Issue published November 15, 2002

T he detailed cellular and molecular mechanisms leading to joint destruction in rheumatoid arthritis, a disease driven by proinflammatory cytokines, are still unknown. To address the question of whether osteoclasts play a pivotal role in this process, transgenic mice that express human TNF (hTNFtg) and that develop a severe and destructive arthritis were crossed with osteopetrotic, c-fos–deficient mice (c-fos^–/–) completely lacking osteoclasts. The resulting mutant mice (c-fos^–/–hTNFtg) developed a TNF-dependent arthritis in the absence of osteoclasts. All clinical features of arthritis, such as paw swelling and reduction of grip strength, progressed equally in both groups. Histological evaluation of joint sections revealed no difference in the extent of synovial inflammation, its cellular composition (except for the lack of osteoclasts), and the expression of matrix metalloprotein-ase-3 (MMP-3) and MMP-13. In addition, cartilage damage, proteoglycan loss, and MMP-3, -9, and -13 expression in chondrocytes were similar in hTNFtg and c-fos^–/–hTNFtg mice. However, despite the presence of severe inflammatory changes, c-fos^–/–hTNFtg mice were fully protected against bone destruction. These data reveal that TNF-dependent bone erosion is mediated by osteoclasts and that the absence of osteoclasts alters TNF-mediated arthritis from a destructive to a nondestructive arthritis. Therefore, in addition to the use of anti-inflammatory therapies, osteoclast inhibition could be beneficial for the treatment of rheumatoid arthritis.

T he pathological modifications of α-synuclein (αS) in Parkinson disease and related diseases are poorly understood. We have detected misfolded αS in situ based on the proteinase K resistance (PK resistance) of αS fibrils, and using specific antibodies against S129-phosphorylated αS as well as oxidized αS. Unexpectedly massive neuritic pathology was found in affected human brain regions, in addition to classical αS pathology. PK resistance and abnormal phosphorylation of αS developed with increasing age in (Thy1)-h[A30P] αS transgenic mice, concomitant with formation of argyrophilic, thioflavin S-positive, and electron-dense inclusions that were occasionally ubiquitinated. αS pathology in the transgenic mice was predominantly in the brainstem and spinal cord. Astrogliosis was found in these heavily affected tissues. Homozygous mice showed the same pathology approximately one year earlier. The transgenic mice showed a progressive deterioration of locomotor function.

A ntigen exposure via airway epithelia is often associated with a failure to prime or with the preferential priming of Th2 cells. We previously reported that the intranasal delivery of a Th1-inducing antigen promoted Th2-dominated responses, rather than the expected Th1 responses. Thus, we proposed that when pulmonary T cell priming is induced, the lung microenvironment might intrinsically favor the generation of Th2 types of responses. To establish a potential mechanism for such preferential priming, we examined the initial interactions between antigens and resident antigen-presenting cells (APCs) within the lung. We show that intranasally delivered antigens are preferentially taken up and can be presented to antigen-specific T cells by a resident population of CD11c^bright APCs. Most of these antigen-loaded APCs remained within lung tissues, and migration into secondary lymphoid organs was not crucial for T cell priming to occur within the pulmonary tract. Furthermore, these pulmonary APCs demonstrated a marked expression of IL-6 and IL-10 within hours of antigen uptake, suggesting that resident tissue APCs have the capacity to promote Th2 T cell differentiation in situ.

T he mouse coat color mutant mahoganoid (md) darkens coat color and decreases the obesity of A^y mice that ectopically overexpress agouti-signaling protein. The phenotypic effects of md are similar to those of the recently identified coat color mutant mahogany (Atrn^mg). We report the positional cloning of mahoganoid, encoding a novel 494–amino acid protein containing a C3HC4 RING (really interesting new gene) domain that may function as an E3 ubiquitin ligase. The mutations in the mahoganoid allelic series (md, md^2J, md^5J) are all due to large retroviral insertions. In md and md^2J, the result is minimal expression of the normal size transcripts in all tissues examined. Unlike Atrn^mg/Atrn^mg animals, we observe no evidence of neurological deficit or neuropathology in md/md mice. Body weight and body mass index (a surrogate for adiposity) measurements of B6.C3H-md-A md/+ and md/md animals on 9% and 45% kcal fat diets indicate that mahoganoid does not suppress body weight in B6.C3H animals in a gene dose-dependent fashion.

