Issue published December 15, 2002

C entral to inflammatory responses are the integrin-mediated adhesive interactions of cells with their ECM-rich environment. We investigated the role of the collagen-binding integrin α₁β₁ in intestinal inflammation using the mouse model of colitis induced by dextran sodium sulfate (DSS). mAb’s directed against murine α₁ were found to significantly attenuate inflammation and injury in DSS-treated wild-type mice; similar protection was seen in mice deficient for α₁β₁ integrin. Blockade or loss of α₁β₁ was also associated with decreased mucosal inflammatory cell infiltrate and cytokine production. Importantly, we demonstrated that development and α₁-mediated inhibition of DSS-induced colitis occurred independently of lymphocytes (Rag-2^–/– mice), and identified the monocyte as a key α₁β₁-expressing cell type involved in the development of colitis in this model. In response to DSS, both α₁ deficiency and anti-α₁ mAb treatment significantly reduced monocyte accumulation and activation within the lamina propria. In summary, the data demonstrate that engagement of leukocyte-associated α₁β₁ receptors with ECM plays a pivotal role in mediating intestinal inflammation via promotion of monocyte movement and/or activation within the inflamed interstitium. Therapeutic strategies designed to disrupt such interactions may prove beneficial in treating intestinal inflammation.

R NA repair has been proposed as a novel gene-based therapeutic strategy. Modified Tetrahymena group I intron ribozymes have been used to mediate trans-splicing of therapeutically relevant RNA transcripts, but the efficiency of the ribozyme-mediated RNA repair process has not been determined precisely and subsequent restoration of protein function has been demonstrated only by indirect means. We engineered a ribozyme that targets the mRNA of a mutant canine skeletal muscle chloride channel (cClC-1) (mutation T268M in ClC-1 causing myotonia congenita) and replaces the mutant-containing 3′ portion by trans-splicing the corresponding 4-kb wild-type sequence. Repair efficiency assessed by quantitative RT-PCR was 1.2% ± 0.1% in a population of treated cells. However, when chloride channel function was examined in single cells, a wide range of electrophysiological activity was observed, with 18% of cells exhibiting significant functional restoration and some cells exhibiting complete rescue of the biophysical phenotype. These results indicate that RNA repair can restore wild-type protein activity and reveal considerable cell-to-cell variability in ribozyme-mediated trans-splicing reaction efficiency.

H istamine is an aminergic neurotransmitter that is localized in the CNS and in peripheral tissues. To date, four histamine receptors have been identified, and the H3 receptor, which was recently cloned, is predominantly expressed in the CNS. The peripheral functions of histamine have been investigated intensively using available molecular and pharmacological tools, and the molecular identification of the H3 receptor opens up new possibilities for investigating the role of histamine in central tissues. To understand the biological function of the histamine presynaptic autoreceptor H3, we inactivated the receptor through homologous recombination. H3^–/– mice manifest mild obese phenotypes that are characterized by increases in body weight, food intake, and adiposity and by reductions in energy expenditure. Consistent with these observations, homozygous null mice have insulin and leptin resistance, increased levels of plasma leptin and insulin, and decreased levels of histamine in the hypothalamic/thalamic region of their brains coupled with increased histamine turnover. The expression of UCP1 in brown adipose tissue and of UCP3 in brown adipose tissue, white adipose tissue, and skeletal muscle is decreased in H3^–/– mutants, and the anorexigenic activity of thioperamide is not observed. These results suggest that neuronal histamine is a mediator of body-weight homeostasis and that neuronal histamine functions through H3 receptors in mice.

P regnancy is associated with changes in host susceptibility to infections and inflammatory disease. We hypothesize that metabolic enzyme trafficking affects maternal neutrophil activation. Specifically, immunofluorescence microscopy has shown that glucose-6-phosphate dehydrogenase (G-6-PDase), the rate-controlling step of the hexose monophosphate shunt (HMS), is located near the cell periphery in control neutrophils but is found near the microtubule-organizing centers in cells from pregnant women. Cytochemical studies confirmed that the distribution of the G-6-PDase antigen is coincident with functional G-6-PDase activity. Metabolic oscillations within activated pregnancy neutrophils are higher in amplitude, though lower in frequency, than activated control neutrophils, suggesting limited HMS activity. Analysis of radioisotope-labeled carbon flux from glucose to CO₂ indicates that the HMS is intact in leukocytes from pregnant women, but its level is not enhanced by cell stimulation. Using extracellular fluorescent markers, activated pregnancy neutrophils were found to release reactive oxygen metabolites (ROMs) at a lower rate than activated control neutrophils. However, basal levels of ROM production in polarized pregnancy neutrophils were greater than in control neutrophils. Microtubule-disrupting agents reversed the observed changes in G-6-PDase trafficking, metabolic oscillations, and ROM production by maternal neutrophils. G-6-PDase trafficking appears to be one mechanism regulating ROM production by maternal neutrophils.

