because both D3 and 204 genes have segments containing the seven‐amino‐acid repeat sequence. The Mouse 202 and 204 genes, however, have an approximately 200‐amino‐ acid carboxyl‐terminal domain that is absent in the LPS‐ inducible macrophage‐derived cDNA. In addition, D3, 202, and 204 can all be distinguished from one another by virtue of unique 3′ noncoding regions 200‐300 base pairs in length. The D3 unique sequence is largely restricted to the smallest of the three size classes of this gene family expressed in macrophages and is not detected in interferon‐ or platelet‐derived growth factor‐stimulated fibroblasts. Overall, three separate mRNAs have now been described, each of which has three or more of a possible seven nucleotide sequence domains. Although the functions) of the members of this gene family remains unknown, the multiple forms inducible by diverse stimuli and their restricted cell type expression suggest diverse and important physiologic roles for their products in inflammation.

Lipid-induced insulin resistance is associated with increased monocyte expression of scavenger receptor CD36 and internalization of oxidized LDL.

Elevated free fatty acids (FFAs) contribute to the development of insulin resistance, type 2 diabetes mellitus (T2DM), and may be atherogenic. We tested the relationship among lipid‐induced insulin resistance, endothelial dysfunction, and monocyte capacity to form foam cells through scavenger receptor A (SRA) and CD36. Ten healthy subjects underwent 24‐h infusion of Intralipid/heparin and saline (0.5 ml/min) on two separate occasions followed by brachial artery reactivity testing and a euglycemic hyperinsulinemic (80 mU/(kg·min)) clamp study to determine insulin sensitivity. Isolation of blood monocytes was performed 24 h after infusion. Surface expression and function of CD36 and SRA to take up oxidized low‐density lipoprotein (oxLDL) was determined by flow cytometry and quantitative confocal imaging. Lipid infusion resulted in a twofold increase in serum FFA levels, reduced whole‐body glucose disposal by ∼20% (P < 0.05), and possibly impaired endothelial‐dependent vasodilation (P = 0.1). Blood monocytes obtained during lipid infusion demonstrated a ∼25% increase in cell surface expression of CD36 (P < 0.05) but no change in SRA expression. Enhanced CD36 expression was associated with a 50% increase in internalization of oxLDL (P < 0.05). The increase in CD36 surface expression during lipid infusion correlated inversely with glucose disposal (P < 0.05) and not with FFA levels or brachial artery dilation. These data support a role for FFAs in induction of insulin resistance and provide a link to atherogenic mechanisms mediated by expression of scavenger receptor CD36.

Comprehensive Structural Model of the Mechanochemical Cycle of a Mitotic Motor Highlights Molecular Adaptations in the Kinesin Family.

Significance Kinesins are a superfamily of ATP-dependent motors that are important for a wide variety of microtubule-based functions in eukaryotic cells. Kinesins have evolved to allow variable tuning of their motor properties, but the link between molecular variation and motor function is largely unknown. To understand this link, we have studied an essential mitotic kinesin, kinesin-5, which is the target of anticancer drugs. We used cryo-electron microscopy to visualize directly sequential conformational changes of structural elements during the motor ATPase cycle. We have identified the contribution of kinesin-5–specific variations to motor function indicating that kinesins indeed are precisely tuned according to cellular function. This insight will be important in designing kinesin-specific inhibitors in different disease contexts. Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and essential cellular activities are poorly understood. It has been assumed that the first identified kinesin—the transport motor kinesin-1—is the mechanistic paradigm for the entire superfamily, but accumulating evidence suggests otherwise. To address the deficits in our understanding of the molecular basis of functional divergence within the kinesin superfamily, we studied kinesin-5s, which are essential mitotic motors whose inhibition blocks cell division. Using cryo-electron microscopy and determination of structure at subnanometer resolution, we have visualized conformations of microtubule-bound human kinesin-5 motor domain at successive steps in its ATPase cycle. After ATP hydrolysis, nucleotide-dependent conformational changes in the active site are allosterically propagated into rotations of the motor domain and uncurling of the drug-binding loop L5. In addition, the mechanical neck-linker element that is crucial for motor stepping undergoes discrete, ordered displacements. We also observed large reorientations of the motor N terminus that indicate its importance for kinesin-5 function through control of neck-linker conformation. A kinesin-5 mutant lacking this N terminus is enzymatically active, and ATP-dependent neck-linker movement and motility are defective, although not ablated. All these aspects of kinesin-5 mechanochemistry are distinct from kinesin-1. Our findings directly demonstrate the regulatory role of the kinesin-5 N terminus in collaboration with the motor’s structured neck-linker and highlight the multiple adaptations within kinesin motor domains that tune their mechanochemistries according to distinct functional requirements.

Vav guanine nucleotide exchange factors link hyperlipidemia and a prothrombotic state

Platelet hyperactivity associated with hyperlipidemia contributes to development of a pro-thrombotic state. We previously showed that oxidized LDL (oxLDL) formed in the setting of hyperlipidemia and atherosclerosis initiated a CD36-mediated signaling cascade leading to platelet hyperactivity. We now show that the guanine nucleotide exchange factors Vav1 and Vav3 were tyrosine phosphorylated in platelets exposed to oxLDL. Pharmacologic inhibition of src family kinases abolished Vav1 phosphorylation by oxLDL in vitro. Coimmunoprecipitations revealed the tyrosine phosphorylated form of src kinase Fyn was associated with Vav1 in platelets exposed to oxLDL. Using a platelet aggregation assay, we demonstrated that Vav1 deficiency, Fyn deficiency, or Vav1/Vav3 deficiency protected mice from diet-induced platelet hyperactivity. Furthermore, flow cytometric analysis revealed that Vav1/Vav3 deficiency significantly inhibited oxLDL-mediated integrin αIIbβIII activation of platelets costimulated with ADP. Finally, we showed with an in vivo carotid artery thrombosis model that genetic deletion of Vav1 and Vav3 together may prevent the development of occlusive thrombi in mice fed a high-fat diet. These findings implicate Vav proteins in oxLDL-mediated platelet activation and suggest that Vav family member(s) may act as critical modulators linking a prothrombotic state and hyperlipidemia.