Issue published September 1, 1999

M any HIV-1 isolates at the late stage of disease are capable of using both CXCR4 and CCR5 in transfected cell lines, and are thus termed dual-tropic. Here we asked whether these dual-tropic variants also use both coreceptors for productive infection in a natural human lymphoid tissue microenvironment, and whether use of a particular coreceptor is associated with viral cytopathicity. We used 3 cloned dual-tropic HIV-1 variants, 89.6 and its chimeras 89-v345.SF and 89-v345.FL, which use both CCR5 and CXCR4 in transfected cell lines. In human lymphoid tissue ex vivo, one variant preferentially used CCR5, another preferentially used CXCR4, and a third appeared to be a true dual-tropic variant. The 2 latter variants severely depleted CD4⁺ T cells, whereas cytopathicity of the virus that used CCR5 only in lymphoid tissue was mild and confined to CCR5⁺/CD4⁺ T cells. Thus, (a) HIV-1 coreceptor usage in vitro cannot be unconditionally extrapolated to natural microenvironment of human lymphoid tissue; (b) dual-tropic viruses are not homogeneous in their coreceptor usage in lymphoid tissue, but probably comprise a continuum between the 2 polar variants that use CXCR4 or CCR5 exclusively; and (c) cytopathicity toward the general CD4⁺ T cell population in lymphoid tissue is associated with the use of CXCR4.

D isorders of hemostasis lead to vascular pathology. Endothelium-derived gene products play a critical role in the formation and degradation of fibrin. We sought to characterize the importance of these locally produced factors in the formation of fibrin in the cardiac macrovasculature and microvasculature. This study used mice with modifications of the thrombomodulin (TM) gene, the tissue-type plasminogen activator (tPA) gene, and the urokinase-type plasminogen activator (uPA) gene. The results revealed that tPA played the most important role in local regulation of fibrin deposition in the heart, with lesser contributions by TM and uPA (least significant). Moreover, a synergistic relationship in fibrin formation existed in mice with concomitant modifications of tPA and TM, resulting in myocardial necrosis and depressed cardiac function. The data were fit to a statistical model that may offer a foundation for examination of hemostasis-regulating gene interactions.

I schemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of tissue injury in organs subjected to ischemia. The mechanism that triggers inflammation and organ injury after ischemia remains to be elucidated, although different causes have been postulated. We investigated the role of apoptosis in the induction of inflammation and organ damage after renal ischemia. Using a murine model, we demonstrate a relationship between apoptosis and subsequent inflammation. At the time of reperfusion, administration of the antiapoptotic agents IGF-1 and ZVAD-fmk (a caspase inactivator) prevented the early onset of not only renal apoptosis, but also inflammation and tissue injury. Conversely, when the antiapoptotic agents were administered after onset of apoptosis, these protective effects were completely abrogated. The presence of apoptosis was directly correlated with posttranslational processing of the endothelial monocyte-activating polypeptide II (EMAP-II), which may explain apoptosis-induced influx and sequestration of leukocytes in the reperfused kidney. These results strongly suggest that apoptosis is a crucial event that can initiate reperfusion-induced inflammation and subsequent tissue injury. The newly described pathophysiological insights provide important opportunities to effectively prevent clinical manifestations of reperfusion injury in the kidney, and potentially in other organs.

T ransgenic (TG) mice with cardiac G_sα overexpression exhibit enhanced inotropic and chronotropic responses to sympathetic stimulation, but develop cardiomyopathy with age. We tested the hypothesis that cardiomyopathy in TG mice with G_sα overexpression could be averted with chronic β-adrenergic receptor (β-AR) blockade. TG mice and age-matched wild-type littermates were treated with the β-AR blocker propranolol for 6–7 months, starting at a time when the cardiomyopathy was developing but was not yet severe enough to induce significant cardiac depression (9.5 months of age), and ending at a time when cardiac depression and cardiomyopathy would have been clearly manifest (16 months of age). Propranolol treatment, which can induce cardiac depression in the normal heart, actually prevented cardiac dilation and the depressed left ventricular function characteristic of older TG mice, and abolished premature mortality. Propranolol also prevented the increase in myocyte cross-sectional area and myocardial fibrosis. Myocyte apoptosis, already apparent in 9-month-old TG mice, was actually eliminated by chronic propranolol. This study indicates that chronic sympathetic stimulation over an extended period is deleterious and results in cardiomyopathy. Conversely, β-AR blockade is salutary in this situation and can prevent the development of cardiomyopathy.

