Mary Jo Staruch

Binding and Functional Properties of Recombinant and Endogenous CXCR3 Chemokine Receptors

IP10 and MIG are two members of the CXC branch of the chemokine superfamily whose expression is dramatically up-regulated by interferon (IFN)-γ. The proteins act largely on natural killer (NK)-cells and activated T-cells and have been implicated in mediating some of the effects of IFN-γ and lipopolysaccharides (LPSs), as well as T-cell-dependent anti-tumor responses. Recently both chemokines have been shown to be functional agonists of the same G-protein-coupled receptor, CXCR3. We now report the pharmacological characterization of CXCR3 and find that, when heterologously expressed, CXCR3 binds IP10 and MIG with Ki values of 0.14 and 4.9 nm, respectively. The receptor has very modest affinity for SDF-1α and little or no affinity for other CXC-chemokines. The properties of the endogenous receptor expressed on activated T-cells are similar. Surprisingly, several CC-chemokines, particularly eotaxin and MCP-4, also compete with moderate affinity for the binding of IP10 to CXCR3. Eotaxin does not activate CXCR3 but, in CXCR3-transfected cells, can block IP10-mediated receptor activation. Eotaxin, therefore, may be a natural CXCR3 antagonist.

CCR5, CXCR4, and CD4 Are Clustered and Closely Apposed on Microvilli of Human Macrophages and T Cells

ABSTRACT The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in small trans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.

Reduction of serum Interleukin-1-like activity after treatment with dexamethasone.

The ability of steroids to modulate the appearance of interleukin‐1(IL‐1) in vivo was evaluated in a model of endotoxin shock. High levels of IL‐1 were found in serum from A/J mice which were sensitized with P. acnes and challenged with bacterial lipopolysaccharide (LPS). The factor appeared in the serum 2–4 hours after LPS challenge and was dependent on the period of P. acnes sensitization and the dose of LPS. Treating the mice with dexamethasone prior to LPS challenge resulted in significantly lower thymocyte proliferative activity in the serum. Three experiments demonstrated that this reduced activity reflects a decrease in IL‐1. 1) The reduced activity was not due to the presence of proliferation inhibitors since mixing the serum from dexamethasone‐treated mice with purified IL‐1 or adding the equivalent amount of steroid directly to thymocyte cultures did not reduce the degree of proliferation. 2) When the serum was fractionated by gel filtration, the proliferative activity for both control and steroid treated sera eluted at 10–16 kilodaltons; however, the activity was nearly 50% less in the sample from steroid‐treated mice. 3) In addition to thymocyte proliferative activity, IL‐1 induces an increase in the serum titer of the acute phase protein known as serum amyloid A. Both serum‐ and gel‐ purified samples were able to induce the SAA, but again the samples from steroid‐treated mice were much less active. We conclude that the factor produced in vivo has the properties of IL‐1 and that the serum titre of the factor is reduced by dexamethasone treatment.

Benzamide derivatives as blockers of Kv1.3 ion channel

The voltage-gated potassium channel, Kv1.3, is present in human T-lymphocytes. Blockade of Kv1.3 results in T-cell depolarization, inhibition of T-cell activation, and attenuation of immune responses in vivo. A class of benzamide Kv1.3 channel inhibitors has been identified. The structure-activity relationship within this class of compounds in two functional assays, Rb_Kv and T-cell proliferation, is presented. In in vitro assays, trans isomers display moderate selectivity for binding to Kv1.3 over other Kv1.x channels present in human brain.

Preparation and in vitro activities of naphthyl and indolyl ether derivatives of the FK-506 related immunosuppressive macrolide ascomycin

Abstract The synthesis of naphthyl- and indolyl-ethers of the immunosuppressive macrolide ascomycin using pentavalent bismuth reagents is described. The in vitro activities of the aryl ether analogs are reported. The indole ether analogs show increased immunosuppressive activity in vitro relative to the parent macrolide.

