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Ling Liu,1 Jirong Lu,1 Barrett W Allan,2 Ying Tang,2 Jonathan Tetreault,1 Chi-kin Chow,1 Barbra Barmettler,2 James Nelson,2 Holly Bina,1 Lihua Huang,3 Victor J Wroblewski,4 Kristine Kikly1 1Biotechnology Discovery Research, Indianapolis, IN, 2Applied Molecular Evolution, Lilly Biotechnology Center, San Diego, CA, 3Bioproduct Research and Development, 4Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA Abstract: Interleukin (IL)-17A exists as a homodimer (A/A) or as a heterodimer (A/F) with IL-17F. IL-17A is expressed by a subset of T-cells, called Th17 cells, at inflammatory sites. Most cell types can respond to the local production of IL-17A because of the near ubiquitous expression of IL-17A receptors, IL-17RA and IL-17RC. IL-17A stimulates the release of cytokines and chemokines designed to recruit and activate both neutrophils and memory T-cells to the site of injury or inflammation and maintain a proinflammatory state. IL-17A-producing pathogenic T-cells contribute to the pathogenesis of autoimmune diseases, including psoriasis, psoriatic arthritis, rheumatoid arthritis, and ankylosing spondylitis. This study describes the generation and characterization of ixekizumab, a humanized IgG4 variant IL-17A-neutralizing antibody. Ixekizumab binds human and cynomolgus monkey IL-17A with high affinity and binds rabbit IL-17A weakly but does not bind to rodent IL-17A or other IL-17 family members. Ixekizumab effectively inhibits the interaction between IL-17A and its receptor in binding assays and potently blocks IL-17A-induced GRO or KC secretion in cell-based assays. In an in vivo mouse pharmcodynamic model, ixekizumab blocks human IL-17A-induced mouse KC secretion. These data provide a comprehensive preclinical characterization of ixekizumab, for which the efficacy and safety have been demonstrated in human clinical trials in psoriasis and psoriatic arthritis.Keywords: ixekizumab, IL-17A monoclonal antibody, anti-IL-17A

Joseph Manetta, Holly Bina, Paul Ryan, Niles Fox, Derrick R Witcher, Kristine Kikly Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA Abstract: B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect. Keywords: autoimmunity, B-cell malignancies, B-cell survival factor, BAFF

Interleukin (IL)-23 exists as a heterodimer consisting of p19 and p40 and is a key cytokine for promoting inflammatory responses in a variety of target organs. IL-23 plays a key role in the differentiation and maintenance of T helper 17 cells, and deregulation of IL-23 can result in autoimmune pathologies of the skin, lungs, and gut. This study describes the generation and characterization of mirikizumab (miri), a humanized IgG4 monoclonal antibody directed against the p19 subunit of IL-23. Miri binds human and cynomolgus monkey IL-23 with high affinity and binds rabbit IL-23 weakly but does not bind to rodent IL-23 or the other IL-23 family members IL-12, IL-27, or IL-35. Miri effectively inhibits the interaction of IL-23 with its receptor, and potently blocks IL-23-induced IL-17 production in cell-based assays while preserving the function of IL-12. In both local and systemic in vivo mouse models, miri blocked IL-23-induced keratin mRNA or IL-17 production, respectively. These data provide a comprehensive preclinical characterization of miri, for which efficacy and safety have been demonstrated in human clinical trials for psoriasis, ulcerative colitis, and Crohn's disease. SIGNIFICANCE STATEMENT: This article describes the generation and characterization of mirikizumab, a high affinity, neutralizing IgG4 variant monoclonal antibody that is under development for the treatment of ulcerative colitis and Crohn's disease. Neutralization of interleukin (IL)-23 is achieved by preventing the binding of IL-23 p19 subunit to the IL-23 receptor and does not affect the IL-12 pathway., (Copyright © 2023 by The Author(s).)

