Issue published September 1, 2004

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C ell-to-cell viral transmission facilitates the propagation of HIV-1 and human T cell leukemia virus type 1. Mechanisms of cell-to-cell transmission by retroviruses were not well understood until the recent description of virological synapses (VSs). VSs function as specialized sites of immune cell-to-cell contact that direct virus infection. Deciphering the molecular mechanisms of VS formation provides a fascinating insight into how pathogens subvert immune cell communication programs and achieve viral spread.

G lomerular filtration in the kidney is a continuous process that acts in concert with tubular reabsorption to prevent derangements of body fluid composition. Filtration is regulated by systemic factors, but it is also controlled by an intrinsic mechanism based on the anatomical connection between the distal nephron and the glomerular arterioles. Facing the threat of urinary salt loss, this mechanism causes vasoconstriction and reduces filtration by generating adenosine through the hydrolysis of nucleotide precursors such as 5′-AMP and possibly ATP .

T here is a wealth of clinical data showing the relationship between diabetes mellitus and atherosclerosis and its clinical complications. To dissect this relationship, investigators have attempted, usually unsuccessfully, to create a small-animal model in which diabetes accelerates vascular lesion development. This effort has often been complicated by development of hyperlipidemia leading to difficulty in differentiating the effects of hyperglycemia from those of lipid abnormalities. A study in the current issue of the JCI provides data on a new mouse model in which atherosclerosis initiation is accelerated in diabetic mice and is reduced by insulin therapy. Moreover, these animals have greater intra-arterial hemorrhage, which might be due to less stable plaques .

P atients with systemic lupus erythematosus (SLE) often develop glomerulonephritis (i.e., inflammation in the glomeruli of the kidney), commonly referred to as lupus nephritis. Patients with lupus nephritis typically have autoantibodies to the complement classical pathway protein C1q. Whether these anti-C1q antibodies play any role in the development of lupus nephritis has been unclear. In this issue of the JCI, a new study demonstrates that anti-C1q antibodies can amplify glomerular injury but only when they are bound within the glomerulus to C1q that has been already brought to that site by other types of glomerular-reactive autoantibodies . These studies are the first, to our knowledge, to provide a causal link between anti-C1q antibodies and target organ damage in SLE.

S ignal transducer and activator of transcription 3 (Stat3) is a transcription factor that is constitutively activated in a variety of human malignancies, including prostate, lung, brain, breast, and squamous cell carcinomas. Inhibition of activated Stat3 leads to decreased proliferation and apoptosis of many cancer-derived cell lines, while the introduction of a constitutively activated form of Stat3 into immortalized human breast epithelial cells and rodent fibroblasts results in cellular transformation. Collectively, these data suggest a role for Stat3 in oncogenesis. A new study from Chan et al. (see related article beginning on page 720) is the first to demonstrate a requirement for Stat3 in de novo epithelial carcinogenesis in vivo. Using the two-step model of chemically induced skin carcinogenesis, the authors demonstrated that mice deficient in Stat3 were completely resistant to skin tumor development.

A mouse model involving the human papillomavirus type-16 oncogenes develops cervical cancers by lesional stages analogous to those in humans. In this study the angiogenic phenotype was characterized, revealing intense angiogenesis in high-grade cervical intraepithelial neoplasias (CIN-3) and carcinomas. MMP-9, a proangiogenic protease implicated in mobilization of VEGF, appeared in the stroma concomitant with the angiogenic switch, expressed by infiltrating macrophages, similar to what has been observed in humans. Preclinical trials sought to target MMP-9 and angiogenesis with a prototypical MMP inhibitor and with a bisphosphonate, zoledronic acid (ZA), revealing both to be antiangiogenic, producing effects comparable to a Mmp9 gene KO in impairing angiogenic switching, progression of premalignant lesions, and tumor growth. ZA therapy increased neoplastic epithelial and endothelial cell apoptosis without affecting hyperproliferation, indicating that ZA was not antimitotic. The analyses implicated cellular and molecular targets of ZA’s actions: ZA suppressed MMP-9 expression by infiltrating macrophages and inhibited metalloprotease activity, reducing association of VEGF with its receptor on angiogenic endothelial cells. Given its track record in clinical use with limited toxicity, ZA holds promise as an “unconventional” MMP-9 inhibitor for antiangiogenic therapy of cervical cancer and potentially for additional cancers and other diseases where MMP-9 expression by infiltrating macrophages is evident.

