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Background: Patient-derived organoid (PDO) models offer potential to transform drug discovery for inflammatory bowel disease (IBD) but are limited by inconsistencies with differentiation and functional characterization. We profiled molecular and cellular features across a range of intestinal organoid models and examined differentiation and establishment of a functional epithelial barrier., Methods: Patient-derived organoids or monolayers were generated from control or IBD patient-derived colon or ileum and were molecularly or functionally profiled. Biological or technical replicates were examined for transcriptional responses under conditions of expansion or differentiation. Cell-type composition was determined by deconvolution of cell-associated gene signatures and histological features. Differentiated control or IBD-derived monolayers were examined for establishment of transepithelial electrical resistance (TEER), loss of barrier integrity in response to a cocktail of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, and prevention of cytokine-induced barrier disruption by the JAK inhibitor, tofacitinib., Results: In response to differentiation media, intestinal organoids and monolayers displayed gene expression patterns consistent with maturation of epithelial cell types found in the human gut. Upon differentiation, both colon- and ileum-derived monolayers formed functional barriers, with sustained TEER. Barrier integrity was compromised by inflammatory cytokines IFN-γ and TNF-α, and damage was inhibited in a dose-dependent manner by tofacitinib., Conclusions: We describe the generation and characterization of human colonic or ileal organoid models capable of functional differentiation to mature epithelial cell types. In monolayer culture, these cells formed a robust epithelial barrier with sustained TEER and responses to pharmacological modulation. Our findings demonstrate that control and IBD patient-derived organoids possess consistent transcriptional and functional profiles that can enable development of epithelial-targeted therapies., (© 2022 Crohn’s & Colitis Foundation. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation.)

IL-33 is a multifunctional cytokine that mediates local inflammation upon tissue damage. IL-33 is known to act on multiple cell types including group 2 innate lymphoid cells (ILC2s), Th2 cells, and mast cells to drive production of Th2 cytokines including IL-5 and IL-13. IL-33 signaling activity through transmembrane ST2L can be inhibited by soluble ST2 (sST2), which acts as a decoy receptor. Previous findings suggested that modulation of IL-13 levels in mice lacking decoy IL-13Rα2, or mice lacking IL-13, impacted responsiveness to IL-33. In this study, we used Il13 -/- mice to investigate whether IL-13 regulates IL-33 activity by modulating the transmembrane and soluble forms of ST2. In Il13 -/- mice, the effects of IL-33 administration were exacerbated relative to wild type (WT). Il13 -/- mice administered IL-33 i.p. had heightened splenomegaly, more immune cells in the peritoneum including an expanded ST2L + ILC2 population, increased eosinophilia in the spleen and peritoneum, and reduced sST2 in the circulation and peritoneum. In the spleen, lung, and liver of mice given IL-33, gene expression of both isoforms of ST2 was increased in Il13 -/- mice relative to WT. We confirmed fibroblasts to be an IL-13-responsive cell type that can regulate IL-33 activity through production of sST2. This study elucidates the important regulatory activity that IL-13 exerts on IL-33 through induction of IL-33 decoy receptor sST2 and through modulation of ST2L + ILC2s., (Copyright © 2021 by The American Association of Immunologists, Inc.)

There continues to be a major need for more effective inflammatory bowel disease (IBD) therapies. IL-13Rα2 is a decoy receptor that binds the cytokine IL-13 with high affinity and diminishes its STAT6-mediated effector functions. Previously, we found that IL-13Rα2 was necessary for IBD in mice deficient in the anti-inflammatory cytokine IL-10. Here, we tested for the first time a therapeutic antibody specifically targeting IL-13Rα2. We also used the antibody and Il13ra2 -/- mice to dissect the role of IL-13Rα2 in IBD pathogenesis and recovery. Il13ra2 - /- mice were modestly protected from induction of dextran sodium sulfate (DSS)-induced colitis. Following a 7-day recovery period, Il13ra2 -/- mice or wild-type mice administered the IL-13Rα2-neutralizing antibody had significantly improved colon health compared to control mice. Neutralizing IL-13Rα2 to increase IL-13 bioavailability promoted resolution of IBD even if neutralization occurred only during recovery. To link our observations in mice to a large human cohort, we conducted a phenome-wide association study of a more active variant of IL-13 (R130Q) that has reduced affinity for IL-13Rα2. Human subjects carrying R130Q reported a lower risk for Crohn's disease. Our findings endorse moving anti-IL-13Rα2 into preclinical drug development with the goal of accelerating recovery and maintaining remission in Crohn's disease patients.

Progressive multifocal leukoencephalopathy (PML) is a rare but serious disease. Caused by the JC virus (JCV), it occurs in individuals with weakened immune systems and is a potential adverse reaction for certain immunomodulatory drugs. The PML Consortium was created to find better methods to predict, prevent, and treat PML. The Consortium brought together the pharmaceutical industry with academic, regulatory, and patient communities to advance research and dialogue on PML through a not-for-profit, collaborative approach involving a grant program, scientific workshops and conferences, and disease awareness efforts. Over nearly a decade, the Consortium contributed to the PML and JCV fields by advancing research, scientific exchange, and awareness of PML. In addition to advancing knowledge and helping to build cross-sector consensus on research priorities, the Consortium's grant program filled a funding gap and brought new investigators into PML and JCV research. Additionally, the Consortium's workshops and conferences created platforms for exchange that drove dialogue on knowledge gaps and future research directions. The Consortium also contributed to the scientific knowledge base with two literature reviews, one on PML treatment studies and a second on T cell deficiencies as a risk factor for PML and the brain as a site for conversion of harmless JCV into a pathogenic virus. Finally, the Consortium addressed a significant information gap with its disease awareness website for healthcare professionals, patients, and caregivers. Beyond its impact on the PML and JCV fields, the PML Consortium is important because it provides a precedent for how the pharmaceutical industry, academic researchers, patient organizations, and government can work together to address rare diseases, in particular rare adverse events. This kind of collaboration could be replicated to speed progress in addressing other rare diseases and adverse events, with significant potential benefits for the scientific, medical, and patient communities. FUNDING: PML Consortium (PML Consortium, Washington, DC).

Background: A major goal of asthma therapy is to achieve disease control, with maintenance of lung function, reduced need for rescue medication, and prevention of exacerbation. Despite current standard of care, up to 70% of patients with asthma remain poorly controlled. Analysis of serum and sputum biomarkers could offer insights into parameters associated with poor asthma control., Objective: To identify signatures as determinants of asthma disease control, we performed proteomics using Olink proximity extension analysis., Methods: Up to 3 longitudinal serum samples were collected from 23 controlled and 25 poorly controlled asthmatics. Nine of the controlled and 8 of the poorly controlled subjects also provided 2 longitudinal sputum samples. The study included an additional cohort of 9 subjects whose serum was collected within 48 hours of asthma exacerbation. Two separate pre-defined Proseek Multiplex panels (INF and CVDIII) were run to quantify 181 separate protein analytes in serum and sputum., Results: Panels consisting of 9 markers in serum (CCL19, CCL25, CDCP1, CCL11, FGF21, FGF23, Flt3L, IL-10Rβ, IL-6) and 16 markers in sputum (tPA, KLK6, RETN, ADA, MMP9, Chit1, GRN, PGLYRP1, MPO, HGF, PRTN3, DNER, PI3, Chi3L1, AZU1, and OPG) distinguished controlled and poorly controlled asthmatics. The sputum analytes were consistent with a pattern of neutrophil activation associated with poor asthma control. The serum analyte profile of the exacerbation cohort resembled that of the controlled group rather than that of the poorly controlled asthmatics, possibly reflecting a therapeutic response to systemic corticosteroids., Conclusions and Clinical Relevance: Proteomic profiles in serum and sputum distinguished controlled and poorly controlled asthmatics, and were maintained over time. Findings support a link between sputum neutrophil markers and loss of asthma control., (© 2018 John Wiley & Sons Ltd.)