The Th17 pathway has been implicated in autoimmune diseases. The retinoic acid receptor-related orphan receptor C2 (RORγt) is a master regulator of Th17 cells and controls the expression of IL-17A. RORγt is expressed primarily in IL-17A-producing lymphoid cells. Here we describe a virtual screen of the ligand-binding pocket and subsequent screen in a binding assay that identified the 1-benzyl-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3- c ]pyran]-2'-carboxamide scaffold as a starting point for optimization of binding affinity and functional activity guided by structure-based design. Compound 12 demonstrated activity in a mouse PK/PD model and efficacy in an inflammatory arthritis mouse model that were used to define the level and duration of target engagement required for efficacy in vivo . Further optimization to improve ADME and physicochemical properties with guidance from simulations and modeling provided compound 22 , which is projected to achieve the level and duration of target engagement required for efficacy in the clinic.

CXCR1 and CXCR2 signaling play a critical role in neutrophil migration, angiogenesis, and tumorigenesis and are therefore an attractive signaling axis to target in a variety of indications. In human, a total of seven chemokines signal through these receptors and comprise the ELR + CXC chemokine family, so named because of the conserved ELRCXC N-terminal motif. To fully antagonize CXCR1 and CXCR2 signaling, an effective therapeutic should block either both receptors or all seven ligands, yet neither approach has been fully realized clinically. In this work, we describe the generation and characterization of LY3041658, a humanized monoclonal antibody that binds and neutralizes all seven human and cynomolgus monkey ELR + CXC chemokines and three of five mouse and rat ELR + CXC chemokines with high affinity. LY3041658 is able to block ELR + CXC chemokine-induced Ca 2+ mobilization, CXCR2 internalization, and chemotaxis in vitro as well as neutrophil mobilization in vivo without affecting other neutrophil functions. In addition to the in vitro and in vivo activity, we characterized the epitope and structural basis for binding in detail through alanine scanning, crystallography, and mutagenesis. Together, these data provide a robust preclinical characterization of LY3041658 for which the efficacy and safety is being evaluated in human clinical trials for neutrophilic skin diseases.

Breaking tolerance is a key event leading to autoimmunity, but the exact mechanisms responsible for this remain uncertain. Here we show that the alarmin IL-33 is able to drive the generation of autoantibodies through induction of the B cell survival factor BAFF. A temporary, short-term increase in IL-33 results in a primary (IgM) response to self-antigens. This transient DNA-specific autoantibody response was dependent on the induction of BAFF. Notably, radiation resistant cells and not myeloid cells, such as neutrophils or dendritic cells were the major source of BAFF and were critical in driving the autoantibody response. Chronic exposure to IL-33 elicited dramatic increases in BAFF levels and resulted in elevated numbers of B and T follicular helper cells as well as germinal center formation. We also observed class-switching from an IgM to an IgG DNA-specific autoantibody response. Collectively, the results provide novel insights into a potential mechanism for breaking immune-tolerance via IL-33-mediated induction of BAFF.

