M H Ginsberg, Z M Ruggeri, A P Varki
P A Edwards
A R Thompson
Patients with a recently identified subepithelial blistering disease have IgG anti-laminin 5 autoantibodies. To determine if such antibodies can be pathogenic in vivo, we developed and characterized rabbit anti-laminin 5 IgG, and passively transferred these antibodies to neonatal mice. Immune rabbit IgG specifically bound human and murine epidermal basement membranes, immunoblotted and immunoprecipitated all laminin 5 subunits from extracts of human and murine keratinocytes, and showed no reactivity to other keratinocyte proteins or epithelial basement membranes that do not contain laminin 5. Mice (n = 29) receiving purified anti-laminin 5 IgG developed, in a dose-related fashion, circulating anti-laminin 5 antibodies, deposits of rabbit IgG and murine C3 in epidermal basement membranes, and subepidermal blisters of skin and mucous membranes. No alterations developed in controls (n = 14) receiving identical amounts of normal rabbit IgG. Passive transfer of anti-laminin 5 (but not control) IgG to neonatal C5- (n = 3) or mast cell-deficient (n = 3) mice produced subepidermal blisters with the same clinical, histologic, and immunopathologic features as those documented in BALB/c mice. These studies establish an animal model of a human blistering disease that can be used to define disease mechanisms and treatment modalities.
Z Lazarova, C Yee, T Darling, R A Briggaman, K B Yancey
We report a detailed analysis of a B cell defect affecting a patient girl born from first cousin parents, characterized by a severe non-X-linked agammaglobulinemia with a total absence of CD19- cells in the periphery. In the bone marrow, CD19 expression was also highly impaired, resulting in the absence of both B and preB compartments. By contrast, CD34+CD10+, CD34psiL+, and some CD19+CD10+ mostly CD34+ early proB cells were present, although diminished. Semiquantitative RT-PCR analysis performed on mononuclear bone marrow cells indicated that lambda-like, VpreB, Rag-1, Rag-2, and TdT transcripts expressed during proB cell stages were found at normal levels whereas E2A, CD10, Syk, Pax-5, CD19, Igalpha, Igbeta, VH-Cmu, and Vkappa-Ckappa transcripts characteristic of later stages were severely depressed. This phenotype resembles that of Pax-5 knock-out mice, but since the coding sequence of the patient Pax-5 cDNA was shown to be normal, the defect might rather result from an altered regulation of this gene. All these data indicate that the patient suffers from a new genetic defect that results in an arrest of differentiation within the proB cell compartment, i.e., earlier than X-linked agammaglobulinemia, before the onset of Ig gene rearrangements.
E Meffre, F LeDeist, G de Saint-Basile, A Deville, M Fougereau, A Fischer, C Schiff
Vasoactive intestinal peptide (VIP), a major neurotransmitter of peripheral nerves, has been suggested to function in host defense by regulating local human immune function. Indirect evidence has been marshaled that VIP can function as a switch factor for IgA in human Ig isotype recombination. In this study we directly tested the ability of VIP to function as a factor driving human B cells into IgA producing cells by assessing its ability to induce switch circular DNA representing direct mu to alpha switching. In addition we determined the generation of alpha germ-line transcripts and measured the level of IgA protein produced. Stimulation with VIP and CD40 mAb induced IgA production by human IgD+ B cells while VIP or CD40 alone failed to do so. Stimulation of purified IgD+ B cells with VIP plus CD40 mAb induced generation of switch circular DNA representing in vitro driven isotype switching from mu to alpha. CD40 mAb alone induced alpha germ-line transcripts but not IgA switch circles. Thus VIP, a neurogenic factor, can induce alpha-specific switching in CD40-activated human B cells and may thereby play an important role in directing the humoral immune response at mucosal surfaces.