Interleukin-4 (IL-4) and IL-13 are critical drivers of immune activation and inflammation in ulcerative colitis, asthma and other diseases. Because these cytokines may have redundant function, dual targeting holds promise for achieving greater efficacy. We have recently described a bifunctional therapeutic targeting IL-4 and IL-13 developed on a novel protein scaffold, generated by combining specific binding domains in an optimal configuration using appropriate linker regions. In the current study, the bifunctional IL-4/IL-13 antagonist was evaluated in the murine oxazolone-induced colitis model, which produces disease with features of ulcerative colitis. The bifunctional IL-4/IL-13 antagonist reduced body weight loss throughout the 7-day course of the model, and ameliorated the increased colon weight and decreased colon length that accompany disease. Colon tissue gene expression was modulated in accordance with the treatment effect. Concentrations of serum amyloid P were elevated in proportion to disease severity, making it an effective biomarker. Serum concentrations of the bifunctional IL-4/IL-13 antagonist were inversely proportional to disease severity, colon tissue expression of pro-inflammatory genes, and serum amyloid P concentration. Taken together, these results define a panel of biomarkers signifying engagement of the IL-4/IL-13 pathway, confirm the T helper type 2 nature of disease in this model, and demonstrate the effectiveness of dual cytokine blockade., (© 2014 John Wiley & Sons Ltd.)

IL-4 and IL-13 comprise promising targets for therapeutic interventions in asthma and other Th2-associated diseases, but agents targeting either IL-4 or IL-13 alone have shown limited efficacy in human clinical studies. Because these cytokines may involve redundant function, dual targeting holds promise for achieving greater efficacy. We describe a bifunctional therapeutic targeting IL-4 and IL-13, developed by a combination of specific binding domains. IL-4-targeted and IL-13-targeted single chain variable fragments were joined in an optimal configuration, using appropriate linker regions on a novel protein scaffold. The bifunctional IL-4/IL-13 antagonist displayed high affinity for both cytokines. It was a potent and efficient neutralizer of both murine IL-4 and murine IL-13 bioactivity in cytokine-responsive Ba/F3 cells, and exhibited a half-life of approximately 4.7 days in mice. In a murine model of ovalbumin-induced ear swelling, the bifunctional molecule blocked both the IL-4/IL-13-dependent early-phase response and the IL-4-dependent late-phase response. In the ovalbumin-induced lung inflammation model, the bifunctional IL-4/IL-13 antagonist reduced the IL-4-dependent rise in serum IgE titers, and reduced IL-13-dependent airway hyperresponsiveness, lung inflammation, mucin gene expression, and serum chitinase responses. Taken together, these findings demonstrate the effective dual blockade of IL-4 and IL-13 with a single agent, which resulted in the modulation of a more extensive range of endpoints than could be achieved by targeting either cytokine alone.

Human studies using Abs to two different, nonoverlapping epitopes of IL-13 suggested that epitope specificity can have a clinically significant impact on clearance of IL-13. We propose that Ab modulation of IL-13 interaction with IL-13Rα2 underlies this effect. Two Abs were administered to healthy subjects and mild asthmatics in separate dose-ranging studies and allergen-challenge studies. IMA-638 allows IL-13 interaction with IL-13Rα1 or IL-13Rα2 but blocks recruitment of IL-4Rα to the IL-13/IL-13Rα1 complex, whereas IMA-026 competes with IL-13 interaction with IL-13Rα1 and IL-13Rα2. We found ∼10-fold higher circulating titer of captured IL-13 in subjects treated with IMA-026 compared with those administered IMA-638. To understand how this difference could be related to epitope, we asked whether either Ab affects IL-13 internalization through cell surface IL-13Rα2. Humans inducibly express cell surface IL-13Rα2 but lack the soluble form that regulates IL-13 responses in mice. Cells with high IL-13Rα2 expression rapidly and efficiently depleted extracellular IL-13, and this activity persisted in the presence of IMA-638 but not IMA-026. The potency and efficiency of this clearance pathway suggest that cell surface IL-13Rα2 acts as a scavenger for IL-13. These findings could have important implications for the design and characterization of IL-13 antagonists.

Rationale: Extensive evidence in animal models supports a role for IL-13 in the pathobiology of asthma. IMA-638 and IMA-026 are fully humanized IgG(1) antibodies that bind to different epitopes and neutralize IL-13 bioactivity., Objectives: We hypothesized that anti-IL-13 treatment would inhibit allergen-induced late-phase asthmatic responses, airway hyperresponsiveness, and inflammation in subjects with asthma., Methods: Fifty-six subjects with mild, atopic asthma were recruited for two double-blind, randomized, placebo-controlled, parallel group trials to compare IMA-638 and IMA-026 IL-13 antibody treatments with placebo treatment. Drug was administered on Days 1 and 8, and allergen challenges were performed on Days 14 and 35. The primary outcome variable was the late-phase area under the curve (AUC), and secondary outcome variables were the early- and late-phase maximum percent fall in FEV(1), early AUC, allergen-induced shift in airway hyperresponsiveness, and sputum eosinophils., Measurements and Main Results: The treatment difference with IMA-638 on Day 14 was -19.1 FEV(1) × hour (95% confidence interval: -36.2, -1.9) for the allergen-induced early AUC and -23.8 FEV(1) × hour (95% confidence interval: -46.4, -1.2) for the late AUC (both P < 0.05), but this effect was lost by Day 35. Treatment with IMA-026 did not attenuate the asthmatic responses on Day 14 or Day 35. There was no effect of either antibody on allergen-induced airway hyperresponsiveness or sputum eosinophils. The frequency of adverse events after administration of the IL-13 antibodies was similar to placebo., Conclusions: IL-13 has a role in allergen-induced airway responses in humans. Further study is required to determine whether anti-IL-13 monoclonal antibodies will be beneficial clinically.

IL-13 is a Th2 cytokine that has been shown to be an important mediator of airway inflammation contributing to asthma lesions. Given its proposed role in asthma, measurements of this cytokine in serum may provide insights into disease mechanisms, progression and pharmacodynamic effects of IL-13 targeted therapeutics. However, current commercially available ELISA immunoassays are frequently unable to detect baseline concentrations of IL-13 in serum from healthy individuals, which are below the limit of detection. Here we describe the use of the novel microparticle-based Erenna IL-13 human immunoassay (Singulex, Inc.), which utilizes proprietary antibodies and single molecule counting technology, to quantify IL-13 from 100 microL of serum from apparently healthy subjects and clinically defined symptomatic and asymptomatic asthma subjects. The lower limit of quantification of the Erenna assay was validated at 0.07 pg/mL and the assay detected baseline concentrations of IL-13 in 98% of serum samples tested. The calibration curve showed good precision over the entire linear range of 0.07-50 pg/mL, with inter-assay imprecision <10% CV except at the lowest concentration tested (<15%). The intra- and inter-assay imprecision of spiked serum samples containing three different IL-13 concentrations (2, 8, and 25 pg/mL) ranged from 2.2-2.4% and 6.1-6.8%, respectively. Using the Erenna IL-13 assay, we observe that serum IL-13 concentrations range from <0.07-1.02 pg/mL in apparently healthy subjects (N=60) with similar ranges in asymptomatic (0.07-0.66 pg/mL, N=26) and symptomatic (<0.07-1.26 pg/mL, N=96) asthma subjects. The Erenna immunoassay improved sensitivity by over two full logs compared to previous ELISA methods, while using smaller sample volumes. In addition, the Erenna assay reliably measured IL-13 in endogenous and spiked human serum samples that were not quantifiable using other methods. Taken together, these results show that this novel assay offers a significant improvement over previous methods for high-sensitive quantitative measurement of IL-13 in human serum samples obtained from both apparently healthy and asthmatic subjects, and can be used in future clinical studies to accurately measure concentrations of this cytokine prior to and following drug therapy in human serum.