Psoriasis patients experience chronic systemic skin inflammation and develop cardiovascular comorbidities that shorten their lifespan. Whether cardiovascular disease is improved by treatment with current biologics that target disease-specific pathways is unclear. KC-Tie2 mice develop psoriasiform skin inflammation with increases in IL-23 and IL-17A and proinflammatory monocytosis and neutrophilia that precedes development of carotid artery thrombus formation. To examine whether targeted blockade of IL-23 or IL-17A in KC-Tie2 psoriasis mice improves cardiovascular outcomes, mice were treated systemically for 6 weeks with antibodies targeting IL-17A, IL-17RA, IL-12/23p40, or IL-23p19. Skin inflammation; thrombosis clotting times; and percentage of splenic monocytes, neutrophils, and CD4 T cells were examined. Skin inflammation significantly improved in KC-Tie2 mice treated with each of the antibodies targeting IL-23, IL-17A, or IL-17RA, consistent with clinical efficacy observed in psoriasis patients. The time to occlusive thrombus formation lengthened in these mice and correlated with attenuated acanthosis. This decrease in skin inflammation paralleled decreases in splenic neutrophils (CD11b + Ly6G + ) but not monocytes (CD11b + Ly6C high ) or T cells (CD4 + ). Our data show that targeted inhibition of IL-23 or IL-17A improves psoriasis-like skin disease and also improves cardiovascular disease in mice., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Hypertensive disorders of pregnancy are a leading cause of maternal and perinatal morbidity and mortality. Early suppression of B-cell lymphopoiesis is necessary for a normal pregnancy. Dysregulation of factors critical to B-cell survival may result in pregnancy complications, including hypertension. In this prospective observational study at a single medical center, serum levels of BAFF (B-cell activating factor) were measured in pregnant participants at each trimester, at delivery, and postpartum and in nonpregnant controls at a single time point. Comparisons were made between nonpregnant and pregnant subjects and between time periods of pregnancy. First-trimester serum BAFF levels were further tested for association with hypertensive disorders of pregnancy. The study included 149 healthy pregnant women, 25 pregnant women with chronic hypertension, and 48 nonpregnant controls. Median first-trimester serum BAFF level (ng/mL) for healthy women (0.90) was lower than median serum BAFF levels for women with chronic hypertension (0.96; P =0.013) and controls (1.00; P =0.002). Serum BAFF levels steadily declined throughout pregnancy, with the median second-trimester level lower than the corresponding first-trimester level (0.77; P =0.003) and the median third-trimester level lower than the corresponding second-trimester level (0.72; P =0.025). The median first-trimester serum BAFF level was elevated in women who subsequently developed hypertension compared with women who remained normotensive (1.02 versus 0.85; P =0.012), with the area under the receiver operating characteristic curve being 0.709. First-trimester serum BAFF level may be an early and clinically useful predictor of hypertensive disorders of pregnancy., (© 2017 American Heart Association, Inc.)

Tissue-specific immune responses play an important role in the pathology of autoimmune diseases. In systemic lupus erythematosus, deposits of IgG-immune complexes and the activation of complement in the kidney have long been thought to promote inflammation and lupus nephritis. However, the events that localize cells in non-lymphoid tertiary organs and sustain tissue-specific immune responses remain undefined. In this manuscript, we show that BAFF promotes events leading to lupus nephritis. Using an inducible model of systemic lupus erythematosus, we found that passive transfer of antinucleosome IgG into AID -/- MRL/lpr mice elevated autoantibody levels and promoted lupus nephritis by inducing BAFF production in the kidneys, and the formation of renal tertiary lymphoid structures (TLSs). Reducing BAFF in vivo prevented the formation of TLSs and lupus nephritis; however, it did not reduce immune cell infiltrates, or the deposits of IgG and complement in the kidney. Mechanistically, lowering BAFF levels also diminished the number of T cells positioned inside the glomeruli and reduced inflammation. Thus, BAFF plays a previously unappreciated role in lupus nephritis by inducing renal TLSs and regulating the position of T cells within the glomeruli., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Objective: Spondyloarthritides (SpA) occur in 1% of the population and include ankylosing spondylitis (AS) and arthropathy of inflammatory bowel disease (IBD), with characteristic spondylitis, arthritis, enthesitis, and IBD. Genetic studies implicate interleukin-23 (IL-23) receptor signaling in the development of SpA and IBD, and IL-23 overexpression in mice is sufficient for enthesitis, driven by entheseal-resident T cells. However, in genetically prone individuals, it is not clear where IL-23 is produced and how it drives the SpA syndrome, including IBD or subclinical gut inflammation of AS. Moreover, it is unclear why specific tissue involvement varies between patients with SpA. We undertook this study to determine the location of IL-23 production and its role in SpA pathogenesis in BALB/c ZAP-70(W163C)-mutant (SKG) mice injected intraperitoneally with β-1,3-glucan (curdlan)., Methods: Eight weeks after curdlan injection in wild-type or IL-17A(-/-) SKG or BALB/c mice, pathology was scored in tissue sections. Mice were treated with anti-IL-23 or anti-IL-22. Cytokine production and endoplasmic reticulum (ER) stress were determined in affected organs., Results: In curdlan-treated SKG mice, arthritis, enthesitis, and ileitis were IL-23 dependent. Enthesitis was specifically dependent on IL-17A and IL-22. IL-23 was induced in the ileum, where it amplified ER stress, goblet cell dysfunction, and proinflammatory cytokine production. IL-17A was pathogenic, while IL-22 was protective against ileitis. IL-22+CD3- innate-like cells were increased in lamina propria mononuclear cells of ileitis-resistant BALB/c mice, which developed ileitis after curdlan injection and anti-IL-22., Conclusion: In response to systemic β-1,3-glucan, intestinal IL-23 provokes local mucosal dysregulation and cytokines driving the SpA syndrome, including IL-17/IL-22-dependent enthesitis. Innate IL-22 production promotes ileal tolerance., (Copyright © 2014 by the American College of Rheumatology.)