A denosine coordinates organ metabolism and blood supply, and it modulates immune responses. In the kidney it mediates the vascular response elicited by changes in NaCl concentration in the macula densa region of the nephron, thereby serving as an important regulator of GFR. To determine whether adenosine formation depends on extracellular nucleotide hydrolysis, we studied NaCl-dependent GFR regulation (tubuloglomerular feedback) in mice with targeted deletion of ecto-5′-nucleotidase/CD73 (e-5′NT/CD73), the enzyme responsible for adenosine formation from AMP. e-5′NT/CD73^–/– mice were viable and showed no gross anatomical abnormalities. Blood pressure, blood and urine chemistry, and renal blood flow were not different between e-5′NT/CD73^+/+ and e-5′NT/CD73^–/– mice. e-5′NT/CD73^–/– mice had a significantly reduced fall in stop flow pressure and superficial nephron glomerular filtration rate in response to a saturating increase of tubular perfusion flow. Furthermore, whereas tubuloglomerular feedback responses did not change significantly during prolonged loop of Henle perfusion in e-5′NT/CD73^+/+ mice, a complete disappearance of the residual feedback response was noted in e-5′NT/CD73^–/– mice over 10 minutes of perfusion. The contractile response of isolated afferent arterioles to adenosine was normal in e-5′NT/CD73^–/– mice. We conclude that the generation of adenosine at the glomerular pole depends to a major extent on e-5′NT/CD73–mediated dephosphorylation of 5′-AMP, presumably generated from released ATP.

I n collapsing focal segmental glomerulosclerosis (FSGS) of HIV-associated nephropathy (HIVAN), podocytes exhibit a high proliferation rate and loss of differentiation markers. We have found previously that the nef gene of HIV-1 is responsible for these changes. Here, we investigated the signaling pathways induced by Nef and its role in the pathogenesis of HIVAN. Using conditionally immortalized podocytes after differentiation, we found that infection of podocytes with nef increased Src kinase activity and signal transducer and activator of transcription 3 (Stat3) phosphorylation and activated the Ras–c-Raf–MAPK1,2 pathway. A dominant negative mutant of Src abolished the Nef effect, whereas inhibition of MAPK1,2 or dominant negative Stat3 reduced Nef effects partially. Reducing the expression of Nef with small interference RNA reversed the Nef effect. Mutation of Nef in the PxxP or R₁₀₅R₁₀₆ motifs diminished Nef signaling and the phenotypic changes in podocytes. Both phospho-MAPK1,2 and phospho-Stat3 staining increased in podocytes of kidneys from HIV-1 transgenic mice compared with their littermates and in podocytes of kidneys from HIVAN patients compared with HIV patients with non-HIVAN kidney diseases or non-HIV patients with idiopathic FSGS, classic FSGS, or minimal-change disease. These data suggest that Nef-induced activation of Stat3 and Ras-MAPK1,2 via Src-dependent pathways is responsible for podocyte proliferation and dedifferentiation, a characteristic finding in collapsing FSGS of HIVAN.