C alcitonin gene–related peptide (CGRP) is a potent vasodilator and relaxes smooth muscle of a variety of tissues, but the effects of CGRP on human myometrial contractions and the changes in CGRP receptors (CGRP-Rs) in human myometrium have not been described. We report that CGRP induced dose-dependent relaxation in spontaneously contracting myometrium from pregnant women. This relaxation effect is diminished in myometrium obtained from patients during labor and in the nonpregnant state. CGRP-induced relaxations are inhibited by a CGRP-R antagonist (CGRP_8-37), a soluble guanylate cyclase inhibitor (LY₈₃₅₈₃), and a nitric oxide synthase inhibitor (L-NAME). Both Western blotting and mRNA analysis showed that CGRP-Rs are present in human myometrium, and that the expression of these receptors is increased during pregnancy and decreased during term labor. Immunofluorescent staining revealed that CGRP-Rs are abundant in the myometrial cells of pregnant women who are not in labor, and are minimal in uterine specimens from women in labor and in the nonpregnant state. We conclude that increased CGRP-Rs in myometrium, and resulting enhanced myometrial sensitivity to CGRP, may play a role in maintaining human myometrium in a quiescent state during pregnancy, and that a decline in the CGRP-Rs at term could contribute to the initiation of labor.

R GS family members are GTPase-activating proteins (GAPs) for heterotrimeric G proteins. There is evidence that altered RGS gene expression may contribute to the pathogenesis of cardiac hypertrophy and failure. We investigated the ability of RGS4 to modulate cardiac physiology using a transgenic mouse model. Overexpression of RGS4 in postnatal ventricular tissue did not affect cardiac morphology or basal cardiac function, but markedly compromised the ability of the heart to adapt to transverse aortic constriction (TAC). In contrast to wild-type mice, the transgenic animals developed significantly reduced ventricular hypertrophy in response to pressure overload and also did not exhibit induction of the cardiac “fetal” gene program. TAC of the transgenic mice caused a rapid decompensation in most animals characterized by left ventricular dilatation, depressed systolic function, and increased postoperative mortality when compared with nontransgenic littermates. These results implicate RGS proteins as a crucial component of the signaling pathway involved in both the cardiac response to acute ventricular pressure overload and the cardiac hypertrophic program.

O ₂ deprivation induces membrane depolarization in mammalian central neurons. It is possible that this anoxia-induced depolarization is partly mediated by an inhibition of K⁺ channels. We therefore performed experiments using patch-clamp techniques and dissociated neurons from mice neocortex. Three types of K⁺ channels were observed in both cell-attached and inside-out configurations, but only one of them was sensitive to lack of O₂. This O₂-sensitive K⁺ channel was identified as a large-conductance Ca²⁺-activated K⁺ channel (BK_Ca), as it exhibited a large conductance of 210 pS under symmetrical K⁺ (140 mM) conditions, a strong voltage-dependence of activation, and a marked sensitivity to Ca²⁺. A low-O₂ medium (PO₂ = 10–20 mmHg) markedly inhibited this BK_Ca channel open probability in a voltage-dependent manner in cell-attached patches, but not in inside-out patches, indicating that the effect of O₂ deprivation on BK_Ca channels of mice neocortical neurons was mediated via cytosol-dependent processes. Lowering intracellular pH (pH_i), or cytosolic addition of the catalytic subunit of a cAMP-dependent protein kinase A in the presence of Mg-ATP, caused a decrease in BK_Ca channel activity by reducing the sensitivity of this channel to Ca²⁺. In contrast, the reducing agents glutathione and DTT increased single BK_Ca channel open probability without affecting unitary conductance. We suggest that in neocortical neurons, (a) BK_Ca is modulated by O₂ deprivation via cytosolic factors and cytosol-dependent processes, and (b) the reduction in channel activity during hypoxia is likely due to reduced Ca²⁺ sensitivity resulting from cytosolic alternations such as in pH_i and phosphorylation. Because of their large conductance and prevalence in the neocortex, BK_Ca channels may be considered as a target for pharmacological intervention in conditions of acute anoxia or ischemia.