In asthma, mast cells infiltrate the airway smooth muscle cell layer and secrete proinflammatory and profibrotic agents that contribute to airway remodeling. To study the effects of mast cell activation on smooth muscle cell-dependent matrix contraction, we developed coculture systems of human airway smooth muscle cells (HASM) with primary human mast cells derived from circulating progenitors or with the HMC-1 human mast cell line. Activation of primary human mast cells by IgE receptor cross-linking or activation of HMC-1 cells with C5a stimulated contraction of HASM-embedded collagen gels. Contractile activity could be transferred with conditioned medium from activated mast cells, implicating involvement of soluble factors. Cytokines and proteases are among the agents released by activated mast cells that may promote a contractile response. Both IL-13 and IL-6 enhanced contraction in this model and the activity of IL-13 was ablated under conditions leading to expression of the inhibitory receptor IL-13Ralpha2 on HASM. In addition to cytokines, matrix metalloproteinases (MMPs), and serine proteases induced matrix contraction. Inhibitor studies suggested that, although IL-13 could contribute to contraction driven by mast cell activation, MMPs were critical mediators of the response. Both MMP-1 and MMP-2 were strongly expressed in this system. Serine proteases also contributed to contraction induced by mast cell-activating agents and IL-13, most likely by mediating the proteolytic activation of MMPs. Hypercontractility is a hallmark of smooth muscle cells in the asthmatic lung. Our findings define novel mechanisms whereby mast cells may modulate HASM-driven contractile responses.

Mast cell-fibroblast interactions may contribute to fibrosis in asthma and other disease states. Fibroblast contraction is known to be stimulated by coculture with the human mast cell line, HMC-1, or by mast cell-derived agents. Matrix metalloproteinases (MMPs) can also mediate contraction, but the MMP-dependence of mast cell-induced fibroblast contractility is not established, and the consequences of mast cell activation within the coculture system have not been fully explored. We demonstrate that activation of primary human mast cells (pHMC) with IgE receptor cross-linking, or activation of HMC-1 with C5a, enhanced contractility of human lung fibroblasts in a three-dimensional collagen lattice system. This enhanced contractility was inhibited by the pan-MMP antagonist, batimastat, and was transferrable in the conditioned medium of activated mast cells. Exogenously added MMPs promoted gel contraction by mediating the proteolytic activation of latent transforming growth factor-beta (TGF-beta). Consistent with this, fibroblast contraction induced by mast cell activation was enhanced by addition of excess latent TGF-beta to the cultures. Batimastat inhibited this response, suggesting that MMPs capable of activating latent TGF-beta were released following mast cell activation in coculture with fibroblasts. Collagen production was also stimulated by activated mast cells in an MMP-dependent manner. MMP-2 and MMP-3 content of the gels increased in the presence of activated mast cells, and inhibition of these enzymes blocked the contractile response. These findings demonstrate the MMP dependence of mast cell-induced fibroblast contraction and collagen production.

Interleukin-13 (IL-13) is a critical mediator of asthma pathology. On B cells, monocytes, epithelial cells, and smooth muscle cells, IL-13 acts through the IL-13Ralpha1/IL-4Ralpha complex to directly induce activation responses that contribute to atopic disease. In human populations, genetic polymorphisms in IL-13, its receptor components, or the essential signaling element STAT6, have all been associated with increased risk of atopy and asthma. Animal studies using IL-13 deficient mice, IL-13 transgenic animals, and IL-13 neutralization strategies have confirmed an essential role for this cytokine in driving major correlates of asthma pathology, including airway hyperresponsiveness (AHR), lung eosinophilia, mucus generation, and fibrosis. Ongoing studies continue to define both overlapping and distinct roles for IL-13 and the related cytokine, IL-4, in promoting asthmatic changes. Furthermore, new evidence concerning the role of the "decoy" receptor, IL-13Ralpha2, has prompted re-evaluation of the receptor forms that underlie the numerous activities of IL-13. In this review, we summarize the essential role of IL-13 in asthma, compare the relative contributions of IL-13 and IL-4 to key aspects of the asthmatic phenotype, and outline novel therapeutic strategies to target this critical cytokine.

Interleukin (IL)-13 is a key cytokine driving allergic and asthmatic responses and contributes to airway inflammation in cynomolgus monkeys after segmental challenge with Ascaris suum antigen. IL-13 bioactivity is mediated by a heterodimeric receptor (IL-13Ralpha1/IL-4Ralpha) and can be inhibited in vitro by targeting IL-13 interaction with either chain. However, in cytokine systems, in vitro neutralization activity may not always predict inhibitory function in vivo. To address the efficacy of two different IL-13 neutralization mechanisms in a primate model of atopic disease, two humanized monoclonal antibodies to IL-13 were generated, with highly homologous properties, differing in epitope recognition. Ab01 blocks IL-13 interaction with IL-4Ralpha, and Ab02 blocks IL-13 interaction with IL-13Ralpha1. In a cynomolgus monkey model of IgE responses to A. suum antigen, both Ab01 and Ab02 effectively reduced serum titers of Ascaris-specific IgE and diminished ex vivo Ascaris-triggered basophil histamine release, assayed 8 weeks after a single administration of antibody. The two antibodies also produced comparable reductions in pulmonary inflammation after lung segmental challenge with Ascaris antigen. Increased serum levels of IL-13, lacking demonstrable biological activity, were seen postchallenge in animals given either anti-IL-13 antibody but not in control animals given human IgG of irrelevant specificity. These findings demonstrate a potent effect of IL-13 neutralization on IgE-mediated atopic responses in a primate system and show that IL-13 can be efficiently neutralized by targeting either the IL-4Ralpha-binding epitope or the IL-13Ralpha1-binding epitope.

Interleukin-13 (IL-13) is a critical mediator of pulmonary pathology associated with asthma. Drugs that block the biological function of IL-13 may be an effective treatment for asthma. IL-13 signals by forming a ternary complex with IL-13Ralpha1 and IL-4R. Genetic variants of IL-13 and of its receptor components have been linked to asthma. One in particular, IL-13R110Q, is associated with increased IgE levels and asthma. We characterized the interactions of the binary complexes composed of IL-13 or IL-13R110Q with IL-13Ralpha1 and the ternary complexes composed of IL-13 or IL-13R110Q and IL-13Ralpha1 with IL-4R using surface plasmon resonance and time-resolved fluorescence resonance energy transfer (TR-FRET). By both biophysical methods, we found no differences between IL-13 and IL-13R110Q binding in either the binary or the ternary complex. IL-4R bound to the IL-13/IL-13Ralpha1 complex with slow on and off rates, resulting in a relatively weak affinity of about 100nM. We developed a TR-FRET assay targeting the interaction between the IL-4R and the binary complex. Two antibodies with known binding epitopes to IL-13 that block binding to either IL-13Ralpha1 or IL-4R inhibited the TR-FRET signal formed by the ternary complex. This assay will be useful to identify and characterize inhibitory molecules of IL-13 function.

Mast cell activation involves the rapid release of inflammatory mediators, including histamine, from intracellular granules. The cells are capable of regranulation and multiple rounds of activation. The goal of this study was to determine if there are changes in the content of pre-formed mast cell mediators after a round of activation. After 24 h, the histamine content of bone marrow-derived mast cells (BMMC), but not that of peritoneal mast cells, exceeded the amount in resting cells. Accumulation of histamine in BMMC peaked at 72 h of activation, and returned toward preactivation levels by 96 h. The increase in histamine content was accompanied by an increase in the gene expression of histidine decarboxylase. No increases in beta hexosaminidase or murine mast cell protease-6 were observed. These findings indicate that BMMC respond to activation by increasing total cell-associated histamine content. This increase may be important to the response of these cells upon subsequent exposure to antigens.

Protein kinase C theta (PKCtheta) is expressed in NK cells, but its functional role has not been defined. Here, we demonstrate involvement of PKCtheta in IL-12-induced NK cell IFN-gamma production. NK cells from PKCtheta(-/-) mice produced less IFN-gamma in response to IL-12 than those from wild-type (WT) mice. IL-12-induced NK cell cytotoxicity was unaffected, and NK cells from PKCtheta(-/-) mice did not display reduced IFN-gamma production in response to IL-18, indicating a specific role for PKCtheta in IL-12-induced IFN-gamma production. Under the conditions tested, T cells did not produce IFN-gamma in response to IL-12 or affect the ability of NK cells to produce the cytokine. PKCtheta deficiency did not affect NK cell numbers, granularity, viability, or cytotoxic activity in response to polyinosinic:polycytydylic acid. NK cells from PKCtheta(-/-) mice exhibited normal expression of IL-12Rbeta1 and STAT4 proteins and normal induction of STAT4 phosphorylation in response to IL-12. Phosphorylation of threonine 538 within the catalytic domain of PKCtheta was detectable in NK cells from WT mice but was not enhanced by IL-12. Transcription of IFN-gamma increased similarly in NK cells from WT and PKCtheta(-/-) mice in response to IL-12, and there was no difference in IFN-gamma mRNA stability. Taken together, these findings indicate a role for PKCtheta in the post-transcriptional regulation of IL-12-induced IFN-gamma production.