T he action of insulin in the central nervous system produces sympathetic nervous system activation (also called sympathoactivation), although the neuronal intracellular mechanisms that mediate this are unclear. We hypothesized that PI3K and MAPK, the major pathways involved in insulin receptor signaling, mediate sympathetic nerve responses to insulin. Intracerebroventricular administration of insulin in rat increased multifiber sympathetic nerve activity to the hindlimb, brown adipose tissue (BAT), adrenal gland, and kidney. Ex vivo biochemical studies of mediobasal hypothalamic tissue revealed that insulin stimulated the association of insulin receptor substrate–1 with the p85α subunit of PI3K and also tyrosine phosphorylation of p42 and p44 subunits of MAPK in the hypothalamus. In order to determine whether PI3K and/or MAPK were involved in insulin-mediated sympathoactivation, we tested the effect of specific inhibitors of PI3K (LY294002 and wortmannin) and MAPK (PD98059 and U0126) on regional sympathetic responses to insulin. Interestingly, regional sympathoactivation to insulin was differentially affected by blockade of PI3K and MAPK. Inhibition of PI3K specifically blocked insulin-induced sympathoactivation to the hindlimb, while inhibition of MAPK specifically blocked insulin-induced sympathoactivation to BAT. Sympathoactivation to corticotrophin-releasing factor, however, was not affected by inhibition of PI3K and MAPK. These data demonstrate that PI3K and MAPK are specific and regionally selective mediators of the action of insulin on the sympathetic nervous system.

D iabetes in humans accelerates cardiovascular disease caused by atherosclerosis. The relative contributions of hyperglycemia and dyslipidemia to atherosclerosis in patients with diabetes are not clear, largely because there is a lack of suitable animal models. We therefore have developed a transgenic mouse model that closely mimics atherosclerosis in humans with type 1 diabetes by breeding low-density lipoprotein receptor–deficient mice with transgenic mice in which type 1 diabetes can be induced at will. These mice express a viral protein under control of the insulin promoter and, when infected by the virus, develop an autoimmune attack on the insulin-producing β cells and subsequently develop type 1 diabetes. When these mice are fed a cholesterol-free diet, diabetes, in the absence of associated lipid abnormalities, causes both accelerated lesion initiation and increased arterial macrophage accumulation. When diabetic mice are fed cholesterol-rich diets, on the other hand, they develop severe hypertriglyceridemia and advanced lesions, characterized by extensive intralesional hemorrhage. This progression to advanced lesions is largely dependent on diabetes-induced dyslipidemia, because hyperlipidemic diabetic and nondiabetic mice with similar plasma cholesterol levels show a similar extent of atherosclerosis. Thus, diabetes and diabetes-associated lipid abnormalities have distinct effects on initiation and progression of atherosclerotic lesions.

N eonatal rodents are more tolerant to hyperoxia than adults. We determined whether maturational differences in lung NF-κB activation could account for the differences. After hyperoxic exposure (O₂ > 95%), neonatal (<12 hours old) lung NF-κB binding was increased and reached a maximum between 8 and 16 hours, whereas in adults no changes were observed. Additionally, neonatal NF-κB/luciferase transgenic mice (incorporating 2 NF-κB consensus sequences driving luciferase gene expression) demonstrated enhanced in vivo NF-κB activation after hyperoxia in real time. In the lungs of neonates, there was a propensity toward NF-κB activation as evidenced by increased lung I-κB kinase protein levels, I-κBα phosphorylation, β-transducin repeat–containing protein levels, and total I-κBα degradation. Increased lung p-JNK immunoreactive protein was observed only in the adult lung. Inhibition of pI-κBα by BAY 11-7085 resulted in decreased Bcl-2 protein levels in neonatal lung homogenates and decreased cell viability in lung primary cultures after hyperoxic exposure. Furthermore, neonatal p50-null mutant (p50^–/–) mice showed increased lung DNA degradation and decreased survival in hyperoxia compared with WT mice. These data demonstrate that there are maturational differences in lung NF-κB activation and that enhanced NF-κB may serve to protect the neonatal lung from acute hyperoxic injury via inhibition of apoptosis.