S Fujieda, J A Waschek, K Zhang, A Saxon
Lipopolysaccharide (LPS), residing in the outer membrane of all gram-negative bacteria, is considered a major initiating factor of the gram-negative septic shock syndrome in humans. LPS forms a complex with the LPS binding protein (LBP) in plasma, and LPS-LBP complexes engage a specific receptor, CD14, on the surface of myeloid cells, leading to the production of potent proinflammatory cytokines. The major goal of this study was to test the importance of the CD14 pathway in vivo in a primate model that is similar to human septic shock. Primates were pretreated with one of two different inhibitory anti-CD14 mAbs, then challenged with intravenous endotoxin (375 microg/kg/h) for 8 h. The anti-CD14 treatment regimens were successful in preventing profound hypotension, reducing plasma cytokine levels (TNF-alpha, IL-1beta, IL-6, and IL-8), and inhibiting the alteration in lung epithelial permeability that occurred in animals treated with LPS and an isotype-matched control antibody. These results demonstrate for the first time the importance of the CD14 pathway in a primate model that is similar to human septic shock. Inhibition of the CD14 pathway represents a novel therapeutic approach to treating this life-threatening condition.
D J Leturcq, A M Moriarty, G Talbott, R K Winn, T R Martin, R J Ulevitch
IL-12 is a cytokine detected in active lesions in multiple sclerosis (MS) and promotes the acquisition of a Th1 cytokine profile by CD4+ T cells. Autoreactive T cells recovered from the central nervous system of animals with experimental autoimmune encephalomyelitis (EAE), a disease model for MS, display this phenotype. We demonstrate that human central nervous system-derived microglia, but not astroglia, can produce IL-12 in vitro. Under basal culture conditions, human adult microglia do not express detectable levels of IL-12, although these cells show some degree of activation as assessed by expression of the immunoregulatory surface molecules HLA-DR and B7 as well as low levels of TNF-alpha mRNA. Following activation with LPS, IL-12 p40 mRNA and p70 protein can be readily detected. IL-12 production is preceded by TNF-alpha production and is inhibited by recombinant soluble human TNF receptor (II)-IgG1 fusion protein (shu-TNF-R). These data indicate regulation of IL-12 by an autocrine-dependent feedback loop, providing an additional mechanism whereby shu-TNF-R, now used in clinical trials in MS, may be exerting its effect.
B Becher, V Dodelet, V Fedorowicz, J P Antel
Breast cancer almost invariably metastasizes to bone in patients with advanced disease and causes local osteolysis. Much of the morbidity of advanced breast cancer is a consequence of this process. Despite the importance of the problem, little is known of the pathophysiology of local osteolysis in the skeleton or its prevention and treatment. Observations in patients with bone metastases suggest that breast cancer cells in bone express parathyroid hormone-related protein (PTHrP) more frequently than in soft tissue sites of metastasis or in the primary tumor. Thus, the role of PTHrP in the causation of breast cancer metastases in bone was examined using human breast cancer cell lines. Four of eight established human breast cancer cell lines expressed PTHrP and one of these cell lines, MDA-MB-231, was studied in detail using an in vivo model of osteolytic metastases. Mice inoculated with MDA-MB-231 cells developed osteolytic bone metastasis without hypercalcemia or increased plasma PTHrP concentrations. PTHrP concentrations in bone marrow plasma from femurs affected with osteolytic lesions were increased 2.5-fold over corresponding plasma PTHrP concentrations. In a separate experiment, mice were treated with either a monoclonal antibody directed against PTHrP(1-34), control IgG, or nothing before tumor inoculation with MDA-MB-231 and twice per week for 26 d. Total area of osteolytic lesions was significantly lower in mice treated with PTHrP antibodies compared with mice receiving control IgG or no treatment. Histomorphometric analysis of bone revealed decreased osteoclast number per millimeter of tumor/bone interface and increased bone area, as well as decreased tumor area, in tumor-bearing animals treated with PTHrP antibodies compared with respective controls. These results indicate that tumor-produced PTHrP can cause local bone destruction in breast cancer metastatic to bone, even in the absence of hypercalcemia or increased circulating plasma concentrations of PTHrP. Thus, PTHrP may have an important pathogenetic role in the establishment of osteolytic bone lesions in breast cancer. Neutralizing antibodies to PTHrP may reduce the development of destructive bone lesions as well as the growth of tumor cells in bone.