Background: IL-13 plays a key regulatory role in asthmatic responses and immunity to parasitic infection. In vivo, IL-13R-alpha2 is a critical modulator of IL-13 bioactivity. When inducibly expressed on the surface of fibroblasts and other cell types under inflammatory conditions, IL-13R-alpha2 contributes to resolution of IL-13 responses. A soluble form of IL-13R-alpha2 (sIL-13R-alpha2) can be detected in murine circulation, and functions as a regulator of IL-13 bioactivity. In humans, sIL-13R-alpha2 has been more difficult to detect. Recently, novel assay systems have been described to quantitate sIL-13R-alpha2 in human circulation, and revealed unexpectedly high levels of sIL-13R-alpha2 in healthy subjects., Objective: To verify sIL-13R-alpha2 quantitation in human plasma samples under stringent conditions of signal verification and false-positive detection., Methods: A standard ELISA protocol was evaluated for specificity using false-positive detection reagents. A more stringent ELISA protocol was developed by optimizing the composition of blocking and dilution buffers., Results: Using the stringent assay protocol, endogenous sIL-13R-alpha2 was undetectable in plasma samples from a total of 120 asthmatics and 20 healthy subjects, and in bronchoalveolar lavage fluid from 10 asthmatics and eight healthy subjects undergoing allergen challenge., Conclusion: These results underscore the necessity to perform rigorous assay controls in the biological matrix to be tested. Because the soluble form could not be demonstrated, our findings question a role for sIL-13R-alpha2 in the regulation of IL-13 bioactivity, and highlight the potentially important contribution of the membrane-bound form of IL-13R-alpha2 in humans.

Background: Airway inflammation is a hallmark feature of asthma and a driver of airway hyperresponsiveness. IL-13 is a key inducer of airway inflammation in rodent models of respiratory disease, but a role for IL-13 has not been demonstrated in primates., Objective: We sought to test the efficacy of a neutralizing antibody to human IL-13 in a cynomolgus monkey model of lung inflammation., Methods: Using cynomolgus monkeys (Macaca fascicularis) that are sensitized to Ascaris suum through natural exposure, we developed a reproducible model of acute airway inflammation after segmental A suum antigen challenge. This model was used to test the in vivo efficacy of mAb13.2, a mouse mAb directed against human IL-13, and IMA-638, the humanized counterpart of mAb13.2. Bronchoalveolar lavage (BAL) cells and BAL fluid were collected before and after antigen challenge and assayed for cellular content by means of differential count., Results: Total BAL cell count, eosinophil number, and neutrophil number were all reduced in animals treated with mAb13.2 or IMA-638 compared with values in control animals that were untreated, given saline, or treated with human IgG of irrelevant specificity. In addition, levels of eotaxin and RANTES in BAL fluid were reduced in anti-IL-13-treated animals compared with levels seen in control animals., Conclusion: These findings support a role for IL-13 in maintaining lung inflammation in response to allergen challenge in nonhuman primates., Clinical Implications: IL-13 neutralization with a specific antibody could be a useful therapeutic strategy for asthma.

IL-13 contributes to airway hyperresponsiveness, mucus secretion, inflammation, and fibrosis, suggesting that it plays a central role in asthma pathogenesis. Neutralization of IL-13 with sIL-13Ralpha2-Fc (sIL-13R) reduces allergen-induced airway responses in rodent models of respiratory disease, but its efficacy in a large animal model has not been previously reported. In this study, we determined whether two different strategies for IL-13 neutralization modified experimental asthma in sheep. Sheep with natural airway hypersensitivity to Ascaris suum antigen were treated intravenously either with sIL-13R, a strong antagonist of sheep IL-13 bioactivity in vitro, or with IMA-638 (IgG1, kappa), a humanized antibody to human IL-13. Higher doses of IMA-638 were used because, although it is a potent antagonist of human IL-13, this antibody has 20 to 30 times lower binding and neutralization activity against sheep IL-13. Control animals received human IgG of irrelevant specificity. Sheep were treated 24 h before inhalation challenge with nebulized A. suum. The effects on antigen-induced early and late bronchial responses, and antigen-induced hyperresponsiveness, were assessed. Both sIL-13R and IMA-638 provided dose-dependent inhibition of the antigen-induced late responses and airway hyperresponsiveness. The highest dose of IMA-638 also reduced the early phase response. These findings suggest that IL-13 contributes to allergen-induced airway responses in this sheep model of asthma, and that neutralization of IL-13 is an effective strategy for blocking these A. suum-induced effects.

IL-4 and IL-13 are potent cytokines that drive production of IgE, which is critical to the development of atopic disease. In this study, we directly compared IgE generation and IgE-dependent mast cell effector function in mouse strains lacking IL-4, IL-13, IL-4 + IL-13, or their common receptor component, IL-4Ralpha. Although serum IgE was undetectable under resting conditions in most animals deficient in one or both cytokines, peritoneal mast cells from mice lacking IL-4 or IL-13 had only partial reductions in surface IgE level. In contrast, peritoneal mast cells from IL-4/13(-/-) and IL-4Ralpha(-/-) animals were severely deficient in surface IgE, and showed no detectable degranulation following treatment with anti-IgE in vitro. Surprisingly, however, intradermal challenge with high concentrations of anti-IgE Ab induced an ear-swelling response in these strains, implying some capacity for IgE-mediated effector function in tissue mast cells. Furthermore, upon specific immunization with OVA, both IL-4/IL-13(-/-) and IL-4Ralpha(-/-) mice produced detectable levels of serum IgE and Ag-specific IgG1, and generated strong ear-swelling responses to intradermal administration of anti-IgE. These findings suggest that a mechanism for IgE production exists in vivo that is independent of IL-4 or IL-13.

In human atopic disease, IgE sensitizes the allergic response, while IgG4 is protective. Because IL-4 and IL-13 trigger switch recombination to both IgE and IgG4, additional agents must regulate the balance between these isotypes to influence susceptibility or tolerance to atopy. In this report, we define in vitro conditions leading to activation or inhibition of human IgE and IgG4 production by IL-21. IL-21 reduced IL-4-driven IgE synthesis by mitogen-stimulated human PBMC. IL-21 inhibition of human IgE production was not a direct effect on B cells, was not seen following B cell activation with IL-13, and was overcome by CD40 ligation. Neither IFN-gamma, IL-10, IL-12, CD40L expression, nor apoptosis was responsible for the inhibitory effect. In contrast, IL-21-stimulated secretion of IgG4 from PBMC. Our findings indicate that IL-21 may influence the production of both human IgE and IgG4, and thus contribute to the regulation of atopic reactions.

IFNalpha/beta, IL-12, and IL-15 regulate NK cell activation and expansion, but signals triggering resolution of the NK response upon induction of adaptive immunity remain to be defined. We now report that IL-21, a product of activated T cells, may serve this function. Mice lacking IL-21R (IL-21R(-/-)) had normal NK cell development but no detectable responses to IL-21. IL-21 enhanced cytotoxic activity and IFNgamma production by activated murine NK cells but did not support their viability, thus limiting their duration of activation. Furthermore, IL-21 blocked IL-15-induced expansion of resting NK cells, thus preventing the initiation of further innate responses. In contrast, IL-21 enhanced the proliferation, IFNgamma production, and cytotoxic function of CD8(+) effector T cells in an allogeneic MLR. These observations suggest that IL-21 promotes the transition between innate and adaptive immunity.