< i>Staphylococcus aureus (SA) is an opportunistic pathogen that affects a variety of organ systems and is responsible for many diseases worldwide. SA express an MHC class II analog protein (Map), which may potentiate SA survival by modulating host immunity. We tested this hypothesis in mice by generating Map-deficient SA (Map^–SA) and comparing disease outcome to wild-type Map⁺SA–infected mice. Map^–SA–infected mice presented with significantly reduced levels of arthritis, osteomyelitis, and abscess formation compared with control animals. Furthermore, Map^–SA–infected nude mice developed arthritis and osteomyelitis to a severity similar to Map⁺SA–infected controls, suggesting that T cells can affect disease outcome following SA infection and Map may attenuate cellular immunity against SA. The capacity of Map to alter T cell function was tested more specifically in vitro and in vivo using native and recombinant forms of Map. T cells or mice treated with recombinant Map had reduced T cell proliferative responses and a significantly reduced delayed-type hypersensitivity response to challenge antigen, respectively. These data suggest a role for Map as an immunomodulatory protein that may play a role in persistent SA infections by affecting protective cellular immunity.

C hronic beryllium disease (CBD) is caused by exposure to beryllium in the workplace, and it remains an important public health concern. Evidence suggests that CD4⁺ T cells play a critical role in the development of this disease. Using intracellular cytokine staining, we found that the frequency of beryllium-specific CD4⁺ T cells in the lungs (bronchoalveolar lavage) of 12 CBD patients ranged from 1.4% to 29% (mean 17.8%), and these T cells expressed a Th1-type phenotype in response to beryllium sulfate (BeSO₄). Few, if any, beryllium-specific CD8⁺ T cells were identified. In contrast, the frequency of beryllium-responsive CD4⁺ T cells in the blood of these subjects ranged from undetectable to 1 in 500. No correlation was observed between the frequency of beryllium-responsive bronchoalveolar lavage (BAL) CD4⁺ T cells as detected by intracellular staining and lymphocyte proliferation in culture after BeSO₄ exposure. Staining for surface marker expression showed that nearly all BAL T cells exhibit an effector memory cell phenotype. These results demonstrate a dramatically high frequency and compartmentalization of antigen-specific effector memory CD4⁺ cells in the lungs of CBD patients. These studies provide insight into the phenotypic and functional characteristics of antigen-specific T cells invading other inaccessible target organs in human disease.

T he physiological relevance of phosphoinositide 3-kinase (PI 3-K) signaling in the liver to fuel homeostasis was investigated. Systemic infusion of an adenovirus encoding a dominant negative mutant of PI 3-K (Δp85) resulted in liver-specific expression of this protein and in inhibition of the insulin-induced activation of PI 3-K in the liver within 3 days, without affecting insulin signaling in skeletal muscle. Hepatic expression of Δp85 led to hyperinsulinemia and to a marked increase in blood glucose concentration in response to oral glucose intake. The increases in both glycogen and glucose 6-phosphate content, as well as in Akt and glycogen synthase activities in the liver, that were induced by glucose intake were markedly impaired in mice expressing Δp85. Despite an upregulation of mRNAs for gluconeogenic enzymes apparent in the liver of these animals, the fasting blood glucose concentration was increased only slightly, and the serum concentrations of gluconeogenic precursors were reduced. However, administration of pyruvate, a substrate for gluconeogenesis, resulted in an exaggerated increase in blood glucose concentration. In the fasted state, the mass of adipose tissue of the mice was about 1.5 times that in control mice. The mice also exhibited marked decreases in the serum concentrations of FFAs and triglyceride and suppression of insulin-induced PI 3-K activation in adipose tissue, probably due to the associated hyperinsulinemia. PI 3-K activity in the liver is thus essential for normal carbohydrate and lipid metabolism in living animals.

