Kevin B. Bacon

CHEMOKINES, LEUKOCYTE TRAFFICKING, AND INFLAMMATION

The relatively recent appreciation of a new class of cytokines, the chemokines, has done much to enhance our understanding of the extracellular signals involved in the movement of various populations of white blood cells. Investigation of the molecular underpinnings of chemokine function and their involvement in inflammatory processes of all kinds is beginning to yield information about the mechanisms of pathogenesis of a number of conditions, as well as providing hope for new therapeutic insights.

The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors

Migration is a basic feature of many cell types in a wide range of species. Since the 1800s, cell migration has been proposed to occur in the nervous and immune systems, and distinct molecular cues for mammalian neurons and leukocytes have been identified. Here we report that Slit, a secreted protein previously known for its role of repulsion in axon guidance and neuronal migration, can also inhibit leukocyte chemotaxis induced by chemotactic factors. Slit inhibition of the chemokine-induced chemotaxis can be reconstituted by the co-expression of a chemokine receptor containing seven transmembrane domains and Roundabout (Robo), a Slit receptor containing a single transmembrane domain. Thus, there is a functional interaction between single and seven transmembrane receptors. Our results reveal the activity of a neuronal guidance cue in regulating leukocyte migration and indicate that there may be a general conservation of guidance mechanisms underlying metazoan cell migration. In addition, we have uncovered an inhibitor of leukocyte chemotaxis, and propose a new therapeutic approach to treat diseases involving leukocyte migration and chemotactic factors.

CpG directly induces T-bet expression and inhibits IgG1 and IgE switching in B cells.

CpG DNA has immunomodulatory effects, such as the suppression of allergic responses mediated by type II T cell help (TH2). Here we report that CpG, but not lipopolysaccharide (LPS), rapidly induces expression of T-bet mRNA in purified B cells. Up-regulation of T-bet by CpG is abrogated in mice deficient in Toll-like receptor 9 (TLR9) and MyD88, but remains intact in B cells deficient in STAT1 (signal transducer and activator of transcription 1). Interleukin 12 (IL-12) alone does not up-regulate T-bet mRNA, but greatly enhances CpG-induced T-bet expression. Furthermore, CpG inhibits immunoglobulin G1 (IgG1) and IgE switching induced by IL-4 and CD40 signaling in purified B cells, and this effect correlates with up-regulation of T-bet. Thus, CpG triggers anti-allergic immune responses by directly regulating T-bet expression via a signaling pathway in B cells that is dependent upon TLR9, independent of interferon-γ (IFN-γ)-STAT1 and synergistic with IL-12.*Note: In the version of this article initially published online, some of the nomenclature was incorrect. This has been corrected for the HTML and print versions of the article.

Discovery of novel and selective IKK-β serine-threonine protein kinase inhibitors. Part 1

IkappaB kinase beta (IKK-beta) is a serine-threonine protein kinase critically involved in the activation of the transcription factor Nuclear Factor kappa B (NF-kappaB) in response to various inflammatory stimuli. We have identified a small molecule inhibitor of IKK-beta. Optimization of the lead compound resulted in improvements in both in vitro and in vivo potency, and provided IKK-beta inhibitors exhibiting potent activity in an acute cytokine release model (LPS-induced TNFalpha).

Chemokines and their receptors in neurobiology: perspectives in physiology and homeostasis

Chemokines are a large family of small secreted proteins (8-14 kDa) associated with the trafficking of leukocytes in physiological immunosurveillance as well as inflammatory cell recruitment in different disease processes. A limited repertoire of chemokines and their specific cognate receptors are detectable in cells of the CNS such as microglia, astrocytes and neurons under physiological conditions. Coupled with distinct patterns of ligand and receptor expression in various pathologies including multiple sclerosis, trauma, neuro-AIDS, Alzheimers disease, stroke, neuro- and glioblastomas, such phenomena have fueled the strong belief that chemokines must fulfill significant and potentially diverse functional roles in the CNS.

A selective novel low‐molecular‐weight inhibitor of IκB kinase‐β (IKK‐β) prevents pulmonary inflammation and shows broad anti‐inflammatory activity

1 Pulmonary inflammatory diseases such as asthma are characterized by chronic, cell‐mediated inflammation of the bronchial mucosa. 2 Recruitment and activation of inflammatory cells is orchestrated by a variety of mediators such as cytokines, chemokines, or adhesion molecules, the expression of which is regulated via the transcription factor nuclear factor kappa B (NF‐κB). 3 NF‐κB signaling is controlled by the inhibitor of kappa B kinase complex (IKK), a critical catalytic subunit of which is IKK‐β. 4 We identified COMPOUND A as a small‐molecule, ATP‐competitive inhibitor selectively targeting IKK‐β kinase activity with a Ki value of 2u2003nM. 5 COMPOUND A inhibited stress‐induced NF‐κB transactivation, chemokine‐, cytokine‐, and adhesion molecule expression, and T‐ and B‐cell proliferation. 6 COMPOUND A is orally bioavailable and inhibited the release of LPS‐induced TNF‐α in rodents. 7 In mice COMPOUND A inhibited cockroach allergen‐induced airway inflammation and hyperreactivity and efficiently abrogated leukocyte trafficking induced by carrageenan in mice or by ovalbumin in a rat model of airway inflammation. 8 COMPOUND A was well tolerated by rodents over 3 weeks without affecting weight gain. 9 Furthermore, in mice COMPOUND A suppressed edema formation in response to arachidonic acid, phorbol ester, or edema induced by delayed‐type hypersensitivity. 10 These data suggest that IKK‐β inhibitors offer an effective therapeutic approach for inhibiting chronic pulmonary inflammation.