The treatment of autoimmune diseases by targeted down-regulation of autoantigen-specific cells has been accomplished by the administration of high doses of autoantigen. We performed direct comparisons between injection of myelin basic protein peptide and administration by several nonparenteral routes to determine whether route impacted benefit in the treatment of murine allergic encephalomyelitis, a model for multiple sclerosis. The range of effective peptide doses spanned over 1000-fold, and route of delivery played a major role in determining optimal dose. The oral route of administration was the least effective, requiring at least 50- to 100-fold more antigen than subcutaneous injection, which in turn required at least 10-fold more antigen than delivery of peptide to the lung using an intratracheal instillation. Intratracheal delivery was also considerably more effective than inhalation of peptide, and, unlike inhalation, resulted in obvious penetration of delivered material deep into the lung. The increase in therapeutic efficacy did not appear to result from slower systemic delivery of antigen. Accumulation of peptide on antigen presenting cells in the spleen and in the brain was less efficient using the intratracheal route of administration compared to subcutaneous injection, implicating a special role for the lung microenvironment in the induction of immune nonresponsiveness., (Copyright 2000 Academic Press.)

Experimental autoimmune encephalomyelitis (EAE) can be effectively treated during disease exacerbation by administration of a peptide corresponding to the major T cell epitope of myelin basic protein (MBP), but the mechanism by which T cell tolerance leads to clinical improvement is not well-defined. Acute exacerbations of EAE are accompanied by an infiltration of blood-borne leukocytes into the brain and spinal cord, where they mediate inflammation and demyelination. To investigate peptide effects on infiltrating cells, we collected cerebrospinal fluid (CSF) from (PL/JxSJL)F1 mice with MBP-induced EAE. Pleiocytosis by lymphocytes, neutrophils, and macrophages was seen throughout the course of relapsing-remitting disease. A single administration of the MBP peptide analog, Ac1-11[4Y], reduced disease severity, accompanied by a dramatic and selective loss of neutrophil pleiocytosis. A longer course of peptide therapy resulted in complete recovery from clinical signs of disease, and decreased pleiocytosis by all cell types. Clinical severity throughout the course of disease and therapy was directly related to the degree of infiltration by neutrophils and macrophages, and the clinical improvement following peptide therapy was accompanied by decreased central nervous system (CNS) expression of chemoattractants for these cell types. These observations support a model of disease exacerbation mediated by phagocytic cellular infiltration under the ultimate control of T cell-derived factors, amenable to treatment by down-regulation of the T cell activation state.

Canis familiaris allergen 1 (Can f 1) and Canis familiaris allergen 2 (Can f 2) are the two major allergens present in dog dander extracts. We now report the isolation of cDNAs encoding both proteins and present their nucleotide and deduced amino acid sequences. Can f 1, produced by tongue epithelial tissue, has homology with the von Ebner's gland (VEG) protein, a salivary protein not previously thought to have allergenic properties. Can f 2, produced by tongue and parotid gland, has homology with mouse urinary protein (MUP), a known allergen. Both VEG protein and MUP are members of the lipocalin family of small ligand-binding proteins. Recombinant forms of Can f 1 and Can f 2 were produced and tested for immunoglobulin E (IgE) reactivity. Among dog-allergic subjects, 45% had IgE directed exclusively to rCan f 1, and 25% had IgE to both rCan f 1 and rCan f 2. In addition, both recombinant proteins were able to cross-link IgE and elicit histamine release from peripheral blood leucocytes in vitro. These findings confirm that Can f 1 and Can f 2 are major and minor dog allergens, respectively, and demonstrate that recombinant forms of dog allergens retain at least some IgE-binding epitopes.

To understand whether the distinct VHDJH gene utilization by natural polyreactive Abs reflects the developmentally restricted Ig VHDJH rearrangements putatively expressed by B-1 cells, we generated 11 (8 IgM, 1 IgG3, 2 IgA1), 7 (6 IgM, 1 IgG1), and 7 (2 IgM, 3 IgG1, 2 IgG3) mAb-producing lines using B-1a (surface CD5+, CD45RAlow), B-1b (surface CD5-, CD45RAlow, CD5 mRNA+), and B-2 (surface CD5-, CD45RAhigh, CD5 mRNA-) cells, respectively, sorted from adult human peripheral blood. Most B-1a and B-1b, but no B-2, cell-derived mAbs were polyreactive; i.e., they bound different self and foreign Ags with different affinities. B-1a and B-2 mAbs preferentially utilized VH4 (p = 0.003) and VH3 (p = 0.010) genes, respectively. All three mAb populations utilized DXP, DLR, DN DH genes, and JH6, but no mAb utilized DHQ52. There were fewer unencoded nucleotide (N) additions in the VHDJH junctions of B-1b (3.00 +/- 2.52, mean +/- SD) than of B-1a (12.45 +/- 3.93, p = 1.23 x 10(-5)) or B-2 (8.29 +/- 4.75, p = 0.020) mAbs. Partly due to the fewer N additions and a paucity of D-D fusions, the B-1b mAb CDR3s were significantly shorter than the B-1a mAb CDR3s (p = 0.013), which contained a nonrandom Tyr distribution (p = 0.003). Finally, all but two B-1 cell-derived mAbs were mutated, in a fashion similar to that of the Ag-selected B-2 mAbs. Thus, in the human adult, B-1 cells that make natural polyreactive Abs may not be representative of the predominantly B-1 developmental waves of colonization of the fetal and neonatal B cell repertoires, and are somatically selected.

Interleukins 3 and 5 and GM-CSF enhance histamine release from basophils triggered by various stimuli. In this report, we describe a subset of allergic patients whose basophils release histamine in response to allergen only when primed with cytokine. In the absence of cytokine, there is no detectable response to allergen. These patients, who represent 4-13% of the allergic population, cannot be distinguished by skin test reactivity or severity of allergic symptoms. Allergen nonreleasers tend to have lower titers of allergen-specific IgE than the majority of atopic subjects, but this difference is not significant (average titer of 29.8 for nonreleasers vs. 188 for typical allergies; p = 0.15). They release histamine normally with anti-IgE and with fMLP, indicating that basophils are responsive to signalling through the IgE receptor, and there is no intrinsic defect in degranulation. Thus, in these patients, the IgE-mediated release of inflammatory mediators from basophils is dependent on, rather than merely enhanced by, T cell cytokines. The relationship between these patients and the previously described anti-IgE 'nonreleasers' is discussed.

As a potent inducing agent for IgE production and differentiation factor for allergen-specific Th2 cells, IL-4 is a key regulatory cytokine both in the pathogenesis of allergic disease and in the ongoing allergic response. The assay of in vitro IL-4 production has often been used to compare the allergen responses of T cells isolated from atopic and non-atopic subjects. Because peripheral blood basophils also have the capacity to respond to specific allergen by producing IL-4, we investigated the relative contribution of these two cell types to IL-4 production in allergen-stimulated primary cultures. Among unfractionated peripheral blood mononuclear cells (PBMC), the major producers of detectable IL-4 in primary in vitro cultures were found to be basophils based on: (i) an allergen dose-response corresponding closely to that required for basophil histamine release and lower than that required for T cell activation; (ii) a rapid time course for IL-4 production (detectable at 3 h), inconsistent with the typical activation requirements of fresh T cells; (iii) the production of comparable levels of IL-4 in cultures stimulated with allergen or anti-IgE; and (iv) the complete loss of detectable IL-4 production following specific depletion of basophils from PBMC. The T cells in these cultures were functionally able to produce IL-4, as demonstrated by mitogen activation of basophil-depleted PBMC. These findings demonstrate that although IL-4 production in primary in vitro cultures can be used as a sensitive indicator of allergen responsiveness, the accurate interpretation of this result requires identification of the responding cell type. Furthermore, these findings raise the possibility that basophil production of IL-4 early in the course of allergen stimulation may shape subsequent T cell responses both in vivo and in vitro.