R econstitution of protective immunity by adoptive transfer of pathogen-specific T cells has been successful in patients with compromised cellular immunity. The in vivo effectiveness of in vitro–expanded CD8 CTLs is variable, however. For example, adoptively transferred Listeria monocytogenes–specific CD8 CTLs only confer protective immunity if challenge infection occurs within 48 hours of T cell infusion. Herein we show that transferred CTLs persist in lymphoid compartments for many weeks, but that their response to bacterial challenge decreases during the first week following transfer. While T cells transferred less than 48 hours before infection proliferate, those transferred 7 days before infection die. Remarkably, treatment of mice with anti-CD40 at the time of T cell infusion reprograms transferred T cells, allowing them to proliferate and confer protective immunity upon bacterial challenge 7 days later. Our study demonstrates, for the first time to our knowledge that CD40-mediated stimuli can influence CD8 T cell activation independent of concurrent antigen exposure. The ability to modulate long-term responsiveness of CD8 T cells with a transient, nonspecific inflammatory stimulus has importation implications for adoptive immunotherapy.

T cell–mediated fulminant hepatitis is a life-threatening event for which the underlying mechanism is not fully understood. Injection of concanavalin A (Con A) into mice recapitulates the histological and pathological sequelae of T cell–mediated hepatitis. In this model, both signal transducer and activator of transcription factor 1 (STAT1) and STAT3 are activated in the liver. Disruption of the STAT1 gene by way of genetic knockout attenuates liver injury, suppresses CD4⁺ and NK T cell activation, and downregulates expression of proapoptotic interferon regulatory factor-1 protein and suppressor of cytokine signaling-1 (SOCS1), but enhances STAT3 activation and STAT3-controlled antiapoptotic signals. Studies from IFN-γ–deficient mice indicate that IFN-γ not only is the major cytokine responsible for STAT1 activation but also partially accounts for STAT3 activation. Moreover, downregulation of STAT3 activation in IL-6–deficient mice is associated with decreased STAT3-controlled antiapoptotic signals and expression of SOCS3, but upregulation of STAT1 activation and STAT1-induced proapoptotic signals and exacerbation of liver injury. Taken together, these findings suggest that STAT1 plays a harmful role in Con A–mediated hepatitis by activation of CD4⁺ and NK T cells and directly inducing hepatocyte death, whereas STAT3 protects against liver injury by suppression of IFN-γ signaling and induction of antiapoptotic protein Bcl-X_L. STAT1 and STAT3 in hepatocytes also negatively regulate one another through the induction of SOCS.

I FN-γ regulates the immunogenicity of target cells by increasing their expression of HLA class I molecules. This facilitates the T cell receptor–mediated recognition by CD8⁺ T cells but decreases target cell sensitivity to lysis by NK cells due to engagement of inhibitory NK receptors. In this study, short-term tumor cell lines from patients with advanced ovarian carcinomas were established. We demonstrate the paradoxical finding that IFN-γ treatment of these short-term ovarian carcinoma cell lines (OVACs) resulted in resistance of tumor cells to lysis by peptide- and allospecific CD8⁺ T cells. Blocking experiments revealed that this phenomenon was dependent on enhanced inhibitory signalling via CD94/NKG2A receptors expressed on the effector cells. This was associated with increased expression of HLA-E mRNA and HLA-G at the protein level in IFN-γ–treated OVACs. Furthermore, pulsing of untreated OVACs with the leader sequence peptide of HLA-G protected these cells from lysis by CTLs, thus mimicking the inhibitory effect of IFN-γ. This study provides evidence that CD94/NKG2A receptors play an important role in regulating T cell activity against tumors and shows that IFN-γ modulation of target cells may shift the balance of triggering and inhibitory signals to T cells, turning off their cytolytic activity.