Obesity continues to be a global health problem, and thus it is imperative that new pathways regulating energy balance be identified. Recently, it was reported: (Hayashi K, Cao T, Passmore H, Jourdan-Le Saux C, Fogelgren B, Khan S, Hornstra I, Kim Y, Hayashi M, Csiszar K. J Invest Dermatol 123: 864-871, 2004) that mice carrying a missense mutation in myelin protein zero-like 3 (Mpzl3rc) have reduced body weight. To determine how Mpzl3 controls energy balance in vivo, we generated mice deficient in myelin protein zero-like 3 (Mpzl3-KO). Interestingly, KO mice were hyperphagic yet had reduced body weight and fat mass. Moreover, KO mice were highly resistant to body weight and fat mass gain after exposure to a high-fat, energy-dense diet. These effects on body weight and adiposity were driven, in part, by a pronounced increase in whole body energy expenditure levels in KO mice. KO mice also had reduced blood glucose levels during an intraperitoneal glucose challenge and significant reductions in circulating insulin levels suggesting an increase in insulin sensitivity. In addition, there was an overall increase in oxidative capacity and contractile force in skeletal muscle isolated from KO mice. Hepatic triglyceride levels were reduced by 92% in livers of KO mice, in part due to a reduction in de novo lipid synthesis. Interestingly, Mpzl3 mRNA expression in liver was increased in diet-induced obese mice. Moreover, KO mice exhibited an increase in insulin-stimulated Akt signaling in the liver, further demonstrating that Mpzl3 can regulate insulin sensitivity in this tissue. We have determined that Mpzl3 has a novel physiological role in controlling body weight regulation, energy expenditure, glycemic control, and hepatic triglyceride synthesis in mice.

IL-23p19 deficient mice have revealed a critical role of IL-23 in the development of experimental autoimmune diseases, such as collagen-induced arthritis (CIA). Neutralizing IL-23 after onset of CIA in rats has been shown to reduce paw volume, but the effect on synovial inflammation and the immunological autoimmune response is not clear. In this study, we examined the role of IL-23 at different stages of CIA and during T cell memory mediated flare-up arthritis with focus on changes in B cell activity and Th1/Th17 modulation. Anti-IL-23p19 antibody (anti-IL23p19) treatment, starting 15 days after the type II collagen (CII)-immunization but before clinical signs of disease onset, significantly suppressed the severity of CIA. This was accompanied with significantly lower CII-specific IgG1 levels and lower IgG2a levels in the anti-IL-23p19 treated mice compared to the control group. Importantly, neutralizing IL-23 after the first signs of CIA did not ameliorate the disease. This was in contrast to arthritic mice that underwent an arthritis flare-up since a significantly lower disease score was observed in the IL-23p19 treated mice compared to the control group, accompanied by lower synovial IL-17A and IL-22 expression in the knee joints of these mice. These data show IL-23-dependent and IL-23-independent stages during autoimmune CIA. Furthermore, the memory T cell mediated flare-up arthritis is IL-23-mediated. These data suggest that specific neutralization of IL-23p19 after onset of autoimmune arthritis may not be beneficial as a therapeutic therapy for patients with rheumatoid arthritis (RA). However, T cell mediated arthritis relapses in patients with RA might be controlled by anti-IL-23p19 treatment.