Background: The production of specific IgE, which underlies the allergic response, may be a normal correlate of the immune response to a certain class of antigen (allergens), or could represent a unique response driven by regulatory signals that are absent in non-allergic individuals. If atopic subjects do possess a regulatory environment favoring IgE production, they may display not only allergen-specific IgE, but also higher levels of total IgE and higher frequencies of IgE-producing B lymphocytes., Objective: To address the contribution of antibody-producing cell number to the circulating IgE titre in atopic vs non-atopic subjects., Methods: Frequency determination by limiting dilution of EBV transformants and Poisson distribution analysis. Titres of total and allergen-specific IgM, IgG, and IgE by specific ELISA., Results: In contrast to findings reported by others, the atopic subjects had a significantly higher frequency of IgE-producing B cells than non-atopics (0.79% of total Ig-producing cells, as compared with 0.17% for the control group; P < 0.01), suggesting that one factor contributing to the high plasma IgE titres in atopic subjects is the high frequency of B lymphocytes with the potential to produce IgE. Although only the atopic subjects produced allergen-specific IgE, the frequency of specific IgE-producing B cells was undetectable in both groups., Conclusion: Atopic subjects have higher frequencies of IgE-producing B cell precursors than non-atopics. A correlation exists between IgE-producing B cell frequency and levels of circulating IgE.

Allergy to the house dust mite Dermatophagoides pteronyssinus is mediated by IgE to the major allergens Der p I and Der p II in the majority of mite-allergic patients. In recent years, standardized preparations of D. pteronyssinus, commercially available from several sources, have become widely used for the diagnosis and immunotherapy of mite allergy. As standardization implies uniformity of allergen composition and potency, we directly compared the absolute and relative quantities of Der p I and Der p II in six different commercial standardized extracts of D. pteronyssinus. Our findings reveal variability in levels of both Der p I and Der p II, producing ratios of Der p I/Der p II ranging from 1.1/1 to 6/1. Although the content of minor allergens in the extracts was not evaluated here, their contribution to the overall reactivity of mite-allergic patients to the commercial extracts was judged to be minimal. This was demonstrated by showing that plasma depleted of reactivity to both Der p I and Der p II had virtually no residual IgE directed against extract components. The variation in the proportion of Der p I and Der p II among different D. pteronyssinus extracts is likely to influence their biological effectiveness. Patients with reactivity against only Der p I or Der p II, who were found to comprise approximately one-third of the mite-allergic population, may not respond optimally to extracts containing relatively low levels of the allergen to which they are sensitive.

Anti-DNA IgA autoantibodies play an important immunopathologic role in SLE patients. To analyze the cellular origin and the VH and VL structure of anti-DNA IgA autoantibodies, we generated five IgA1 mAbs to DNA using B lymphocytes from three SLE patients. Two mAbs bound to ssDNA only and one to both ssDNA and dsDNA (monoreactive antibodies). The remaining two mAbs bound to DNA (one to ssDNA and the other to both ssDNA and dsDNA) and to other self and foreign Ag (polyreactive antibodies). The IgA mAb relative avidity for DNA ranged from 7.5 x 10(-8) to 8.0 x 10(-10) g/microliters. The anti-DNA IgA mAb used VH segments of the VHI(VI-3b), VHII (VH2-MC2), VHIII (WHG16G and VH26c), and VHIV (V71-2) families in conjunction with V kappa I, V kappa IIIb, or V lambda I segments. All IgA mAb VH segments were juxtaposed with JH4b segments. The heavy chain CDR3 sequences were divergent in composition and length. When compared with those of the closest reported germ line genes, the IgA mAb VH and VL gene sequences displayed a number of differences. That these differences represented somatic point mutations was formally proved in both the monoreactive IgA mAb 412.67.F1.3 and the polyreactive IgA mAb 412.66.F1 VH segments by differential PCR amplification and cloning and sequencing of genomic DNA from the mAb-producing cell lines and autologous polymorphonuclear cells. The sequences of the germ line genes that putatively gave rise to the mAb 412.67.F1.3 and mAb 412.66.F1 VH segments were identical with those of the WHG16G and VH26c genes, respectively. In not only the monoreactive mAb 412.67.F1.3 but also the polyreactive mAb 412.66.F1 and mAb 448.9G.F1 VH segments, the higher concentration of replacement (R) mutations and the higher R:S (silent) mutation ratios in the complementarity-determining region (infinity; 19:0) than in the framework region (1.0) (p = 0.00001, chi 2 test) were highly consistent with selection by Ag. In the five IgA mAb VH and VL segments, the putative and verified somatic point mutations yielded 68 amino acid replacements, of which 38 were nonconserved. Twenty of these yielded positively charged or polar residues that play a major role in DNA binding, including seven Arg, five Lys, three Tyr, two Gln, two His, and a Thr. The conserved amino acid changes included seven Asn. These findings suggest that anti-DNA IgA autoantibodies use a broad selection of VH and VL genes and enhance their fit for Ag by undergoing somatic hypermutation and Ag selection.(ABSTRACT TRUNCATED AT 400 WORDS)

The interaction of fluorescein-labeled nerve growth factor (NGF) with human melanoma cells (A875) has been studied in order to assess better methodology for rigorous NGF binding studies. The NGF was modified at a single carboxyl group with iodoacetamidofluorescein after reaction with carbodiimide and cystamine. The modified NGF showed full binding competence in competition with radiolabeled NGF and full biological activity in neurite outgrowth assays compared to native NGF. Binding to unfixed, viable cells was assayed using flow cytometry. This method offers the advantage that unbound ligand need not be separated from that which is cell-associated, thus avoiding perturbation of the binding equilibrium, and accurate, extensive statistical analysis is possible. Binding of fluorescein-NGF was mainly specific and saturable, with analysis by three methods of data treatment indicating a Kd of 0.8 to 3 nM at 4 degrees C. Time-based data acquisition allowed a continuous time course for binding to be generated. Binding reached a steady-state level within 5 min of exposure of the cells to the ligand. Kinetic and steady-state results obtained using fluorescein-NGF agree well with previous data produced by 125I-NGF binding studies. The main limitation of the flow cytometric method in the NGF system is the relative lack of sensitivity compared to the binding of radiolabeled NGF, partially due to unusual quenching of the fluorophore bound to NGF.

Polyreactive (natural) antibodies are primarily IgM and account for a major proportion of circulating Ig in humans. They use various V gene segments, in general, in germ line (unmutated) configuration. To analyze the VH regions of polyreactive antibodies, with particular attention at their somatically mutated status, we generated five IgG (three IgG1 and two IgG3) mAb (using B cells from a healthy subject, a patient with insulin-dependent diabetes mellitus and a patient with SLE), which bound with various efficiencies a number of different self and foreign Ag. Gene cloning experiments showed that the VH region sequences were unique to each IgG mAb. The H chain complementary determining region (CDR3) of two IgG (mAb10 and mAb426.4.2F20) displayed an identical stretch of five amino acids (RFLEW), but the other three IgG mAb CDR3 were divergent in both length and composition. The VH gene sequences of two IgG, mAb426.4.2F20 and mAb410.7.F91, were 99% identical to those of the germ line VH4.11 and VH4.21 genes, respectively. Those of the remaining three IgG mAb displayed a number of differences (93.6 to 95.9% identity) when compared with the germ line VH4.18, VH4.11, and hv1263 gene sequences. These and the VH4.21 gene have been found to encode polyreactive IgM and IgA and, in mutated configuration, monoreactive high affinity autoantibodies and antibodies induced by foreign Ag. When compared with the respective framework region, the CDR of three IgG mAb VH segment sequences displayed a significantly higher: 1) frequency of total nucleotide differences (6.1 x 10(-2) vs 4.5 x 10(-2) difference/base); 2) frequency of putative nucleotide changes yielding amino acid replacements (5.6 x 10(-2) vs 1.4 x 10(-2) replacement change/base); and 3) ratio of overall putative replacement to silent (R:S) mutations (11.0 vs 0.4). Thus, the distribution and nature of the nucleotide differences were consistent with a process of somatic mutation and Ag-dependent clonal selection. This was formally proved in IgG mAb426.12.3F1.4 and IgG mAb10 by differentially targeted polymerase chain reaction amplification and cloning and sequencing of the germ line genes that gave rise to the expressed VH segments, using DNA from polymorphonuclear cells of the same subjects whose B cells were used for the generation of these IgG mAb. Somatic mutations might have been responsible for bringing about polyreactivity in originally monoreactive antibodies or, more likely, they accumulated in originally polyreactive antibodies, which after undergoing a process of Ag selection, retained polyreactivity and may have or may have not acquired a higher affinity for the selecting Ag.