R elapsing polychondritis (RP) differs from rheumatoid arthritis (RA) in that primarily cartilage outside diarthrodial joints is affected. The disease usually involves trachea, nose, and outer ears. To investigate whether the tissue distribution of RP may be explained by a specific immune response, we immunized rats with cartilage matrix protein (matrilin-1), a protein predominantly expressed in tracheal cartilage. After 2–3 weeks, some rats developed a severe inspiratory stridor. They had swollen noses and/or epistaxis, but showed neither joint nor outer ear affection. The inflammatory lesions involved chronic active erosions of cartilage. Female rats were more susceptible than males. The disease susceptibility was controlled by both MHC genes (f, l, d, and a haplotypes are high responders, and u, n, and c are resistant) and non-MHC genes (the LEW strain is susceptible; the DA strain is resistant). However, all strains mounted a pronounced IgG response to cartilage matrix protein. The initiation and effector phase of the laryngotracheal involvement causing the clinical symptoms were shown to depend on αβ T cells. Taken together, these results represent a novel model for RP: matrilin-1–induced RP. Our findings also suggest that different cartilage proteins are involved in pathogenic models of RP and RA.

T he mechanisms underlying the initiation of virus-induced autoimmune disease are not well understood. Theiler’s murine encephalomyelitis virus–induced demyelinating disease (TMEV-IDD), a mouse model of multiple sclerosis, is initiated by TMEV-specific CD4⁺ T cells targeting virally infected central nervous system–resident (CNS-resident) antigen-presenting cells (APCs), leading to chronic activation of myelin epitope–specific CD4⁺ T cells via epitope spreading. Here we show that F4/80⁺, I-A^s+, CD45⁺ macrophages/microglia isolated from the CNS of TMEV-infected SJL mice have the ability to endogenously process and present virus epitopes at both acute and chronic stages of the disease. Relevant to the initiation of virus-induced autoimmune disease, only CNS APCs isolated from TMEV-infected mice with preexisting myelin damage, not those isolated from naive mice or mice with acute disease, were able to endogenously present a variety of proteolipid protein epitopes to specific Th1 lines. These results offer a mechanism by which localized virus-induced, T cell–mediated inflammatory myelin destruction leads to the recruitment/activation of CNS-resident APCs that can process and present endogenous self epitopes to autoantigen-specific T cells, and thus provide a mechanistic basis by which epitope spreading occurs.

M ammalian and venom secreted phospholipases A₂ (sPLA₂s) have been associated with a variety of biological effects. Here we show that several sPLA₂s protect human primary blood leukocytes from the replication of various macrophage and T cell–tropic HIV-1 strains. Inhibition by sPLA₂s results neither from a virucidal effect nor from a cytotoxic effect on host cells, but it involves a more specific mechanism. sPLA₂s have no effect on virus binding to cells nor on syncytia formation, but they prevent the intracellular release of the viral capsid protein, suggesting that sPLA₂s block viral entry into cells before virion uncoating and independently of the coreceptor usage. Various inhibitors and catalytic products of sPLA₂ have no effect on HIV-1 infection, suggesting that sPLA₂ catalytic activity is not involved in the antiviral effect. Instead, the antiviral activity appears to involve a specific interaction of sPLA₂s to host cells. Indeed, of 11 sPLA₂s from venom and mammalian tissues assayed, 4 venom sPLA₂s were found to be very potent HIV-1 inhibitors (ID₅₀ < 1 nM) and also to bind specifically to host cells with high affinities (K_0.5 < 1 nM). Although mammalian pancreatic group IB and inflammatory-type group IIA sPLA₂s were inactive against HIV-1 replication, our results could be of physiological interest, as novel sPLA₂s are being characterized in humans.