Objective: The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohn's disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 β-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17-dependent inflammatory arthritis developed after dectin 1-mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-β-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process., Methods: SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti-IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies., Results: After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohn's disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell- and IL-23-dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies., Conclusion: Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic β-glucan or mannan exposure., (Copyright © 2012 by the American College of Rheumatology.)

Autoimmune inflammatory responses and the diseases that develop as a consequence are now thought to be driven through a novel non-Th(1) pathway. IL-23, together with additional factors including TGF-beta1 and IL-6, collectively generate and sustain a distinct CD4(+) 'Th(17) inflammation effector' T-cell subset characterized by its production of inflammatory chemokines and cytokines, including IL-17. With this paradigm shift in understanding of autoimmune inflammation pathogenesis comes exciting opportunities to identify and to target therapeutically molecules within the IL-23/Th(17) axis that are key to disease development.

The B7 homolog B7-H3 is important for the regulation of immune responses though its functions in vivo are controversial. We report the first clinical and experimental data concerning expression and function of B7-H3 in alloresponses. Immunohistological and molecular analyses showed B7-H3 expression by cells mediating rejection of human and mouse allografts. To analyze the significance of B7-H3 in rejecting allografts, we generated B7-H3-/- mice and showed that targeting of B7-H3 was synergistic with other forms of immune modulation; e.g. a regimen of rapamycin gave 12-14 days of survival in wild-type controls but led to permanent cardiac and islet allograft survival in B7-H3-/- mice. Cardiac allografts in treated B7-H3-/- mice showed markedly decreased production of key cytokine, chemokine and chemokine receptor mRNA transcripts as compared to wild-type controls. The incidence of chronic rejection in two different cardiac allograft models was also inhibited in B7-H3-/- as compared to wild-type recipients. Lastly, in addition to the expected antigen-presenting cell expression of B7-H3, CD4 and CD8 T cells showed B7-H3 induction upon cell activation, and both dendritic cell- and T cell-expressed B7-H3 each enhanced T cell proliferation in vitro and in vivo. We conclude that B7-H3 promotes T cell-mediated immune responses and the development of acute and chronic allograft rejection.

Caspase-8 is a proximal effector protein of the tumor necrosis factor receptor family death pathway. In the present human postmortem study, we observed a significantly higher percentage of dopaminergic (DA) substantia nigra pars compacta neurons that displayed caspase-8 activation in Parkinson's disease (PD) patients compared with controls. In an in vivo experimental PD model, namely subchronically 1,2,3,6-tetrahydropyridine-treated mice, we also show that caspase-8 is indeed activated after exposure to this toxin early in the course of cell demise, suggesting that caspase-8 activation precedes and is not the consequence of cell death. However, cotreatment of 1-methyl-4-phenylpyridinium-intoxicated primary DA cultures with broad-spectrum and specific caspase-8 inhibitors did not result in neuroprotection but seemed to trigger a switch from apoptosis to necrosis. We propose that this effect is related to ATP depletion and suggest that the use of caspase inhibitors in pathologies linked to intracellular energy depletion, such as PD, should be cautiously evaluated.