T he cancer-testis antigen NY-ESO-1 is one of the most promising candidates for generic vaccination of cancer patients. Here we analyzed the CD8⁺ T cell response to a NY-ESO-1 peptide vaccine composed of the two previously defined peptides 157-165 and 157-167, administered with GM-CSF as a systemic adjuvant. The NY-ESO-1 peptide vaccine elicited a CD8⁺ T cell response directed against multiple distinct epitopes in the 157-167 region, as revealed by using A2/peptide multimers incorporating overlapping A2 binding peptides in this region. However, only a minor fraction of the elicited CD8⁺ T cells, namely those recognizing the peptide 157-165 with sufficiently high functional avidity, recognized the naturally processed target on NY-ESO-1⁺ tumor cells. In contrast, the majority of peptide 157-165–specific CD8⁺ T cells exhibited lower functional avidity and no tumor reactivity. In addition, vaccine-elicited CD8⁺ T cells specific for other overlapping epitopes in the 157-167 region failed to significantly recognize NY-ESO-1–expressing tumor targets. Thus, because of the complexity of the CD8⁺ T cell repertoire that can be elicited by vaccination with synthetic peptides, a precise definition of the targeted epitope, and hence, of the corresponding peptide to be used as immunogen, is required to ensure a precise tumor targeting.

I gG Fc receptors (FcγRs, especially FcγRIII) and complement (in particular, C5a anaphylatoxin) are critical effectors of the acute inflammatory response to immune complexes (ICs). However, it is unknown whether and how these two key components can interact with each other in vivo. We use here a mouse model of the acute pulmonary IC hypersensitivity reaction to analyze their potential interaction. FcγRIII and C5aR are coexpressed on alveolar macrophages (AMs), and both FcγRIII and C5aR mutant mice display impaired immune responses. We find that recombinant human C5a (rhC5a) can control inverse expression of various FcγRs, and costimulation of ICs with rhC5a results in strong enhancement of FcγRIII-triggered cellular activation in vitro and in vivo. Moreover, we show here that early IC-induced bioactive C5a, and its interaction with C5aR, causes induction of activating FcγRIII and suppression of inhibitory FcγRII on AMs that appears crucial for efficient cytokine production and neutrophil recruitment in lung pathology. Therefore, C5a, which is a potent chemoattractant, has a broader critical function in regulating the inhibitory/activating FcγRII/III receptor pair to connect complement and FcγR effector pathways in immune inflammation.

H epatocellular carcinoma is strongly associated with chronic infection by the hepatitis B virus (HBV) and has poor prognosis due to intrahepatic metastasis. HBx is often the only HBV protein detected in hepatic tumor cells; however, its contribution to tumor invasion and metastasis has not been established so far. In this work, we show that HBx enhances tumor cell invasion, both in vivo and in vitro. The increased invasive capacity induced by HBx is mediated by an upregulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) expression, which in turn activates matrix metalloproteinase-2. Induction of both MT1-MMP expression and cell invasion by HBx is dependent on cyclooxygenase-2 (COX-2) activity. In addition, HBx upregulates the expression of COX-2, which is mediated by the transcriptional activation of the COX-2 gene promoter in a nuclear factor of activated T cell–dependent (NF-AT–dependent) manner. These results demonstrate the ability of HBx to promote tumor cell invasion by a mechanism involving the upregulation of MT1-MMP and COX-2 and provide new insights into the mechanism of action of this viral protein and its involvement in tumor metastasis and recurrence of hepatocellular carcinoma.

D iabetes is caused by an absolute (type 1) or relative (type 2) deficiency of insulin-producing β cells. The mechanisms governing replication of terminally differentiated β cells and neogenesis from progenitor cells are unclear. Mice lacking insulin receptor substrate-2 (Irs2) develop β cell failure, suggesting that insulin signaling is required to maintain an adequate β cell mass. We report that haploinsufficiency for the forkhead transcription factor Foxo1 reverses β cell failure in Irs2^–/– mice through partial restoration of β cell proliferation and increased expression of the pancreatic transcription factor pancreas/duodenum homeobox gene-1 (Pdx1). Foxo1 and Pdx1 exhibit mutually exclusive patterns of nuclear localization in β cells, and constitutive nuclear expression of a mutant Foxo1 is associated with lack of Pdx1 expression. We show that Foxo1 acts as a repressor of Foxa2-dependent (Hnf-3β–dependent) expression from the Pdx1 promoter. We propose that insulin/IGFs regulate β cell proliferation by relieving Foxo1 inhibition of Pdx1 expression in a subset of cells embedded within pancreatic ducts.