A nti-C1q autoantibodies are present in sera of patients with several autoimmune diseases, including systemic lupus erythematosus (SLE). Strikingly, in SLE the presence of anti-C1q is associated with the occurrence of nephritis. We have generated mouse anti–mouse C1q mAb’s and used murine models to investigate whether anti-C1q autoantibodies actually contribute to renal pathology in glomerular immune complex disease. Administration of anti-C1q mAb JL-1, which recognizes the collagen-like region of C1q, resulted in glomerular deposition of C1q and anti-C1q autoantibodies and mild granulocyte influx, but no overt renal damage. However, combination of JL-1 with a subnephritogenic dose of C1q-fixing anti–glomerular basement membrane (anti-GBM) antibodies enhanced renal damage characterized by persistently increased levels of infiltrating granulocytes, major histological changes, and increased albuminuria. This was not observed when a non–C1q-fixing anti-GBM preparation was used. Experiments with different knockout mice showed that renal damage was dependent not only on glomerular C1q and complement activation but also on Fcγ receptors. In conclusion, anti-C1q autoantibodies deposit in glomeruli together with C1q but induce overt renal disease only in the context of glomerular immune complex disease. This provides an explanation why anti-C1q antibodies are especially pathogenic in patients with SLE.

S ubspecies of Trypanosoma brucei cause severe brain diseases after penetration of the blood-brain barrier. We investigated whether cytokines that modulate inflammatory cell infiltration into the brain also influence T. brucei neuroinvasion. Migration of a rodent pathogenic T. brucei strain from the cerebral blood vessels into the brain parenchyma was impeded in IFN-γ^–/–, IFN-γ receptor^–/– (IFN-γR^–/–), IL-12p40^–/–, and recombinant activating gene–1^–/– (RAG-1^–/–) mice as compared with their WT littermates despite higher levels of parasitemia in the mutant strains. Parasites accumulated in the perivascular compartment, confined between the endothelial and the parenchymal basement membranes, in certain areas of the brains of IFN-γ^–/–, IFN-γR^–/–, and RAG-1^–/– mice. This accumulation occurred around endothelial basement membranes containing the laminin α4 chain, while blood vessels showing robust laminin α5 chain immunostaining were not associated with parasite infiltration. The number of CD4⁺ and CD8⁺ T cells infiltrating the brain parenchyma was also reduced in the IFN-γ^–/– and IFN-γR^–/– mice. Our findings suggest that lymphocyte-derived IFN-γ facilitates trypanosome penetration across cerebral blood vessels and that the site of penetration is determined by the composition of the basement membranes of these vessels.

P orcine endogenous retrovirus (PERV) is a potential pathogen in clinical xenotransplantation; transmission of PERV in vivo has been suggested in murine xenotransplantation models. We analyzed the transmission of PERV to human cells in vivo using a model in which immunodeficient NOD/SCID transgenic mice were transplanted with porcine and human lymphohematopoietic tissues. Our results demonstrate, we believe for the first time, that human and pig cells can coexist long-term (up to 25 weeks) without direct PERV infection of human cells. Despite the transplantation of porcine cells that did not produce human-tropic PERV, human cells from the chimeric mice were frequently found to contain PERV sequences. However, this transmission was due to the pseudotyping of PERV-C (a virus without human tropism) by xenotropic murine leukemia virus, rather than to de novo generation of human-tropic PERV. Thus, pseudotyping might account for the PERV transmission previously observed in mice. The absence of direct human cell infection following long-term in vivo coexistence with large numbers of porcine cells provides encouragement regarding the potential safety of using pigs that do not produce human-tropic PERV as source animals for transplantation to humans.

H epatic immunobiology is paradoxical: although the liver possesses unusual tolerogenic properties, it is also the site of effective immune responses against multiple pathogens and subject to immune-mediated pathology. The mechanisms underlying this dichotomy remain unclear. Following previous work demonstrating that the liver may act as a site of primary T cell activation, we demonstrate here that the balance between immunity and tolerance in this organ is established by competition for primary activation of CD8⁺ T cells between the liver and secondary lymphoid tissues, with the immune outcome determined by the initial site of activation. Using a transgenic mouse model in which antigen is expressed within both liver and lymph nodes, we show that while naive CD8⁺ T cells activated within the lymph nodes were capable of mediating hepatitis, cells undergoing primary activation within the liver exhibited defective cytotoxic function and shortened half-life and did not mediate hepatocellular injury. The implications of these novel findings may pertain not only to the normal maintenance of peripheral tolerance, but also to hepatic allograft tolerance and the immunopathogenesis of chronic viral hepatitis.

