Meredith Halks-Miller

CD4-independent association between HIV-1 gp120 and CXCR4: functional chemokine receptors are expressed in human neurons

BACKGROUND Chemokines are a family of proteins that chemoattract and activate immune cells by interacting with specific receptors on the surface of their targets. We have shown previously that chemokine receptors including the interleukin-8 receptor B (CXCR2) and the Duffy blood group antigen are expressed on subsets of neurons in various regions of the adult nervous system. RESULTS Using a combination of immunohistochemical staining and receptor binding studies, we show that hNT cells, which are differentiated human neurons derived from the cell line NTera2, express functional chemokine receptors of the C-X-X and C-C types. These chemokine receptors include CXCR2, CXCR4, CCR1 and CCR5. We demonstrate high-affinity binding of both types of chemokines to hNT neurons and dose-dependent chemotactic responses to these chemokines in differentiated, but no t undifferentiated, NTera 2 cells. In addition, we show that the envelop glycoprotein from the T-cell-tropic human immunodeficiency virus 1 (HIV-1) strain IIIB is a CD4-independent, dose-dependent inhibitor of the binding of stromal cell-derived factor 1 to its receptor, CXCR4. CONCLUSIONS These data support recent findings that members of the chemokine family, including CCR5 and LESTR/Fusin (CXCR4), function as coreceptors in combination with CD4 for HIV-1 invasion. This is the first report of functional expression of chemokine receptors on human neurons. Furthermore, our studies provide for direct CD4-independent association of the viral envelope protein of the HIV-1 strain III with the chemokine receptor CXCR4.

The CC chemokine I-309 inhibits CCR8-dependent infection by diverse HIV-1 strains.

Using a chemokine receptor model based on known receptor sequences, we identified several members of the seven transmembrane domain G-protein superfamily as potential chemokine receptors. The orphan receptor ChemR1, which has recently been shown to be a receptor for the CC chemokine I-309, scored very high in our model. We have confirmed that I-309, but not a number of other chemokines, can induce a transient Ca2+ flux in cells expressing CCR8. In addition, the human erythroleukemic cell line K562 responded chemotactically in a dose-responsive manner to this chemokine. Since several chemokine receptors have been shown to be required as coreceptors for HIV-1 infection, we asked whether human immunodeficiency virus type 1 (HIV-1) could efficiently utilize CCR8. Here we show that the CCR8 receptor can serve as a coreceptor for diverse T-cell tropic, dual-tropic, and macrophage-tropic HIV-1 strains and that I-309 was a potent inhibitor of HIV-1 envelope-mediated cell-cell fusion and virus infection. Furthermore, we show by flow cytometry and immunohistochemistry that antibodies generated against the CCR8 receptor amino-terminal peptide cross-reacted with U-87 MG cells stably expressing CCR8, THP-1 cells, HL-60 cells, and human monocytes, a target cell for HIV-1 infectivity in vivo.

Urokinase-Type Plasminogen Activator Plays a Critical Role in Angiotensin II–Induced Abdominal Aortic Aneurysm

Abstract— We have previously demonstrated that urokinase-type plasminogen activator (uPA) is highly expressed in the aneurysmal segment of the abdominal aorta (AAA) in apolipoprotein E–deficient (apoE−/−) mice treated with angiotensin II (Ang II). In the present study, we tested the hypothesis that uPA is essential for AAA formation in this model. An osmotic minipump containing Ang II (1.44 mg/kg per day) was implanted subcutaneously into 7- to 11-month-old male mice for 1 month. Ang II induced AAA in 9 (90%) of 10 hyperlipidemic mice deficient in apoE (apoE−/−/uPA+/+ mice) but in only 2 (22%) of 9 mice deficient in both apoE and uPA (apoE−/−/uPA−/− mice) (P <0.05). Although the expansion of the suprarenal aorta was significantly less in apoE−/−/uPA−/− mice than in apoE−/−/uPA+/+ mice, the aortic diameters of the aorta immediately above or below the suprarenal aorta were similar between the 2 groups. Ang II induced AAA in 7 (39%) of 18 strain-matched wild-type C57 black/6J control mice. The incidence was significantly higher in atherosclerotic apoE-deficient (apoE−/−) mice, in which 8 (100%) of 8 mice developed AAA. Only 1 (4%) of 27 uPA−/− mice developed AAA after Ang II treatment. We conclude the following: (1) uPA plays an essential role in Ang II–induced AAA in mice with or without preexisting hyperlipidemia and atherosclerosis; (2) uPA deficiency does not affect the diameter of the nonaneurysmal portion of the aorta; and (3) atherosclerosis and/or hyperlipidemia promotes but is not essential for Ang II–induced AAA formation in this model.