T A Guise, J J Yin, S D Taylor, Y Kumagai, M Dallas, B F Boyce, T Yoneda, G R Mundy
Nitric oxide (NO), a free radical that is negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by an NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to examine iNOS induction in the rat diaphragm after Escherichia Coli LPS inoculation (1.6 mg/kg i.p.), and its involvement in diaphragmatic contractile dysfunction. Inducible NOS protein and activity could be detected in the diaphragm as early as 6 h after LPS inoculation. 6 and 12 h after LPS, iNOS was expressed in inflammatory cells infiltrating the perivascular spaces of the diaphragm, whereas 12 and 24 h after LPS it was expressed in skeletal muscle fibers. Inducible NOS was also expressed in the left ventricular myocardium, whereas no expression was observed in the abdominal, intercostal, and peripheral skeletal muscles. Diaphragmatic force was significantly decreased 12 and 24 h after LPS. This decrease was prevented by inhibition of iNOS induction by dexamethasone or by inhibition of iNOS activity by N(G)-methyl-L-arginine. We conclude that iNOS was induced in the diaphragm after E. Coli LPS inoculation in rats, being involved in the decreased muscular force.
J Boczkowski, S Lanone, D Ungureanu-Longrois, G Danialou, T Fournier, M Aubier
Smooth muscle cell proliferation and migration is important in arteriosclerosis. In this process, cytokines and growth factors are upregulated and bind to their respective receptors, which in turn stimulate mitogen-activated protein (MAP) kinases. MAP kinases then relay signals to the nucleus that activate quiescent smooth muscle cells. Phosphatases downregulate MAP kinases. We investigated the role of a dual-specificity tyrosine phosphatase, MAP kinase phosphatase-1 (MKP-1), in smooth muscle cell proliferation. MKP-1 expression was high in arterial tissue by Northern analysis, and MKP-1 message was detected mainly in the arterial smooth muscle layer by in situ hybridization. After balloon injury of the rat carotid artery, expression of MKP-1 decreased greatly, whereas that of MAP kinases, especially p44 MAP kinase, increased. The time course of the reduction in MKP-1 message correlated with increased tyrosine phosphorylation and elevated p44 MAP kinase enzymatic activity. In rat arterial smooth muscle cells overexpressing MKP-1, growth was arrested in the G1 phase and entry into the S phase was blocked. A reduction in MKP-1 expression may contribute in part to proliferation of smooth muscle cells after vascular injury, possibly through a decrease in dephosphorylation of p44 MAP kinase.
K Lai, H Wang, W S Lee, M K Jain, M E Lee, E Haber
This study demonstrates that rat islet beta cells constitutively express an apoptotic program which is activated when mRNA or protein synthesis is blocked. Apoptotic beta cells were detectable by electron microscopy after treatment with actinomycin D or cycloheximide. With a fluorescence microscopic assay both agents were found to increase the number of apoptotic beta cells dose- and time-dependently, up to 70% after 1 wk of culture; virtually no apoptotic beta cells occurred in control preparations or in conditions leading to primary necrosis. Thus, survival of beta cells seems dependent on synthesis of proteins which suppress an endogenous suicide program. This mechanism explains earlier observed effects of glucose on survival of cultured beta cells. Glucose is known to dose-dependently increase the percentage of beta cells in active biosynthesis and the percentage that survives during culture. It is now demonstrated that the glucose-induced survival of beta cells cultured for 1 wk results from a dose-dependent reduction in the percentage of beta cells dying in apoptosis (49% at 3 mM glucose, 40% at 6 mM, 9% at 10 mM). Thus, intercellular differences in glucose sensitivity appear responsible for the heterogeneity in beta cell sensitivity to apoptotic conditions. These data indicate that glucose promotes survival of beta cells by activating synthesis of proteins which suppress apoptosis. The present model allows for further investigation of the regulation of apoptosis in beta cells and the identification of agents which induce or prevent beta cell death.