F amilial hypercholesterolemia (FH) is characterized by a raised concentration of LDL in plasma that results in a significantly increased risk of premature atherosclerosis. In FH, impaired removal of LDL from the circulation results from inherited mutations in the LDL receptor gene or, more rarely, in the gene for apo B, the ligand for the LDL receptor. We have identified two unrelated clinically homozygous FH patients whose cells exhibit no measurable degradation of LDL in culture. Extensive analysis of DNA and mRNA revealed no defect in the LDL receptor, and alleles of the LDL receptor or apo B genes do not cosegregate with hypercholesterolemia in these families. FACS^® analysis of binding and uptake of fluorescent LDL or anti–LDL receptor antibodies showed that LDL receptors are on the cell surface and bind LDL normally, but fail to be internalized, suggesting that some component of endocytosis through clathrin-coated pits is defective. Internalization of the transferrin receptor occurs normally, suggesting that the defective gene product may interact specifically with the LDL receptor internalization signal. Identification of the defective gene will aid genetic diagnosis of other hypercholesterolemic patients and elucidate the mechanism by which LDL receptors are internalized.

T he intestinal absorption of many nutrients and drug molecules is mediated by ion-driven transport mechanisms in the intestinal enterocyte plasma membrane. Clearly, the establishment and maintenance of the driving forces — transepithelial ion gradients — are vital for maximum nutrient absorption. The purpose of this study was to determine the nature of intracellular pH (pH_i) regulation in response to H⁺-coupled transport at the apical membrane of human intestinal epithelial Caco-2 cells. Using isoform-specific primers, mRNA transcripts of the Na⁺/H⁺ exchangers NHE1, NHE2, and NHE3 were detected by RT-PCR, and identities were confirmed by sequencing. The functional profile of Na⁺/H⁺ exchange was determined by a combination of pH_i, ²²Na⁺ influx, and EIPA inhibition experiments. Functional NHE1 and NHE3 activities were identified at the basolateral and apical membranes, respectively. H⁺/solute-induced acidification (using glycylsarcosine or β-alanine) led to Na⁺-dependent, EIPA-inhibitable pH_i recovery or EIPA-inhibitable ²²Na⁺ influx at the apical membrane only. Selective activation of apical (but not basolateral) Na⁺/H⁺ exchange by H⁺/solute cotransport demonstrates that coordinated activity of H⁺/solute symport with apical Na⁺/H⁺ exchange optimizes the efficient absorption of nutrients and Na⁺, while maintaining pH_i and the ion gradients involved in driving transport.

T o understand the role of Na⁺/H⁺ exchanger 1 (NHE1) in intracellular pH (pH_i) regulation and neuronal function, we took advantage of natural knockout mice lacking NHE1, the most ubiquitously and densely expressed NHE isoform in the central nervous system (CNS). CA1 neurons from both wild-type (WT) and NHE1 mutant mice were studied by continuous monitoring of pH_i, using the fluorescent indicator carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. In the nominal absence of CO₂/HCO₃^–, steady-state pH_i was higher in WT neurons than in mutant neurons. Using the NH₄Cl prepulse technique, we also show that H⁺ flux in WT neurons was much greater than in mutant neurons. The recovery from acid load was blocked in WT neurons, but not in mutant neurons, by removal of Na⁺ from the extracellular solution or by using 100 μM 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate (HOE 694) in HEPES buffer. Surprisingly, in the presence of CO₂/HCO₃^–, the difference in H⁺ flux between WT and mutant mice was even more exaggerated, with a difference of more than 250 μM/s between them at pH 6.6. H⁺ flux in CO₂/HCO₃^– was responsive to diisothiocyanato-stilbene-2,2′-disulfonate (DIDS) in the WT but not in the mutant. We conclude that (a) the absence of NHE1 in the mutant neurons tended to cause lower steady-state pH_i and, perhaps more importantly, markedly reduced the rate of recovery from an acid load; and (b) this difference in the rate of recovery between mutant and WT neurons was surprisingly larger in the presence, rather than in the absence, of HCO₃^–, indicating that the presence of NHE1 is essential for the regulation and/or functional expression of both HCO₃^–-dependent and -independent transporters in neurons.