T issue-type plasminogen activator (tPA) is one of the major components in the matrix proteolytic network whose role in the pathogenesis of renal interstitial fibrosis remains largely unknown. Here, we demonstrate that ablation of tPA attenuated renal interstitial fibrotic lesions in obstructive nephropathy. Mice lacking tPA developed less morphological injury and displayed a reduced deposition of interstitial collagen III and fibronectin as well as total tissue collagen in the kidneys after sustained ureteral obstruction, when compared with their wild-type counterparts. Deficiency of tPA selectively blocked tubular epithelial-to-myofibroblast transition (EMT), but did not affect myofibroblastic activation from interstitial fibroblasts. A marked decrease in matrix metalloproteinase-9 (MMP-9) induction was found in the obstructed kidneys of tPA^–/– mice, which led to a dramatic preservation of the structural and functional integrity of tubular basement membrane (TBM). In vitro, tPA induced MMP-9 gene expression and protein secretion in renal interstitial fibroblasts. Thus, increased tPA is detrimental in renal interstitial fibrogenesis through a cascade of events that lead to MMP-9 induction, TBM destruction, and promotion of EMT. Our findings establish a crucial and definite importance of EMT in the pathogenesis of renal interstitial fibrosis at the whole-animal level.

A part from its role in the synthesis of protein and nitric oxide (NO), and in ammonia detoxification, the amino acid arginine exerts an immunosupportive function. We have studied the role of arginine in immune defense mechanisms in the developing postnatal immune system. In suckling mice, arginine is produced in the small intestine. In F/A-2^+/+ transgenic mice, which overexpress arginase in their enterocytes, circulating and tissue arginine concentrations are reduced to 30–35% of controls. In these mice, the development and composition of the T cell compartment did not reveal abnormalities. However, in peripheral lymphoid organs and the small intestine, B cell cellularity and the number and size of Peyer’s patches were drastically reduced, and serum IgM levels were significantly decreased. These phenotypes could be traced to an impaired transition from the pro– to pre–B cell stage in the bone marrow. Cytokine receptor levels in the bone marrow were normal. The development of the few peripheral B cells and their proliferative response after in vitro stimulation was normal. The disturbance in B cell maturation was dependent on decreased arginine levels, as this phenotype disappeared upon arginine supplementation and was not seen in NO synthase– or ornithine transcarbamoylase–deficient mice. We conclude that arginine deficiency impairs early B cell maturation.

T he efficacy of anti-tumor IgG reflects the balance between opposing signals mediated by activating and inhibitory Fcγ receptors (FcγRs) expressed by effector cells. Here, we show that human malignant melanoma cells express the inhibitory low-affinity Fcγ receptor FcγRIIB1 in 40% of tested metastases. When melanoma cells were grafted in nude mice, a profound inhibition of FcγRIIB1 tumor growth that required the intracytoplasmic region of the receptor was observed. IgG immune complexes (ICs) may be required for this inhibition, since sera from nude mice bearing tumors contained IgG that decreased the proliferation of FcγRIIB1-positive cells in vitro, and tumor development of FcγRIIB1-positive melanoma lines was not inhibited in antibody-defective severe combined immunodeficiency (SCID) mice. Passive immunization of SCID mice with anti–ganglioside G_D2 antibody resulted in significant inhibition of growth of FcγRIIB1-positive tumors in an intracytoplasmic-dependent manner. Altogether, these data suggest that human melanoma cells express biologically active inhibitory FcγRIIB1, which regulates their development upon direct interaction with anti-tumor antibodies. Therefore, FcγR expression on human tumors may be one component of the efficacy of antibody-mediated therapies, and FcγR-positive tumors could be the most sensitive candidates for such treatments.

T here are two well-described syndromes caused by tumor production of parathyroid hormone-related peptide (PTHrP), namely osteolytic bone disease associated with breast cancer and humoral hypercalcemia of malignancy (HHM) that occurs with or without bone metastasis. Both syndromes have been shown experimentally to be inhibited by neutralizing antibodies to PTHrP. In a search for small-molecule inhibitors of PTHrP production or effects, we have identified guanine-nucleotide analogs as compounds that inhibit PTHrP expression by human tumor cells associated with these syndromes. We show in nude athymic murine models that these compounds reduce PTHrP-mediated osteolytic lesions associated with metastatic human breast-cancer cells as well as the degree of hypercalcemia caused by excessive PTHrP production by a squamous-cell carcinoma of the lung. These results suggest that the PTHrP gene promoter may be a suitable target for treating the skeletal effects of malignancy.