A tacrolimus-related immunosuppressant with reduced toxicity

BACKGROUND Tacrolimus (FK506) has potent immunosuppressive properties reflecting its ability to block the transcription of lymphokine genes in activated T cells through formation of a complex with FK506 binding protein-12, which inhibits the phosphatase activity of calcineurin. The clinical usefulness of tacrolimus is limited, however, by severe adverse effects, including neurotoxicity and nephrotoxicity. Although this toxicity, like immunosuppression, appears mechanistically related to the calcineurin inhibitory action of the drug, a large chemistry effort has been devoted to search for tacrolimus analogs with reduced toxicity but preserved immunosuppressive activity that might have enhanced therapeutic utility. METHODS Here, we report on the identification of such an analog, which was synthetically derived from ascomycin (ASC), the C21 ethyl analog of tacrolimus, by introducing an indole group at the C32 position. The profile of biological activity of indolyl-ASC was characterized in rodent models of immunosuppression and toxicity. RESULTS Indolyl-ASC was found to exhibit an immunosuppressive potency equivalent to that of tacrolimus in T-cell activation in vitro and in murine transplant models, even though indolyl-ASC bound about 10 times less to intracellular FK506 binding protein-12 than tacrolimus or ASC. Further evaluation of indolyl-ASC revealed that it is threefold less potent than tacrolimus in inducing hypothermia, a response that may reflect neurotoxicity, and in causing gastrointestinal transit alterations in mice. Moreover, indolyl-ASC was at least twofold less nephrotoxic than tacrolimus upon 3-week oral treatment in rats. CONCLUSIONS Altogether, these data indicate a modest but definite improvement in the therapeutic index for indolyl-ASC compared with tacrolimus in rodent models.

Hypothemycin inhibits the proliferative response and modulates the production of cytokines during T cell activation.

Hypothemycin, a resorcylic acid lactone antibiotic, was identified as active in a screen for inhibitors of T cell activation. It was found to inhibit the proliferation of mouse and human T cells stimulated with anti-CD3 mAb + PMA and of human PBMC stimulated with anti-CD3 mAb alone. This inhibition was partially reversed by exogenous IL-2 indicating that it is not due to non-specific toxicity. Hypothemycin potently suppressed the production of IL-2 (IC50: 9 nM) but affected IL-2-induced proliferation to a lesser extent (IC50: 194 nM). Hypothemycin also inhibited IL-6, IL-10, IFN-gamma and TNF-alpha production. By contrast, it markedly enhanced the production of IL-4, IL-5 and IL-13. These effects were seen both at the mRNA and protein secretion levels. Analysis of the effect of hypothemycin on CD69 induction suggested that it disrupts calcineurin-independent rather than calcineurin-dependent signaling. Furthermore, hypothemycin was able to inhibit the phosphorylation of ERK1/2 induced by PMA treatment of T cells. Therefore, hypothemycin represents an inhibitor of T cell activation with a novel mode of action and unique modulatory activity on cytokine production.

Preparation and in vitro activity of aryl ether derivatives of the FK-506 related immunosuppressive macrolides ascomycin and L-683,742

Abstract The arylation of the immunosuppressive macrolides ascomycin and L-683,742 using pentavalent bismuth reagents is described. The in vitro activities of the aryl ether analogs are reported.

Control of the mitogenicity of muramyl dipeptide

Abstract Muramyl dipeptide (MDP) has been shown to be the smallest subunit of bacterial peptidoglycan which exhibits adjuvanticity in vivo . Among its numerous in vitro activities is the ability to enhance the rate of DNA synthesis of murine splenocytes. In the present study, the dividing splenocytes were shown to be B cells which required prolonged incubation with MDP before becoming committed to division. T cells contributed minimally, if at all, to the DNA synthesis response observed nor did they function as helpers. Macrophage depleted lymphocytes responded poorly but could be reconstituted with fresh macrophages or 2-mercaptoethanol. X-ray irradiation of the macrophages reduced their ability to serve as accessory cells. Because of the biological similarity between bacterial endotoxin (LPS) and MDP, the possibility was tested that the gene(s) controlling the mitogenic response to MDP were either linked to or identical with the genes controlling the response to LPS. The results obtained from the comparison of selected mouse strains and from backcross analysis indicated that the two responses are not controlled by closely linked genes. These same strains of mice were compared with respect to their ability to respond to MDP as an adjuvant for the secondary antibody response to bovine albumin in vivo . It was found that the intensity of the in vivo adjuvant response was not correlated with the intensity of the in vitro mitogen response. We conclude from these observations that the ability of MDP to act as a mitogen makes little or no contribution to its adjuvanticity in vivo .

C32-O-imidazol-2-yl-methyl ether derivatives of the immunosuppressant ascomycin with improved therapeutic potential

A series of C32-O-aralkyl ether derivatives of the FK-506 related macrolide ascomycin have been prepared based on an earlier reported C32-O-cinnamyl ether design. In the present study, the nature of the aryl tethering group was varied in an attempt to improve oral activity. An imidazol-2-yl-methyl tether was found to be superior among those investigated and has resulted in an ascomycin analog, L-733,725, with in vivo immunosuppressive activity comparable to FK-506 but with an improved therapeutic index.