C alcitonin (CT) is a known inhibitor of bone resorption. Calcitonin gene–related peptide-α (CGRPα), produced by alternative RNA processing of the CT/CGRP gene, has no clearly defined role in bone. To better understand the physiologic role of the CT/CGRP gene we created a mouse in which the coding sequences for both CT and CGRPα were deleted by homologous recombination. The CT/CGRP^–/– knockout (KO) mice procreated normally, there were no identifiable developmental defects at birth, and they had normal baseline calcium-related chemistry values. However, KO animals were more responsive to exogenous human parathyroid hormone as evidenced by a greater increase of the serum calcium concentration and urine deoxypyridinoline crosslinks, an effect reversed by CT and mediated by a greater increase in bone resorption than in controls. Surprisingly, KO mice have significantly greater trabecular bone volume and a 1.5- to 2-fold increase in bone formation at 1 and 3 months of age. This effect appears to be mediated by increased bone formation. In addition, KO mice maintain bone mass following ovariectomy, whereas wild-type mice lose approximately one-third of their bone mass over 2 months. These findings argue for dual roles for CT/CGRP gene products: prevention of bone resorption in hypercalcemic states and a regulatory role in bone formation.

W e examined the in vivo function of the angiotensin II type 1 receptor (Agtr1) on macrophages in renal fibrosis. Fourteen days after the induction of unilateral ureteral obstruction (UUO), wild-type mice reconstituted with marrow lacking the Agtr1 gene (Agtr1^–/–) developed more severe interstitial fibrosis with fewer interstitial macrophages than those in mice reconstituted with Agtr1^+/+ marrow. These differences were not observed at day 5 of UUO. The expression of profibrotic genes — including TGF-β1, α1(I) collagen, and α1(III) collagen — was substantially higher in the obstructed kidneys of mice with Agtr1^–/– marrow than in those with Agtr1^+/+ marrow at day 14 but not at day 5 of UUO. Mice with Agtr1^–/– marrow were characterized by reduced numbers of peripheral-blood monocytes and macrophage progenitors in bone marrow. In vivo assays revealed a significantly impaired phagocytic capability in Agtr1^–/– macrophages. In vivo treatment of Agtr1^+/+ mice with losartan reduced phagocytic capability of Agtr1^+/+ macrophages to a level comparable to that of Agtr1^–/– macrophages. Thus, during urinary tract obstruction, the Agtr1 on bone marrow–derived macrophages functions to preserve the renal parenchymal architecture, and this function depends in part on its modulatory effect on phagocytosis.

P reformed and elicited Ab’s against the Galα1,3Gal terminating carbohydrate chains (αGal Ab’s) are the primary cause of hyperacute and acute vascular xenograft rejection in pig-to-primate transplantation. αGal Ab’s are produced by long-lived Ab-producing cells that are not susceptible to pharmacological immunosuppression. We reasoned that antigen-specific elimination of αGal Ab’s might be achieved in vivo by systemic administration of nonimmunogenic polyvalent αGal structures with high avidity for αGal Ab’s. We devised GAS914, a soluble trisaccharide-polylysine conjugate of approximately 500 kDa that effectively competes for αGal binding by αGal IgM (IC₅₀, 43 nM) and IgG (IC₅₀, 28 nM) in vitro. Injections of GAS914 in cynomolgus monkeys, at the dose of 1 mg/kg, resulted in the immediate decrease of more than 90% of circulating αGal Ab’s and serum anti-pig cytotoxicity. In baboons, repeated injections of GAS914 effectively reduced both circulating αGal Ab’s and cytotoxicity over several months. Studies with [¹⁴C]GAS914 in rhesus monkeys and Gal^–/– mice indicate that GAS914 binds to circulating αGal Ab’s and that the complex is quickly metabolized by the liver and excreted by the kidney. Remarkably, posttreatment αGal Ab titers never exceeded pretreatment levels and no sensitization to either αGal or the polylysine backbone has been observed. Furthermore there was no apparent acute or chronic toxicity associated with GAS914 treatment in primates. We conclude that GAS914 may be used therapeutically for the specific removal of αGal Ab’s.