N itric oxide (NO) derived from the inducible isoform of NO synthase (iNOS) is an inflammatory product implicated both in secondary damage and in recovery from brain injury. To address the role of iNOS in experimental traumatic brain injury (TBI), we used 2 paradigms in 2 species. In a model of controlled cortical impact (CCI) with secondary hypoxemia, rats were treated with vehicle or with 1 of 2 iNOS inhibitors (aminoguanidine and L-N-iminoethyl-lysine), administered by Alzet pump for 5 days and 1.5 days after injury, respectively. In a model of CCI, knockout mice lacking the iNOS gene (iNOS^–/–) were compared with wild-type (iNOS^+/+) mice. Functional outcome (motor and cognitive) during the first 20 days after injury, and histopathology at 21 days, were assessed in both studies. Treatment of rats with either of the iNOS inhibitors after TBI significantly exacerbated deficits in cognitive performance, as assessed by Morris water maze (MWM) and increased neuron loss in vulnerable regions (CA3 and CA1) of hippocampus. Uninjured iNOS^+/+ and iNOS^–/– mice performed equally well in both motor and cognitive tasks. However, after TBI, iNOS^–/– mice showed markedly worse performance in the MWM task than iNOS^+/+ mice. A beneficial role for iNOS in TBI is supported.

T -helper type 2 (Th2) cytokines have been implicated in the pathogenesis of the pulmonary inflammatory response and altered bronchial responsiveness in allergic asthma. To elucidate the mechanism of Th2-dependent mediation of altered airway responsiveness in the atopic asthmatic state, the expression and actions of specific cytokines were examined in isolated rabbit and human airway smooth muscle (ASM) tissues and cultured cells passively sensitized with sera from atopic asthmatic patients or nonatopic/nonasthmatic (control) subjects. Relative to control tissues, the atopic asthmatic sensitized ASM exhibited significantly enhanced maximal isometric contractility to acetylcholine and attenuated relaxation responses to isoproterenol. These proasthmatic changes in agonist responsiveness were ablated by pretreating the atopic sensitized tissues with either an IL-5 receptor blocking antibody (IL-5ra) or the human recombinant IL-1 receptor antagonist (IL-1ra), whereas an IL-4 neutralizing antibody had no effect. Moreover, relative to controls, atopic asthmatic sensitized ASM cells demonstrated an initial, early (after 3 hours of incubation) increased mRNA expression and protein release of IL-5. This was followed (after 6 hours of incubation) by an enhanced mRNA expression and release of IL-1β protein, an effect that was inhibited in sensitized cells pretreated with IL-5ra. Extended studies demonstrated that naive ASM exposed to exogenously administered IL-5 exhibited an induced upregulated mRNA expression and protein release of IL-1β associated with proasthmatic-like changes in ASM constrictor and relaxant responsiveness, and that these effects were ablated in tissues pretreated with IL-1ra. Taken together, these observations provide new evidence that (a) the Th2 cytokine IL-5 and the pleiotropic proinflammatory cytokine IL-1β are endogenously released by atopic asthmatic sensitized ASM and mechanistically interact to mediate the proasthmatic perturbations in ASM responsiveness; and (b) the nature of this interaction is given by an initial endogenous release of IL-5, which then acts to induce the autologous release of IL-1β by the sensitized ASM itself, resulting in its autocrine manifestation of the proasthmatic phenotype.