The recruitment and cleavage of pro-caspase-8 to produce the active form of caspase-8 is a critical biochemical event in death receptor-mediated apoptosis. However, the source of pro-caspase-8 available for activation by apoptotic triggers is unknown. In human fibroblasts and mouse clonal striatal cells, confocal microscopy revealed that pro-caspase-8 immunofluorescence was colocalized with cytochrome c in mitochondria and was also distributed diffusely in some nuclei. Biochemical analysis of subcellular fractions indicated that pro-caspase-8 was enriched in mitochondria and in nuclei. Pro-caspase-8 was found in the intermembrane space, inner membrane, and matrix of mitochondria after limited digestion of mitochondrial fractions, and this distribution was confirmed by immunogold electron microscopy. Pro-caspase-8 and cytochrome c were released from isolated mitochondria that were treated with an inhibitor of the ADP/ATP carrier atractyloside, which opens the mitochondria permeability transition pore. Release was blocked by the mitochondria permeability transition pore inhibitor cyclosporin A (CsA). After clonal striatal cells were exposed for 6 h to an apoptotic inducer tumor necrosis factor-alpha (TNF-alpha), mitochondria immunoreactive for cytochrome c and pro-caspase-8 became clustered at perinuclear sites. Pro-caspase-8 and cytochrome c levels decreased in mitochondrial fractions and increased, along with pro-caspase-8 cleavage products, in the cytoplasm of the TNF-alpha-treated striatal cells. CsA blocked the TNF-alpha-induced release of pro-caspase 8 but not cytochrome c. Internucleosomal DNA fragmentation started at 6 h and peaked 12 h after TNF-alpha treatment. These results suggest that pro-caspase-8 is predominantly localized in mitochondria and is released upon apoptotic stimulation through a CsA-sensitive mechanism.

The siglecs (sialic acid-binding immunoglobulin-like lectins) mediate sialic acid-dependent cellular interactions and may in some cases signal through SH2-binding domains. In addition to the previously characterized siglecs, sialoadhesin, CD22, CD33 and myelin-associated glycoprotein, several new ones, siglec-5, siglec-7 and siglec-8, have recently been cloned. Although these novel receptors have generated considerable interest as therapeutic targets because of their expression pattern on immune cells, very little is known about how their lectin activity is regulated. Previous studies with sialoadhesin, CD22 and CD33 have shown that siglec glycosylation has significant effects on binding. To determine any differences in the glycan composition of siglec-5, siglec-7 and siglec-8 that may modify their function, we released and characterized the N-linked oligosaccharide distribution in these three glycoproteins. The glycan pools from siglec-5 and siglec-7 contained a larger proportion of sialylated and core-fucosylated biantennary, triantennary and tetra-antennary oligosaccharides, whereas the carbohydrate mixture released from siglec-8 is noticeably less sialylated and is more abundant in 'high-mannose'-type glycans. In addition, we show that, in contrast with CD22 and CD33, mutating the conserved potentially N-linked glycosylation site in the first domain has no effect on binding mediated by siglec-5 or siglec-7.

We have identified a type I cytokine receptor, which we have termed novel interleukin receptor (NILR), that is most related to the IL-2 receptor beta chain (IL-2Rbeta) and physically adjacent to the IL-4 receptor alpha chain gene on chromosome 16. NILR mRNA is most highly expressed in thymus and spleen, and is induced by phytohemagglutinin in human peripheral blood mononuclear cells. NILR protein was detected on human T cell lymphotropic virus type I-transformed T cell lines, Raji B cells, and YT natural killer-like cells. Artificial homodimerization of the NILR cytoplasmic domain confers proliferation to Ba/F3 murine pro-B cells but not to 32D myeloid progenitor cells or CTLL-2 murine helper T cells. In these latter cells, heterodimerization of IL-2Rbeta and the common cytokine receptor gamma chain (gamma(c)) cytoplasmic domains allows potent proliferation, whereas such heterodimerization of NILR with gamma(c) does not. This finding suggests that NILR has signaling potential but that a full understanding of its signaling partner(s) is not yet clear. Like IL-2Rbeta, NILR associates with Jak1 and mediates Stat5 activation.