Expression of Interleukin-6 in Atherosclerotic Lesions of Male ApoE-Knockout Mice Inhibition by 17β-Estradiol

Increased levels of interleukin-6 (IL-6) have been proposed to contribute to a number of pathological disorders, including osteoporosis and Alzheimers disease. In human atherosclerotic lesions, IL-6 protein and mRNA have been detected, although the role of IL-6 in plaque formation is unknown. We have examined the expression pattern of IL-6 mRNA and secreted protein in male apolipoprotein E-knockout (apoE-KO) mice aortas. Furthermore, we have evaluated the effects of 17beta-estradiol (E2), a vasculoprotective sex steroid hormone, on the secretion of this inflammatory cytokine from isolated male apoE-KO mice aortas. The expression of IL-6 mRNA was detected by reverse transcription-polymerase chain reaction in the apoE-KO mouse aortas but not in the aortas of age-matched control mice. Similarly, the secretion of IL-6 protein from isolated apoE-KO aortic segments was significantly greater than that from aortas of age-matched control animals. The secretion of IL-6 from isolated aortic rings of apoE-KO mice ranging in age from 6 to 48 weeks showed a significant, positive correlation with percent lesion area measured in the same tissue. Immunohistochemical staining of apoE-KO mouse aortic tissue sections demonstrated colocalization of IL-6 expression with macrophages. Treatment of male apoE-KO mice with E2 for 3 weeks resulted in a statistically significant 50% reduction in IL-6 secretion from ex vivo aortic tissue segments. There was no significant change in total serum cholesterol and triglyceride levels in the E2-treated group compared with placebo-treated controls. These data demonstrate that (1) IL-6 mRNA and protein are expressed in the atherosclerotic plaques of apoE-KO mice aortas and (2) IL-6 production is suppressed by E2 treatment, which may contribute to the antiatherosclerotic effects of E2 in the apoE-KO mouse model of atherosclerosis.

Beneficial role of the GPR30 agonist G-1 in an animal model of multiple sclerosis.

The beneficial effects of estrogens in multiple sclerosis are thought to be mediated exclusively by the classical nuclear estrogen receptors ERalpha and ERbeta. However, recently many reports revealed that estrogens are able to mediate rapid signals through a G protein-coupled receptor (GPCR), known as GPR30. In the present study, we set out to explore whether effects mediated through this receptor were anti-inflammatory and could account for some of the beneficial effects of estrogen. We demonstrate that GPR30 is expressed in both human and mouse immune cells. Furthermore a GPR30-selective agonist, G-1, previously described by us, inhibits the production of lipopolysaccharide (LPS)-induced cytokines such as TNF-alpha and IL-6 in a dose-dependent manner in human primary macrophages and in a murine macrophage cell line. These effects are likely mediated solely through the estrogen-specific receptor GPR30 since the agonist G-1 displayed an IC(50) far greater than 10 microM on the classical nuclear estrogen receptors as well as a panel of 25 other GPCRs. Finally, we show that the agonist G-1 is able to reduce the severity of disease in both active and passive EAE models of multiple sclerosis in SJL mice and that this effect is concomitant with a G-1-mediated decrease in proinflammatory cytokines, including IFN-gamma and IL-17, in immune cells harvested from these mice. The effect of G-1 appears indirect, as the GPR30 agonist did not directly influence IFN-gamma or IL-17 production by purified T cells. These data indicate that G-1 may represent a novel therapeutic agent for the treatment of chronic autoimmune, inflammatory diseases.

Angiotensin II increases urokinase-type plasminogen activator expression and induces aneurysm in the abdominal aorta of apolipoprotein E-deficient mice.

Urokinase-type plasminogen activator (uPA) is increased in human abdominal aortic aneurysm (AAA). Chronic infusion of angiotensin II (Ang II) results in AAA in apolipoprotein E-deficient mice. We tested the hypothesis that Ang II infusion results in an elevation of uPA expression contributing to aneurysm formation. Ang II or vehicle was infused by osmotic pumps into apoE-KO mice. All mice treated with Ang II developed a localized expansion of the suprarenal aorta (75% increase in outer diameter), accompanied by an elevation of blood pressure (22 mmHg), compared to the vehicle-treated group. Histological examination of the dilated aortic segment revealed similarities to human AAA including focal elastin fragmentation, macrophage infiltration, and intravascular hemorrhage. Ang II treatment resulted in a 13-fold increase in the expression of uPA mRNA in the AAA segment in contrast to a twofold increase in the atherosclerotic aortic arch. Increased uPA protein was detected in the abdominal aorta as early as 10 days after Ang II infusion before significant aorta expansion. Thus, Ang II infusion results in macrophage infiltration, increased uPA activity, and aneurysm formation in the abdominal aorta of apoE-KO mice. These data are consistent with a causal role for uPA in the pathogenesis of AAA.