Chemokines as mediators of allergic inflammation.

The selective distribution of reactive leukocytes to foci of inflammation or lymphoid organs is thought to rely on the generation of highly specific attractive forces which can enhance or subvert the physiological trafficking process. It is becoming increasingly apparent that the selective trafficking of leukocytes is governed by both the release of soluble mediators, or chemoattractants, as well as the matrix upon or through which the cells must traverse. A balance exists between endogenous cellular adhesion receptors (as well as extracellular matrix proteins) and other inducible adhesion receptors which can be up-regulated on this docking station. This dynamic environment provides a prominent signal for leukocyte extravasation from the blood or lymph vessel lumenal surface through to the tissue space. This report reviews current thinking on the delicate interplay between a superfamily of chemoattractant cytokines, the chemokines, and the various classes of cellular adhesion molecules. In it we highlight the idea that the balance between basal and inducible regulators of cell adhesion and migration is critical. Should it be disrupted, the signals responsible for induction and maintenance of an inflammatory response and those responsible for its resolution become disregulated, resulting in inflammatory pathology.

CD40-dependent and -independent activation of human tonsil B cells by CpG oligodeoxynucleotides.

Immunostimulatory CpG oligodeoxynucleotide (CpG‐ODN) sequences are known to directly activate Bu2004cells. We investigated the expression of the CpG receptor, Toll‐like receptoru20049 (TLR9), in humantonsil Bu2004cells, and determined functional responses following stimulation by a well‐characterized stimulatory CpG‐containing ODN sequence in the human immune system, ODNu20042006. Tonsil Bu2004cells were found to express high amounts of TLR9 mRNA and protein, and exposure of Bu2004cells to CpG‐ODN but not to an inactive control ODN induced a concentration‐ and time‐dependent up‐regulation of the activation markers CD23, CD25, CD40, CD54, CD80, CD86 and HLA‐DR. However, significant induction of proliferation and the release of IL‐6, IL‐10, IgG and IgM were only noted when Bu2004cells were co‐incubated with irradiated CD40L‐expressing CHO cells. Endogenous IL‐10 was identified as a critical mediator of Ig production, whereas all activating effects were independent of IL‐6. Further, CpG‐ODN counteracted IgE production induced by IL‐4. Collectively, these findings suggest a synergistic role of the TLR9/CD40 system and a critical role for the immunomodulatory cytokine IL‐10 in the orchestration of CpG‐ODN‐induced responses in Bu2004lymphocytes.

Growth Inhibition of Multiple Myeloma Cells by a Novel IκB Kinase Inhibitor

Involvement of nuclear factor-κB (NF-κB) in cell survival and proliferation of multiple myeloma has been well established. In this study we observed that NF-κB is constitutively activated in all human myeloma cell lines, thus confirming the previous studies. In addition, we found the phosphorylation of p65 subunit of NF-κB in addition to the phosphorylation of IκBα and the activation of NF-κB DNA binding and that various target genes of NF-κB including bcl-xL, XIAP, c-IAP1, cyclin D1, and IL-6 are up-regulated. We then examined the effect of a novel IκB kinase inhibitor, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile (ACHP). When myeloma cells were treated with ACHP, the cell growth was efficiently inhibited with IC50 values ranging from 18 to 35 μmol/L concomitantly with inhibition of the phosphorylation of IκBα/p65 and NF-κB DNA-binding, down-regulation of the NF-κB target genes, and induction of apoptosis. In addition, we observed the treatment of ACHP augmented the cytotoxic effects of vincristine and melphalan (l-phenylalanine mustard), conventional antimyeloma drugs. These findings indicate that IκB kinase inhibitors such as ACHP can sensitize myeloma cells to the cytotoxic effects of chemotherapeutic agents by blocking the antiapoptotic nature of myeloma cells endowed by the constitutive activation of NF-κB.

Chemokine Receptor CCR3 Function Is Highly Dependent on Local pH and Ionic Strength

The CC chemokine receptor 3 (CCR3) plays an important role in the regulation of the migration of eosinophils, a leukocyte population involved in many inflammatory pathologies including asthma. CCR3 binds to the CC chemokine eotaxin, a promigratory cytokine originally isolated as the key component in a model of eosinophil-induced airway inflammation. We show here that eotaxin/CCR3 binding interactions exhibit a marked sensitivity to relatively small changes in the extracellular environment. In particular, modest variations in the pH and the level of sodium chloride over a range of physiologic and near physiologic conditions had dramatic effects on eotaxin binding and CCR3-mediated cytoplasmic Ca2+ mobilization. These biochemical indices were reflected at the functional level as well; small changes in pH and salt also resulted in striking changes in the migration of primary human eosinophils in vitro. These results reveal that relatively small perturbations in extracellular buffer conditions can yield widely disparate interpretations of CCR3 ligand binding and affinities and suggest that modulation of the tissue microenvironment might be utilized to control the affinity and efficacy of chemokine-mediated cell migration.