The delineation of distinct subsets committed to the production of antibodies with different antigen-binding activities supports the view of a compartmentalization and specialization of function in the B cell repertoire and is consistent with the hypothesis of a developmentally layered immune system; as originally proposed by Herzenberg and Herzenberg. On the basis of the data by Solvason and Kearney in the human fetus and our data in the adult, and in agreement with the findings of Herzenberg et al. and Hardy et al. in the mouse, we propose that the human B cell repertoire includes at least three distinct B cell subsets: B-1a cells, which develop from progenitors in the fetal splanchnic district, namely the omentum, and are maintained in adult life by virtue of their self-replenishing nature; B-1b cells, progenitors of which can be found in the splanchnic district and, perhaps, adult bone marrow; and, finally, B-2 cells, which arise in the fetal liver and are continuously replenished in adult life by progenitors in the bone marrow (Figure 5). The different B cells types are distinguished by their differential expression of surface CD5 and, perhaps, CD11b and CD14, their differential expression of CD5 mRNA, and the different classes and specificities of the Ig they produce (Figure 5). B-1 lymphocytes play a major role in autoimmunity and constitute the physiological equivalent of the neoplastic forms in various lymphoproliferative disorders, such as CLL and SLL, which are often associated with the production of monoclonal antibodies to self antigens. Human B-1a (CD5+ B) and B-1b (CD5- CD45RAlo B) cells are responsible for the production of natural (polyreactive and monoreactive) antibodies in the fetus, neonate, and adult, and can give rise to the autoantibody-producing cells characteristic of several autoimmune disease states. Our recent findings suggest that while in healthy subjects the majority of natural polyreactive antibodies is encoded in V genes in germline configuration, some polyreactive antibodies are encoded in somatically mutated V genes, in a fashion consistent with an antigen-driven process of selection of such mutations. The nature of the antigen(s) involved in these selection processes remains to be determined. Under possibly different circumstances, the application of an antigen-driven process of clonal selection to B-1a and/or B-1b cells, previously committed to natural antibody production, can result in the generation of monoreactive high affinity and possibly pathogenic autoantibodies (Figures 5A and 5B).(ABSTRACT TRUNCATED AT 400 WORDS)

The production of "natural" autoantibodies or antibodies, i.e., Ig that bind a variety of self- and/or exogenous Ag and arise independently of known immunization, is though to be a feature of CD5+ B lymphocytes. To determine whether other lymphocyte subsets exist that might be committed to the production of natural antibodies, human peripheral blood B cells were sorted on the basis of surface CD5 expression and differential expression of surface CD45RA (CD5+CD45RAintermediate(int), CD5-CD45RAlow(lo), and CD5-CD45RAhigh(hi)), and analyzed for the type of Ig produced after EBV infection and culture. Like their CD5+ counterparts, most CD5-CD45RAlo B lymphocytes were precursors of cells producing IgM, a major proportion of which displayed the Ag-binding features of natural antibodies. In contrast, CD5-CD45RAhi B cells comprised a high frequency of IgG-producing cell precursors, possibly including memory B lymphocytes. Six of seven IgM mAb generated from sorted CD5-CD45RAlo B cells and three of four IgM mAb from sorted CD5+ B cells were polyreactive, binding with different affinities (Kd, 10(-5) to 10(-8) M) to two or more Ag; the remaining mAb from CD5-CD45RAlo and the mAb from CD5+ B cells each bound to a single Ag (Kd, 10(-7) to 10(-8) M), beta-galactosidase and ssDNA, respectively. CD5-CD45RAlo B cells account for 4.1 +/- 1.2% (mean +/- SD in 11 healthy subjects; CD5+ B cells, 23.3 +/- 6.9%) of total B lymphocytes and display the features of quiescent cells. In a given individual, the number of CD5-CD45RAlo B cells remains constant over time. CD5-CD45RAlo and CD5+ B cells bear surface CD11b and CD14, at densities and/or frequencies apparently higher than those of CD5-CD45RAhi B lymphocytes. Despite their surface CD5- phenotype, CD45RAlo B cells express CD5+ mRNA at levels comparable with those of CD5+ B lymphocytes, whereas CD5-CD45RAhi B cells express only trace amounts of CD5 mRNA. The commitment to natural antibody production and the degree of CD5 mRNA expression suggest that the newly defined CD5-CD45RAlo B cell subset is related to CD5+ B lymphocytes, and may constitute the human homologue of the mouse Ly-1-"sister" B cell population.

The murine immune response to lymphocytic choriomeningitis virus (LCMV) infection involves the activation of CD8+, class I MHC-restricted and virus-specific CTL. At times coinciding with CTL activation, high levels of IL-2 gene expression and production occur, the IL-2R is expressed, and T cell blastogenesis and proliferation are induced. We have previously found that, although both CD4+ and CD8+ T cell subsets transcribe IL-2, the CD4+ subset appears to be the major producer of IL-2 whereas the CD8+ subset appears to be the major proliferating population when the subsets are separated after activation in vivo. The studies presented here were undertaken to examine the contribution made by the CD4+ subset to lymphocyte proliferation in vivo. Responses to LCMV infection were examined in intact mice and in mice depleted of CD4+ or CD8+ subsets by antibody treatments in vivo. Protocols were such that in vivo treatments with anti-CD4 or anti-CD8 depleted the respective subset by greater than 90%. In situ hybridizations demonstrated that the IL-2 gene was expressed in non-B lymphocytes isolated from either CD4+ cell-depleted or CD8+ cell-depleted mice on day 7 post-infection with LCMV. When placed in culture, however, cells from CD8+ cell-depleted mice produced significantly higher levels of detectable IL-2 than did cells isolated from CD4+ cell-depleted mice on day 7 post-infection. IL-2 was apparently produced in vivo in mice depleted of either CD4+ or CD8+ cells, as expression of the gene for the p55 chain of the IL-2R, IL-2 responsiveness, and lymphocyte proliferation were observed with cells isolated from both sets of mice. Lymphocyte proliferation was shown to be sustained in mice depleted of CD4+ cells in vivo by three criteria: 1) non-B lymphocytes isolated from infected mice depleted of CD4+ cells underwent more DNA synthesis than did those isolated from uninfected mice or from infected mice depleted of CD8+ cells; 2) leukocyte yields were expanded during infection of CD4+ cell-depleted mice; and 3) CD8+ cell numbers were increased during infection of CD4+ cell-depleted mice. The majority of non-B lymphocytes having the characteristics of blast lymphocytes was recovered in the CD8+ populations isolated from infected CD4+ cell-depleted mice. These findings suggest that the requirement for the CD4+ subset to sustain CD8+ lymphocyte proliferation in vivo is limited, and that CD4+ and CD8+ cell types can function independently in many aspects of their responses to viral infections.

A comparison has been made of various methods for measuring binding of nerve growth factor (NGF) to PC12 cells in suspension, on plates, and by a combination of the two. Results indicated that the extensive washing in the plate binding assay removed some cell surface ligand, underestimated the fast receptor binding, and overestimated the proportion of internalized ligand. In addition, the binding and internalization by a nonresponding PC12 mutant cell line has been studied. The nonresponding mutants had fewer total NGF receptors (10-50%) than normal cells in any binding assay. However, when measured in the suspension assay, the mutant cells showed both fast and slow binding receptors, in proportion approximately equivalent to those found on native PC12 cells. The PC12 nonresponders in suspension were also found to internalize and degrade low levels of NGF, in proportion to their reduced receptor number. Different results concerning PC12 wild type and mutant cells that have been reported in the literature may be due to the particular binding assay protocol that was used.