M yelodysplasia is a hematological disease in which genomic abnormalities accumulate in a hematopoietic stem cell leading to severe pancytopenia, multilineage differentiation impairment, and bone marrow (BM) apoptosis. Mortality in the disease results from pancytopenia or transformation to acute myeloid leukemia. There are frequent cytogenetic abnormalities, including deletions of chromosomes 5, 7, or both. Recurring chromosomal translocations in myelodysplasia are rare, but the most frequent are the t(3;3)(q21;q26) and the inv(3)(q21q26), which lead to the inappropriate activation of the EVI1 gene located at 3q26. To better understand the role of EVI1 in this disease, we have generated a murine model of EVI1-positive myelodysplasia by BM infection and transplantation. We find that EVI1 induces a fatal disease of several stages that is characterized by severe pancytopenia. The disease does not progress to acute myeloid leukemia. Comparison of in vitro and in vivo results suggests that EVI1 acts at two levels. The immediate effects of EVI1 are hyperproliferation of BM cells and downregulation of EpoR and c-Mpl, which are important for terminal erythroid differentiation and platelet formation. These defects are not fatal, and the mice survive for about 10 months with compensated hematopoiesis. Over this time, compensation fails, and the mice succumb to fatal peripheral cytopenia.

C onstitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a wide spectrum of human malignancies. Here, we have assessed the effect of Stat3 deficiency on skin tumor development using the 2-stage chemical carcinogenesis model. The epidermis of Stat3-deficient mice showed a significantly reduced proliferative response following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) because of a defect in G1-to-S-phase cell cycle progression. Treatment with the tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) resulted in a significant increase in the number of keratinocyte stem cells undergoing apoptosis in the bulge region of hair follicles of Stat3-deficient mice compared with nontransgenic littermates. Notably, Stat3-deficient mice were completely resistant to skin tumor development when DMBA was used as the initiator and TPA as the promoter. Abrogation of Stat3 function using a decoy oligonucleotide inhibited the growth of initiated keratinocytes possessing an activated Ha-ras gene, both in vitro and in vivo. In addition, injection of Stat3 decoy into skin tumors inhibited their growth. To our knowledge, these data provide the first evidence that Stat3 is required for de novo epithelial carcinogenesis, through maintaining the survival of DNA-damaged stem cells and through mediating and maintaining the proliferation necessary for clonal expansion of initiated cells during tumor promotion. Collectively, these data suggest that, in addition to its emerging role as a target for cancer therapy, Stat3 may also be a target for cancer prevention strategies.

J unctional adhesion molecule-A (JAM-A) is a transmembrane adhesive protein expressed at endothelial junctions and in leukocytes. In the present work, we found that DCs also express JAM-A. To evaluate the biological relevance of this observation, Jam-A^–/– mice were generated and the functional behavior of DCs in vitro and in vivo was studied. In vitro, Jam-A^–/– DCs showed a selective increase in random motility and in the capacity to transmigrate across lymphatic endothelial cells. In vivo, Jam-A^–/– mice showed enhanced DC migration to lymph nodes, which was not observed in mice with endothelium-restricted deficiency of the protein. Furthermore, increased DC migration to lymph nodes was associated with enhanced contact hypersensitivity (CHS). Adoptive transfer experiments showed that JAM-A^–deficient DCs elicited increased CHS in Jam-A^+/+ mice, further supporting the concept of a DC-specific effect. Thus, we identified here a novel, non-redundant role of JAM-A in controlling DC motility, trafficking to lymph nodes, and activation of specific immunity.