Charles Lam

Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages

We have previously reported that mice lacking inducible nitric oxide synthase (NOS2) developed enhanced Th1 cell responses. We now investigated the mechanism by which NO modulates Th1 cells differentiation. Peritoneal macrophages from NOS2‐deficient mice infected with Leishmania major in vivo or stimulated with IFN‐γ or lipopolysaccharide (LPS) in vitro produced significantly higher levels of IL‐12 than those from heterozygous or wild‐type mice. A macrophage cell line, J774, produced significant amounts of IL‐12 following activation with LPS, or LPS plus IFN‐γ. This could be markedly enhanced by the NOS inhibitor L‐NG monomethyl arginine (L‐NMMA), but profoundly inhibited by the NO‐generating compound S‐nitroso‐N‐acetyl‐penicillamine (SNAP). The effect of NO in this system is selective, since SNAP enhanced and L‐NMMA decreased TNF‐α synthesis by LPS‐activated J774 cells. The differential effect of NO on IL‐12 and TNF‐α is at the transcriptional level and is activation dependent. Since IL‐12 is a major inducer of Th1 cells which produce IFN‐γ that can activate macrophages to produce IL‐12, our data demonstrate that NO can be an inhibitor of this feedback loop, preventing the excessive amplification of Th1 cells which are implicated in a range of immunopathologies.

Conformationally constrained no synthase inhibitors: Rigid analogs of L-N-iminoethylornithine

The synthesis of eight rigid analogs of L-N-iminoethylornithine (L-NIO) is described. The compounds have been evaluated for their inhibition of inducible nitric oxide synthase. Preliminary structure-activity relationships are discussed.

6-[2-(adamantylidene)-hydroxybenzoxazole]-O-sulfamate, a steroid sulfatase inhibitor for the treatment of androgen- and estrogen-dependent diseases.

Steroid sulfatase (STS) offers a new target for the treatment of steroid hormone-dependent diseases, such as breast and prostate cancer and androgen-dependent skin diseases. We here characterize a novel non-estrogenic inhibitor of the enzyme, namely 6-[2-(adamantylidene)-hydroxybenzoxazole]-O-sulfamate (AHBS), with special attention to its potential use in the treatment of acne. The compound blocks STS activity in homogenates of human skin with IC(50)=16 nM. Following a single oral dose (5 mg/kg) in rats, the compound blocks STS in the skin by 95% at 8 h, followed by recovery of activity over 5 days. Following topical application to the skin, both in vitro and in vivo, AHBS passes through the stratum corneum leading to inhibition of STS activity in the dermal compartment with rapid onset and long duration. Topical application of AHBS to Göttingen minipigs for a period of 2 weeks does not induce symptoms of ichthyosis as seen in STS-deficient human subjects, but leads to a reduction of sebum secretion to the skin surface. Based on these data, clinical studies with AHBS in acne patients are warranted, in order to verify the hypothesis on the importance of the sulfatase pathway in androgen-dependent skin diseases.

INTERLEUKIN-8 PRIMING OF HUMAN NEUTROPHILS IS NOT ASSOCIATED WITH PERSISTENTLY ALTERED CALCIUM FLUXES BUT IS ADDITIVE WITH LIPOPOLYSACCHARIDE

Interleukin‐8 (IL‐8) priming was studied in neutrophils to examine its dependency on altered calcium fluxes and for similarity to lipopolysaccharide (LPS). IL‐8 caused a rapid rise in [Ca2+]i that returned to baseline values by 20 min. Peak [Ca2+]i transients in response to N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) were unaltered in IL‐8‐primed compared with unprimed cells. In comparison to LPS and tumor necrosis factor (TNF), IL‐8 was a much weaker priming agent as measured by either O2 – or H2O2 production. Despite their large disparity in potency, IL‐8 and LPS priming were additive using fMLP, a receptor‐dependent stimulator, and synergistic using the post‐receptor, protein kinase C activator, phorbol 12‐myristate 13‐acetate (PMA) to trigger the respiratory burst. In contrast, IL‐8 and TNF priming were synergistic for fMLP (P = 0.05), but completely nonadditive when PMA was used as the neutrophil stimulant (P = 0.05 for subadditivity). Thus, lasting alterations in [Ca2+]i are not a necessary characteristic of IL‐8‐primed cells. IL‐8 and LPS appear to prime by non‐overlapping pathways, whereas IL‐8 and TNF appear to share mechanisms distal to protein kinase C activation. IL‐8 and LPS may independently contribute to neutrophil‐mediated host defense or injury by priming through distinct pathways. J. Leukoc. Biol. 64: 511–518; 1998.

Purification, crystallization and preliminary X-ray diffraction analysis of recombinant human neutrophil-activating peptide 2 (rhNAP-2)

The potent activator and chemoattractant for human neutrophils, neutrophil‐activating peptide 2 (NAP‐2), has been cloned and expressed in Escherichia coli. The protein has been purified to homogeneity (> 98%) by a series of chromatographic techniques, including reversed phase HPLC. The biological activity of recombinant human NAP‐2 (rhNAP‐2), characterized by the induction of elastase release from human neutrophils, was found to be comparable to natural NAP‐2. rhNAP‐2 has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P222 with unit cell dimensions of a = 30.8 Å, b = 39.5 Å and c = 95.3 Å. A packing density of 3.8 Å3/Da with a solvent content of approximately 68% is obtained when one molecule per asymmetric unit is assumed. The crystals were shown to diffract to beyond 2.0 Å on a conventional X‐ray source. They are stable to X‐rays for several days and are thus suitable for high resolution structure determination.

Elucidation of Structure Function Relationships in the IL-8 Family by X-ray Crystallography

After its identification in 1987 as a novel neutrophil-activating cytokine, interleukin-8 (IL-8) was the center of interest of a large variety of interdisciplinary scientists for study of its action in vitro and in vivo (1,2,3,4). Soon it became clear that interleukin-8 plays a critical role in a series of diseases which are all characterized by strong neutrophil involvement. Rheumatoid artrithis, psoriasis, polytrauma, septicemia, adult respiratory distress syndrome (ARDS), asthma, emphysema, myocardial infarction, nephritis, inflammatory bowel disease, and gout are diseases which affect millions of people, and IL-8 is implicated in all these disease states. Therefore, inhibition of interleukin-8 itself, or blocking of the interaction with its seven transmembrane domain cellular receptor represents an attractive and logical approach for treatment of these inflammatory states. Possibilities for interference include neutralising antibodies, small molecules binding to the chemokine at critical positions, (e.g. domains involved in dimerization, heparin binding or receptor binding) or molecules which bind to the receptor. An additional approach would be the use of peptides based on the IL-8 structure or mutant IL-8 molecules to act as antagonists. As an alternative to screening for inhibitors in a series of biological assays, a rational way of finding an inhibitory principle involves the elucidation of structure/function relationships. This is usually done by production of mutant proteins of the target molecule and subsequent determination of biological activities. As only the primary structures of the mutants are known this might be called a “one-dimensional structure activity relationship”. We are attempting to go one step further and actually determine the three-dimensional structure of mutant IL-8 derivatives with interesting and clear changes in biological activities and, based on structure/function relationships in three dimensions, get a more detailed insight into structural requirements for specific biological activities.