A Hoorens, M Van de Casteele, G Klöppel, D Pipeleers
The NH2-terminal domain of sterol-regulatory element binding protein-1a (SREBP-1a) activates transcription of genes encoding enzymes of cholesterol and fatty acid biosynthesis in cultured cells. This domain is synthesized as part of a membrane-bound precursor that is attached to the nuclear envelope and endoplasmic reticulum. In sterol-depleted cells a two-step proteolytic process releases this NH2-terminal domain, which enters the nucleus and activates transcription. Proteolysis is suppressed by sterols, thereby suppressing transcription. In the current experiments we produce transgenic mice that overexpress a truncated version of human SREBP-1a that includes the NH2-terminal domain but lacks the membrane attachment site. This protein enters the nucleus without a requirement for proteolysis, and therefore it cannot be down-regulated. Expression was driven by the phosphoenolpyruvate carboxykinase (PEPCK) promoter, which gives high level expression in liver. When placed on a low carbohydrate/high protein diet to induce the PEPCK promoter, the transgenic mice developed progressive and massive enlargement of the liver, owing to the engorgement of hepatocytes with cholesterol and triglycerides. The mRNAs encoding 3-hydroxy-3-methylglutaryl CoA (HMG CoA) synthase, HMG CoA reductase, squalene synthase, acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase-1 were all elevated markedly, as was the LDL receptor mRNA. The rates of cholesterol and fatty acid synthesis in liver were elevated 5- and 25-fold, respectively. Remarkably, plasma lipid levels were not elevated. The amount of white adipose tissue decreased progressively as the liver enlarged. These studies indicate that the NH2-terminal domain of SREBP-1a can produce major effects on lipid synthesis and storage in the liver.
H Shimano, J D Horton, R E Hammer, I Shimomura, M S Brown, J L Goldstein
The deposition of anti-dsDNA antibodies in the glomerulus is believed to play a critical role in the pathogenesis of nephritis in SLE. However, an absolute correlation between serum levels of anti-dsDNA antibodies and renal disease has not been found. Recently a glomerular binding assay (GBA) has been developed to detect IgG binding to isolated rat glomeruli. We have used the GBA to study sera from four groups of SLE patients: (A) + anti-dsDNA antibodies, active nephritis; (B) - anti-dsDNA antibodies, active nephritis; (C) + anti-dsDNA antibodies, no nephritis; and (D) - anti-dsDNA antibodies, no nephritis. The serum anti-dsDNA antibodies in group A and group C patients could not be distinguished on the basis of isotype, charge, or cross-reactivity with histones. Nevertheless, the mean intensity of glomerular immunofluorescence was significantly higher in group A than in the three other patient groups and distinguished between patients with serum anti-dsDNA antibodies who had nephritis and those without clinically apparent nephritis. GBA reactivity was unaffected by DNase treatment of sera, but was partially inhibited by preincubation with dsDNA. These findings are consistent with the hypothesis that some anti-dsDNA antibodies cross-react with glomerular components and that the presence of this cross-reactivity is associated with, and may be responsible for, the development of nephritis. In addition, we have identified a group of SLE patients with renal disease and typical renal histopathology and immune deposits who do not have serum anti-dsDNA antibodies or antibodies that directly bind to glomeruli in the GBA. The mechanism of renal immune deposition in these patients remains to be determined.
L Budhai, K Oh, A Davidson
The amino acids encoded at the junctions of T cell receptor (TCR) V and J genes directly interact with MHC bound peptides. However, the regulation of the human TCRBJ gene repertoire has been difficult to analyze, because of the potentially complex number of BJ gene rearrangements. To overcome this problem, we developed a PCR-ELISA method to study BJ gene expression, and compared peripheral T lymphocytes from 12 pairs of monozygotic twins, including 6 rheumatoid arthritis (RA) discordant pairs, and 5 normals. Analyses of the TCRBV5, 13 and 17 gene families, which have been reported to be increased in RA patients, showed: (a) the three TCRBV transcripts have common features of BJ gene usage; (b) TCR transcripts from each TCRBV family display a distinctive BJ gene profile, which is displayed better by CD4+ than CD8+ lymphocytes; (c) the BJ gene repertoires of monozygotic twins are more similar than those of unrelated individuals; and (d) the inflammation of RA does not induce specific changes in the genetically determined pattern of BJ expression. These results indicate that the frequency of expression particular TCRBV-TCRBJ recombinants in human lymphocytes is controlled genetically, and is maintained despite the presence of a chronic inflammatory disease.
T Nanki, H Kohsaka, N Mizushima, W E Ollier, D A Carson, N Miyasaka
It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fusion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP (deltaPLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of activated T cells completely prevented subsequent clinical paralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Prevention of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived functional peptide-MHC complexes in vivo, as well as by a significant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resistant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 administration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyelinating disease.