The IFN inducer, poly(I:C), elicits acute NK cell blastogenesis and proliferation in vivo. The role of IL-2 in mediating this proliferation was investigated in the studies presented here. Blast NK cells were isolated from poly(I:C)-treated, T cell-deficient athymic mice. Dividing cells, incorporating [3H]thymidine, were enriched in the J11d- low density populations isolated from poly(I:C)-treated mice, and were characterized as NK by the following criteria: (a) they were eliminated by treatment with anti-AGM1 in vivo; and (b) they directly mediated lysis of NK-sensitive target cells in a single cell cytotoxicity assay with autoradiography. These poly(I:C)-induced blast NK cells were responsive to IL-2, but, when compared with in vivo activated T cells, responsiveness required 1,000-fold higher concentrations of the factor. The technique of in situ hybridization was used to evaluate induction of IL-2 gene expression after poly(I:C) treatment in vivo. Treatment of euthymic, athymic, and severe combined immunodeficient mice with poly(I:C) activated IL-2 gene expression in a small percentage of spleen leukocytes. The transcription-positive cells were enriched in low density cell populations. These findings demonstrate that IL-2 transcription occurs after IFN induction in vivo, and suggest that an endogenous source of IL-2 exists other than the mature T cell. To assess the IL-2 dependence of in vivo NK cell expansion, poly(I:C)-treated athymic mice were given cyclosporin A (CsA), an agent that regulates IL-2 production at the level of gene transcription. The drug resulted in an 85-100% reduction in the percentages of cells transcribing IL-2. In contrast, CsA administration did not block IFN-enhanced NK cell cytolytic activity, expansion of large granular lymphocyte numbers, or NK cell proliferation. These findings demonstrate that although the proliferation of blast NK cells can be supported by IL-2, IL-2 is not an important mediator of IFN-induced NK cell expansion. Moreover, they establish that the acute proliferation of NK cells in response to IFNs is CsA insensitive.

The growth factor, IL-2, was administered to mice to evaluate the in vivo responsiveness of NK cells to this factor. The immediate effects of this factor on NK cells were determined by examining cytotoxic activity at 18-24 h after a single treatment with rIL-2. Although moderate doses of rIL-2 (3 x 10(4) U) could be shown to activate existing cytotoxic cells on a per cell basis, higher doses (10(6) U) were required to elicit blast size killer cells. The elicited killer cells were characterized as NK cells by the following criteria: (a) they were readily induced in athymic mice; (b) they mediated killing of NK-sensitive YAC-1 target cells but not NK-resistant P815 target cells; and (c) they expressed the NK cell determinants asialo ganglio-n-tetraosylceramide and NK1.1, but not the T cell determinants CD3, L3T4, or Lyt-2. High-dose IL-2 treatment induced not only the appearance of blast size NK cells, but also the expansion of this population. After treatments, the number of large granular lymphocytes and the number of NK1.1+ cells were increased at least twofold. Analysis of DNA content within the NK1.1+ cell subset demonstrated that IL-2 preferentially drove NK1.1+ cells into S and G2/M phases of the cell cycle. The in vivo elicited blast lymphocytes were examined by Northern blot analysis and in situ hybridization for expression of the IL-2-R p55 alpha chain gene. As previous work from this laboratory has demonstrated that NK cells proliferate in response to IFNs and IFN inducers in vivo, blast lymphocytes were also prepared after IFN treatments. The NK cells were not induced to express detectable levels of the alpha chain gene under any of the conditions examined. Blast T lymphocytes, isolated at times during viral infections when IL-2 production can be demonstrated in vitro, were induced to transcribe the alpha chain gene. Treatments of euthymic mice with high-dose IL-2 also induced transcription of the alpha chain gene in 41% of the non-B blast lymphocytes, but only background percentages of the NK1.1+ cells expressed the alpha chain gene. Transcription of the alpha chain gene was not induced in the NK cell-abundant athymic mice after IL-2 treatment. All of the in vivo elicited blast lymphocytes were induced to express IFN-gamma. Taken together, these data definitively demonstrate that IL-2 can induce NK cell proliferation and expansion in vivo. They also show that exposure to IL-2 in vivo, either by administration or endogenous production of the factor, induces transcription of the IL-2-R alpha chain gene in populations of cells containing T cell subsets. The results suggest, however, that murine NK cells are not induced to express high levels of the alpha chain gene in response to IL-2 in vivo.

The immunosuppressive agent, cyclosporin A (CsA) blocks production of IL-2 by lymphocytes in vitro, and impairs immune responses in vivo. During infection of mice with lymphocytic choriomeningitis virus (LCMV), IL-2 is produced by spleen lymphocytes with a time course corresponding to that of T cell activation and proliferation, but distinct from NK cell activation and proliferation. To evaluate the requirement for IL-2 in supporting lymphocyte proliferation in vivo, and to investigate the mechanisms of CsA-induced immunosuppression, the effects of CsA on LCMV-elicited responses were examined. CsA had profound effects on lymphocyte expansion and CTL activation on day 7 postinfection, the peak of the T cell response to LCMV. Proliferation of both the CD4+ and CD8+ T cell subsets was affected. Inhibition of T cell expansion was accompanied by the inhibition of IL-2 production and IL-2 responsiveness. In situ hybridization revealed a 50% reduction in the percentage of cells transcribing IL-2, suggesting that CsA blocked IL-2 production at the level of gene transcription. Transcripts of the gene for the IL-2R p55 chain are also normally elevated during infection, and CsA treatment resulted in an 80% reduction in the percentage of cells transcribing this gene. A reduced responsiveness of freshly isolated cells to rIL-2 in vitro correlated with the reduction of IL-2 receptor gene transcription positive cells. In contrast to effects of the drug on T cells, the level of NK cell activation was not decreased as a result of CsA treatment. These observations suggest that the IL-2 produced by lymphocytes in vivo in response to virus infection is required to promote the T cell response to LCMV, but do not support a role for IL-2 in NK cell activation under the conditions examined. Furthermore, the data demonstrate the profound inhibition of lymphocyte proliferation induced by CsA treatment during an in vivo immune response.

During infection of mice with lymphocytic choriomeningitis virus (LCMV), activation and proliferation of NK cells occurs early, followed by the activation and proliferation of CTL. To investigate the role of endogenously produced growth factors in mediating proliferation of these effector cell types, the transcription of IL-2 during infection was studied. We report that IL-2 is transcribed in vivo by mouse spleen cells during infection with LCMV. The time course of transcription corresponds to CTL activation, to the accumulation of Lyt-2+ and L3T4+ T cells in the spleen, to the incorporation of [3H]TdR by B cell-depleted spleen lymphocytes, and to production of IL-2 by these cells. At the peak of CTL activation and proliferation, both Lyt-2+ and L3T4+ populations transcribed IL-2. The results strongly support a role for IL-2 in mediating CTL proliferation during LCMV infection. Furthermore, the results demonstrate that Lyt-2+ cells can transcribe helper factors such as IL-2 in vivo, which may act to promote endogenous effector cell proliferation.

Nerve growth factor (NGF) is a polypeptide hormone involved in development of the sympathetic and central nervous systems. The detection and measurement of NGF in clinical samples would be useful in evaluating its role in various disease states. In this report, NGF activity and protein levels have been investigated in human amniotic fluid and cerebrospinal fluid samples. In amniotic fluid, NGF activity was found at levels ranging from less than 10 pM to nanomolar. The activity in all samples was blocked by polyclonal and monoclonal antibodies to mouse NGF. The finding of NGF in clinically obtainable samples raises the possibility of correlating NGF levels with a variety of disorders in which changes in NGF levels or activity have been implicated.

Binding and internalization of nerve growth factor (NGF) by responsive cells is a complex process. We have incubated rat pheochromocytoma cells (PC12) with 125I-NGF at 37 degrees C and measured the association of ligand after removal of subsets of bound ligand by different methods. Chase with unlabeled NGF at either 4 or 37 degrees C, acid stripping, nonionic detergent stability, and combinations of these protocols were utilized. These variations of the binding assay were able to distinguish ligand bound to fast versus slow cell surface receptors, NGF bound to slow receptors at the cell surface versus cell interior, and soluble ligand versus cytoskeletally attached NGF. Quantitative and temporal relations among five cellular pools were defined. Experiments with the inhibitors chloroquine, cytochalasin B, and colchicine defined pools of NGF in terms of the route through the cell from the plasma membrane to the lysosome. Chloroquine caused accumulation of NGF only in the pool that was not associated with the cytoskeleton, implicating the involvement of this pool in supplying ligand to the lysosome. Results with cytochalasin B and colchicine suggest that both microfilaments and microtubules are involved in pathways leading to NGF degradation. A semiquantitative model for the movement of NGF through the cell is presented based on these observations.