Accelerated atherosclerosis and premature calcified cartilaginous metaplasia in the aorta of diabetic male Apo E knockout mice can be prevented by chronic treatment with 17β-estradiol

Epidemiological data indicate that estrogens significantly reduce the risk of morbidity and mortality due to cardiovascular diseases in postmenopausal women. Although numerous animal studies demonstrated inhibition of early atheromatous lesion formation by estrogen treatment in several species, information about the potential benefits of estrogens on complex, advanced atherosclerotic lesions is still lacking. The present study was designed to test whether chronic treatment with 17 beta-estradiol affects hyperglycemia-induced premature advanced lesion formation in 40-week-old male apolipoprotein E-deficient (Apo E-KO) mice. In order to accelerate advanced lesion formation, we treated male Apo E-KO mice with streptozotocin (STZ) at the age of 6 weeks. Two weeks later the STZ-treated mice received a slow release pellet containing either 17 beta-estradiol or placebo. STZ treatment caused sustained hyperglycemia without changes in serum total cholesterol or triglyceride levels compared to citrate control mice. STZ-treated Apo E-KO mice developed significantly more lesions in some (but not all) parts of the aorta and its main branches, and caused premature calcified cartilaginous metaplasia in the lesions of the proximal aorta. Chronic treatment with 17 beta-estradiol lead to a significant decrease in blood glucose and triglyceride levels, reduced the lesion area in all vascular segments studied and prevented cartilaginous metaplasia in STZ-treated Apo E-KO mice. The results of this study show that STZ treatment leads to significant acceleration of atherosclerotic lesion formation and premature occurrence of calcified cartilaginous areas in Apo E-KO mice, which could be effectively prevented by chronic estrogen treatment.

The type IV phosphodiesterase specific inhibitor mesopram inhibits experimental autoimmune encephalomyelitis in rodents

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease with pathological features reminiscent of those seen in multiple sclerosis and thus serves as an animal model for this disease. Inhibition of type IV phosphodiesterase (PDE IV) in animals with this disease has been shown to result in amelioration of disease symptoms. Here we describe the immunomodulatory activity of the novel potent and selective PDE IV inhibitor mesopram. In vitro, mesopram selectively inhibits the activity of type 1 helper T (Th1) cells without affecting cytokine production or proliferation of type 2 helper T (Th2) cells. Administration of mesopram to rodents inhibits EAE in various models. Clinically, EAE is completely suppressed by mesopram in Lewis rats. This is accompanied by a reduction of inflammatory lesions in spinal cord and brain. RT-PCR analysis revealed a marked reduction in the expression of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in the brains of these animals. Furthermore, the ex vivo production of Th1 cytokines by activated spleen cells derived from mesopram-treated animals is significantly reduced compared to vehicle-treated controls. Amelioration of the clinical symptoms is also observed during chronic EAE in mesopram-treated SJL mice as well as in relapsing-remitting EAE in SWXJ mice using a therapeutic treatment regimen. These data demonstrate the anti-inflammatory activity of mesopram and provide a rationale for its clinical development.

CCR1 is an early and specific marker of Alzheimer's disease.

Chemokines are a diverse group of small proteins that effect cell signaling by binding to G‐protein–coupled, seven‐trans‐membrane receptors. Our group had found previously that the chemokine receptor CCR1 was present in neurons and dystrophic processes in a small sample of Alzheimers disease cases. This expanded immunohistochemical study shows that the number of CCR1‐positive plaque‐like structures in the hippocampus and entorhinal cortex is highly correlated to dementia state as measured by the clinical dementia rating score. CCR1 immunoreactivity is found in dystrophic, neurofilament‐positive, synaptophysin‐negative neurites that are associated with senile plaques containing amyloid beta peptides of the 1‐42 species (Aβ42). CCR1 was not, however, associated with diffuse deposits of Aβ42. There was limited expression of CCR1 in neurofibrillary tangle‐bearing neuritic processes. Astrocytes and microglia were typically negative for CCR1. Human brains from age‐matched, nondemented individuals rarely displayed either CCR1 or Aβ42 immunoreactivity. Seven other types of dementing neurodegenerative diseases were examined, and all failed to demonstrate CCR1 immunopositivity unless Aβ42‐positive plaques were also present. Thus, neuronal CCR1 is not a generalized marker of neurodegeneration. Rather, it appears to be part of the neuroimmune response to Aβ42‐positive neuritic plaques.

Reduction of cardiac functional reserve and elevation of aortic stiffness in hyperlipidemic Yucatan minipigs with systemic and coronary atherosclerosis

To test the hypothesis that cardiac functional reserve is reduced in animals with severe atherosclerosis, Yucatan minipigs were fed a high-cholesterol diet (Chol) for 8 months. Half of them was made diabetic, an additional risk factor for atherosclerosis, with streptozotocin (STZ). Another group of age-matched minipigs were fed a normal diet as controls. At the end of the treatment period, animals were instrumented for the measurement of cardiovascular hemodynamic parameters under isoflurane anesthesia. Cardiac functional reserve was measured by the magnitude of the inotropic response to isoproterenol stress. Hyperlipidemic minipigs developed severe atherosclerotic plaques in the aorta, coronary and iliac artery, accompanied by an increase in the aortic stiffness indexed by increases in pulse wave velocity and augmentation index. These vascular changes were more severe in STZ-induced insulin-dependent diabetes mellitus. The isoproterenol-induced increase in left ventricular contractility (dP/dt) and relaxation (-dP/dt) and, consequently, cardiac output were also significantly reduced in both the Chol groups with or without STZ, compared to control group. Thus, cardiac functional reserve measured by isoproterenol-stimulated responses was reduced in atherosclerotic minipigs, which was further diminished in diabetes.