E A Elliott, H I McFarland, S H Nye, R Cofiell, T M Wilson, J A Wilkins, S P Squinto, L A Matis, J P Mueller
Recent work from one laboratory has shown, in both nonobese diabetic mice and humans, an association between insulin-dependent diabetes mellitus (IDDM) and quantitative difference in MHC class I molecule expression. This reported decrease in MHC class I molecule expression is very controversial in the nonobese diabetic mouse model of IDDM, but to our knowledge, it has not been evaluated by another group in human IDDM. To evaluate this question, we studied 30 patients with IDDM and 30 age- and sex-matched normal controls. MHC class I molecule expression was measured by flow cytometry with conformational-dependent MHC class I mAbs. The mean antigen density of MHC class I molecule expression in IDDM vs. normal control is 454+/-34 vs. 440+/-28 for lymphocytes and 1,440+/-117 vs. 1,494+/- 117 for monocytes, both P > 0.05. Three conformational-dependent MHC class I antibodies showed consistent results. To estimate the biological variation of MHC class I molecule expression in normal controls, we also studied 10 age- and sex-matched normal control pairs. Using X +/-SD of the percentage difference of mean antigen density in the normal control pairs as our definition of normal, we found that 70% (21/30) of IDDM patients had normal, 13% (4/30) of IDDM patients had decreased, and 17% (5/30) of IDDM patients had increased MHC class I molecule expression on lymphocytes. All IDDM patients showed normal MHC class I expression on monocytes. In conclusion, we find that there is no consistent decrease in MHC class I molecule expression on either lymphocytes or monocytes from patients with IDDM. The MHC class I molecule expression observed in IDDM patients is largely within the expected biological variation of MHC class I molecule expression that has been observed in normal controls.
W Hao, P Gladstone, S Engardt, C Greenbaum, J P Palmer
The propeptide sequences of the vitamin K-dependent clotting factors serve as a recognition site for the enzyme gamma-glutamylcarboxylase, which catalyzes the carboxylation of glutamic acid residues at the NH2 terminus of the mature protein. We describe a mutation in the propeptide of Factor IX that results in warfarin sensitivity because of reduced affinity of the carboxylase for the Factor IX precursor. The proband has a Factor IX activity level of > 100% off warfarin and < 1% on warfarin, at a point where other vitamin K-dependent factors were at 30-40% activity levels. Direct sequence analysis of amplified genomic DNA from all eight exons and exon-intron junctions showed a single guanosine-->adenosine transition at nucleotide 6346 resulting in an alanine to threonine change at residue -10 in the propeptide. To define the mechanism by which the mutation resulted in warfarin sensitivity, we analyzed wild-type and mutant recombinant peptides in an in vitro carboxylation reaction. The peptides that were analyzed included the wild-type sequence, the Ala-10-->Thr sequence, and Ala-10-->Gly, a substitution based on the sequence in bone gamma-carboxyglutamic acid protein. Measurement of C02 incorporation at a range of peptide concentrations yielded a Vmax of 343 cpm/min/reaction for the wild-type peptide, and Vmax values of 638 and 726 for A-10T and A-10G respectively, a difference of only twofold. The Km values, on the other hand, showed a 33-fold difference between wild-type and the variants, with a value of 0.29 microM for wild-type, and 10.9 and 9.50 microM, respectively, for A-10T and A-10G. Similar kinetic experiments showed no substantial differences between wild-type and mutant peptides in kinetic parameters of the carboxylase-peptide complexes for reduced vitamin K. We conclude that the major defect resulting from the Factor IX Ala-l0-->Thr mutation is a reduction in affinity of the carboxylase for the mutant propeptide. These studies delineate a novel mechanism for warfarin sensitivity. In addition, the data may also explain the observation that bone Gla protein is more sensitive to warfarin than the coagulation proteins.
K Chu, S M Wu, T Stanley, D W Stafford, K A High
The finding that intracellular expression of the beta-amyloid protein (Abeta) under a neuron-specific promoter led progressively to degeneration and death of neurons in the brains of transgenic mice provides a unique opportunity to utilize this animal model to both understand the mechanism that underlies neuronal cell death and define the complexity of events which may ensue. We observed a correlation between Abeta accumulation in selective neurons and activation of p53, a protein that has been implicated in the induction of apoptosis. Histological and immunohistochemical evaluations of adjacent brain sections suggest that expression of p53 is accompanied by nuclear DNA fragmentation. In certain regions with marked neuronal cell death, extracellular deposition of A(beta) became evident, together with the local activation of astrocytes. Interestingly, the neuritic structures underlying the Abeta deposits showed altered synaptophysin immunoreactivity and morphologic evidence for damage. This transgenic mouse model suggests that intracellular generation of the Abeta protein not only leads to the death of the neuron but may also functionally impair neighboring neurons as well. It further offers a mechanism whereby neuritic plaques may be derived.
F M LaFerla, C K Hall, L Ngo, G Jay
In the last few years, mutiple protein target antigens for immunorecognition by T cells have been identified on human melanoma. How melanoma lesions escape from functional antigen-specific immune recognition remains poorly understood. We have identified the concomitant loss of the immunodominant T cell-defined MART-1/Melan-A antigen and downregulation of the TAP-1 gene in a recurrent metastatic melanoma that was resected in 1993. This phenotype was not observed for an earlier autologous melanoma lesion resected in 1987. The "antigen loss" could be restored in the variant tumor cell line by simultaneously providing both the MART-1/Melan-A gene (by retroviral transfer) and the TAP-1 gene (by a bioballistic approach) resulting in tumor cell sensitivity to MART-1/Melan-A-specific cytotoxic T lymphocytes. This suggests that tumor escape from immune surveillance may have occurred in vivo as a sequential result of (a) antigen loss, and (b) downregulation of the peptide-transporter protein TAP-1 expression by this patient's tumor over a 6-yr period from 1987 to 1993. These results suggest that the characterization of the T cell response to melanoma in individual patients and definition of the immunologically relevant genetic defects in tumors may be required to select the most effective therapeutic strategies for a given patient.
M J Maeurer, S M Gollin, D Martin, W Swaney, J Bryant, C Castelli, P Robbins, G Parmiani, W J Storkus, M T Lotze
End organ ischemia, fragmentation of elastic membranes, and aneurysm formation in patients with giant cell vasculitis results from an inflammation destroying the mural layers of large and medium sized arteries. Although the inflammatory infiltrate extends through all layers of the affected blood vessel, the most pronounced changes involve the intima and the internal elastic lamina. Analysis of the functional profile of tissue infiltrating CD68+ cells demonstrates that different subsets of macrophages can be distinguished. TGFbeta1-expressing CD68+ cells coproduce IL-1beta and IL-6, are negative for inducible nitric oxide synthase (iNOS), and exhibit a strong preference for localization in the adventitia. The adventitial homing of TGFbeta1+ CD68+ cells places them in the vicinity of IFN-gamma secreting CD4+ T cells which also accumulate in the exterior layer of the artery. Conversely, iNOS expressing CD68+ cells are negative for TGFbeta and are almost exclusively found in the intimal layer of the inflamed artery. The intimal-medial junction is the preferred site for 72-kD collagenase expressing CD68+ cells. Thus, TGFbeta1-producing macrophages colocalize with activated CD4+ T cells and home to an area of inflammation which is distant from the site of tissue damage but critical in regulating cellular influx, suggesting that TGFbeta1 functions as a proinflammatory mediator in this disease. iNOS- and 72-kD collagenase-producing macrophages accumulate at the center of pathology implying a role of these products in tissue destruction. These data indicate that the microenvironment controls the topographical arrangement as well as the functional commitment of macrophages.
C M Weyand, A D Wagner, J Björnsson, J J Goronzy
Cardiac glycosides exert a positive inotropic effect by inhibiting sodium pump (Na,K-ATPase) activity, decreasing the driving force for Na+-Ca++ exchange, and increasing cellular content and release of Ca++ during depolarization. Since the inotropic response will be a function of the level of expression of sodium pumps, which are alpha(beta) heterodimers, and of Na+-Ca++ exchangers, this study aimed to determine the regional pattern of expression of these transporters in the heart. Immunoblot assays of homogenate from atria, ventricles, and septa of 14 nonfailing human hearts established expression of Na,K-ATPase alpha1, alpha2, alpha3, beta1, and Na+-Ca++ exchangers in all regions. Na,K-ATPase beta2 expression is negligible, indicating that the human cardiac glycoside receptors are alpha1beta1, alpha2beta1, and alpha3beta1. alpha3, beta1, sodium pump activity, and Na+-Ca++ exchanger levels were 30-50% lower in atria compared to ventricles and/or septum; differences between ventricles and septum were insignificant. Functionally, the EC50 of the sodium channel activator BDF 9148 to increase force of contraction was lower in atria than ventricle muscle strips (0.36 vs. 1.54 microM). These results define the distribution of the cardiac glycoside receptor isoforms in the human heart and they demonstrate that atria have fewer sodium pumps, fewer Na+-Ca++ exchangers, and enhanced sensitivity to inotropic stimulation compared to ventricles.
J Wang, R H Schwinger, K Frank, J Müller-Ehmsen, P Martin-Vasallo, T A Pressley, A Xiang, E Erdmann, A A McDonough
Chronic lymphocytic leukemia (CLL) usually involves the expansion of a clone of CD5+ B cells synthesizing IgM antibodies. These B cells appear to be blocked at the antigen receptor-expressing stage of B cell differentiation and are thought not to undergo an isotype class switch to IgG or IgA production. In vivo and in vitro studies suggest, however, that in some instances terminal differentiation and isotype switching can occur. To test the hypothesis that in vivo isotype class switching occurs in IgM+ B-type CLL cells, we analyzed the PBMC of 19 CLL patients for the presence of transcripts encoding the rearranged CLL V(H)DJ(H) associated with either gamma or alpha H chains. The molecular data indicate that approximately 50% of B-CLL patients have amplifications of IgM+ B cells that undergo an isotype class switch. Switching to IgA appears to occur more often than to IgG; also, switching can involve different IgG subclasses in individual patients. In many instances, these CLL-related gamma and alpha transcripts are much more plentiful than those of normal B cells that produce the same isotype. These switched transcripts do not reveal evidence for the accumulation of significant numbers of new V(H) gene mutations. The cellular data indicate that B cells with lesser amounts of surface membrane IgD and higher IgM/IgD ratios are more likely to undergo this switching process. Furthermore, B cells expressing IgG and IgA of the same idiotype or V(H) family and the same CDR3 length as those of the CLL IgM+ clone can be identified in the blood of patients studied using multiparameter immunofluorescence analyses. Collectively, these data suggest that not all members of a B-CLL clone are frozen at the surface membrane Ig-expressing stage of B cell maturation, and that some members can switch to the production of non-IgM isotypes. The occurrence of switching without the accumulation of V gene mutations indicates that the processes of differentiation and diversification are not linked.
F Fais, B Sellars, F Ghiotto, X J Yan, M Dono, S L Allen, D Budman, K Dittmar, J Kolitz, S M Lichtman, P Schulman, M Schuster, V P Vinciguerra, K Rai, F K Stevenson, P K Gregersen, M Ferrarini, N Chiorazzi
Elevated vascular endothelial growth factor (VEGF) levels are required for ocular and tumor angiogenesis in animal models. Ischemic hypoxia is strongly correlated with increased VEGF expression in these systems and is considered a physiologically relevant stimulus. Because ischemic hypoxia is often followed by reperfusion and reactive oxygen intermediate (ROI) generation, we examined the potential role of ROI in the control of VEGF gene expression. Human retinal pigment epithelial cells exposed to superoxide or hydrogen peroxide rapidly increased VEGF mRNA levels. Superoxide-associated mRNA increases were dose dependent, blocked by antioxidants, and associated with elevated VEGF protein levels in conditioned media. Increases in VEGF mRNA levels were also observed in cultured human melanoma and rat glioblastoma cells with superoxide or hydrogen peroxide. Cycloheximide prevented the ROI-associated increases in VEGF mRNA. Transcriptional inhibition with actinomycin D revealed an inducible increase in VEGF mRNA half-life, but nuclear run-on experiments showed no increase in VEGF transcriptional rate. Reoxygenation of human retinal pigment epithelial cells in vitro and ocular reperfusion in vivo increased retinal VEGF mRNA levels. Antioxidants prevented the reperfusion-associated VEGF mRNA increases in retina. We conclude that ROIs increase VEGF gene expression in vitro and during the reperfusion of ischemic retina in vivo. The ROI-associated increases are mediated largely through increases in VEGF mRNA stability.
M Kuroki, E E Voest, S Amano, L V Beerepoot, S Takashima, M Tolentino, R Y Kim, R M Rohan, K A Colby, K T Yeo, A P Adamis
Bee venom phospholipase A2 (PLA) is the major allergen in bee sting allergy. It displays three peptide and a glycopeptide T cell epitopes, which are recognized by both allergic and non-allergic bee venom sensitized subjects. In this study PLA- and PLA epitope-specific T cell and cytokine responses in PBMC of bee sting allergic patients were investigated before and after 2 mo of rush immunotherapy with whole bee venom. After successful immunotherapy, PLA and T cell epitope peptide-specific T cell proliferation was suppressed. In addition the PLA- and peptide-induced secretion of type 2 (IL-4, IL-5, and IL-13), as well as type 1 (IL-2 and IFN-gamma) cytokines were abolished, whereas tetanus toxoid-induced cytokine production and proliferation remained unchanged. By culturing PBMC with Ag in the presence of IL-2 or IL-15 the specifically tolerized T cell response could be restored with respect to specific proliferation and secretion of the type 1 T cell cytokines, IL-2 and IFN-gamma. In contrast, IL-4, IL-5, and IL-13 remained suppressed. Treatment of tolerized T cells with IL-4 only partially restored proliferation and induced formation of distinct type 2 cytokine pattern. In spite of the allergen-specific tolerance in T cells, in vitro produced anti-PLA IgE and IgG4 Ab and their corresponding serum levels slightly increased during immunotherapy, while the PLA-specific IgE/IgG4 ratio changed in favor of IgG4. These findings indicate that bee venom immunotherapy induces a state of peripheral tolerance in allergen-specific T cells, but not in specific B cells. The state of T cell tolerance and cytokine pattern can be in vitro modulated by the cytokines IL-2, IL-4, and IL-15, suggesting the importance of microenvironmental cytokines leading to success or failure in immunotherapy.
C A Akdis, M Akdis, T Blesken, D Wymann, S S Alkan, U Müller, K Blaser
A severe complication of dengue virus infection, dengue hemorrhagic fever (DHF), is hypothesized to be immunologically mediated and virus-specific cytotoxic T lymphocytes (CTLs) may trigger DHF. It is also likely that dengue virus-specific CTLs are important for recovery from dengue virus infections. There is little available information on the human CD8+ T cell responses to dengue viruses. Memory CD8+CTL responses were analyzed to determine the diversity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult volunteers who had received monovalent, live-attenuated candidate vaccines of the four dengue serotypes. All the donors had specific T cell proliferation to dengue and to other flaviviruses that we tested. CTLs were generated from the stimulated PBMC of all donors, and in the seven donors tested, dengue virus-specific CD8+CTL activity was demonstrated. The nonstructural (NS3 and NS1.2a) and envelope (E) proteins were recognized by CD8+CTLs from six, five, and three donors, respectively. All donors recognized either NS3 or NS1.2a. In one donor who received a dengue 4 vaccine, CTL killing was seen in bulk culture against the premembrane protein (prM). This is the first demonstration of a CTL response against the prM protein. The CTL responses using the PBMC of two donors were serotype specific, whereas all other donors had serotype-cross-reactive responses. For one donor, CTLs specific for E, NS1.2a, and NS3 proteins were all HLA-B44 restricted. For three other donors tested, the potential restricting alleles for recognition of NS3 were B38, A24, and/or B62 and B35.These results indicate that the CD8+CTL responses of humans after immunization with one serotype of dengue virus are diverse and directed against a variety of proteins. The NS3 and NS1.2a proteins should be considered when designing subunit vaccines for dengue.
A Mathew, I Kurane, A L Rothman, L L Zeng, M A Brinton, F A Ennis