We have developed a modified method of immobilized metal-ion affinity chromatography (IMAC) that can be used for the purification of histidine-tagged proteins from conditioned medium containing free copper ions. Classical methods of IMAC purification, using resins such as Ni-NTA, have proven inefficient for this type of purification and require multiple steps due to the interference of divalent copper ions with the binding of His-tagged protein to the charged resin. In contrast, this modified IMAC procedure, using chelating Sepharose instead of Ni-NTA, enables efficient purification from copper-containing medium in a single step. This method appears to rely upon a preferential interaction of protein-copper complexes with immobilized chelating resin. We have utilized this method to purify active, His-tagged murine interleukin 12 from the conditioned medium of Drosophila S2 cells coexpressing recombinant p40 and His-tagged p35 subunits and for the purification of the extracellular domain of the erythropoietin receptor. This method should be applicable to the purification of a wide variety of His-tagged fusion proteins expressed in Drosophila cells and in other systems where free metal ions are present., (Copyright 2000 Academic Press.)

Background: Eosinophils, basophils, and mast cells are believed to be the central tenet cells in allergic conditions including allergic rhinitis, asthma, and eczema. The molecular mechanisms underlying the recruitment of these cells to sites of allergic inflammation are poorly understood., Objectives: Our aim was to identify a common adhesion molecule that could potentially be responsible for mediating the recruitment of the allergic cell types to the lungs and other sites of allergy., Methods: We have cloned a sialoadhesin molecule from a human eosinophil library with the use of expressed sequence tag technology and characterized its expression on allergic cells by the use of flow cytometry and specific mAbs., Results: With the use of expressed sequence tag sequencing, we have identified a novel siglec molecule, SAF-2. SAF-2 has homology with other sialoadhesin family members (CD33 and siglec-5) and belongs to a subgroup of the Ig superfamily. SAF-2 is a 431-amino acid protein composed of 3 Ig domains with a 358-amino acid extracellular domain and a 47-amino acid tail. SAF-2 is highly restricted to eosinophils, basophils, and mast cells. Antibodies to SAF-2 do not modulate Ca(++) mobilization or chemotaxis of human eosinophils induced by eotaxin., Conclusion: SAF-2 is a highly restricted sialoadhesin molecule, which may be useful in the detection and/or modulation of allergic cells.

Polycystic kidney disease (PKD) is characterized by the development of large renal cysts and progressive loss of renal function. Although the cause of the development of renal cysts is unknown, recent evidence suggests that excessive apoptosis occurs in PKD. With the use of terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, we have confirmed the presence of apoptotic bodies in cystic kidneys of congenital polycystic kidney (cpk) disease mice carrying a homozygous mutation at 3 wk of age. Apoptosis was localized primarily to the interstitium with little evidence of cell death in cyst epithelium or noncystic tubules. In addition, we observed that the expression of various caspases, bax and bcl-2, was upregulated in cystic kidneys. With the use of various substrates in enzyme activity assays, we have demonstrated a greater than sevenfold increase in caspase 4 activity and a sixfold increase in caspase 3 activity. These data suggest that there is a caspase-dependent apoptosis pathway associated with PKD and support the hypothesis that apoptotic cell death contributes to cyst formation in PKD.

A number of studies have provided evidence that neuronal cell loss after stroke involves programmed cell death or apoptosis. In particular, recent biochemical and immunohistochemical studies have demonstrated the expression and activation of intracellular proteases, notably caspase-3, which act as both initiators and executors of the apoptotic process. To further elucidate the involvement of caspases in neuronal cell death induced by focal stroke we developed a panel of antibodies and investigated the spatial and temporal pattern of both caspase-8 and caspase-3 expression. Our efforts focused on caspase-8 because its "apical" position within the enzymatic cascade of caspases makes it a potentially important therapeutic target. Constitutive expression of procaspase-8 was detectable in most cortical neurons, and proteolytic processing yielding the active form of caspase-8 was found as early as 6 hr after focal stroke induced in rats by permanent middle cerebral artery occlusion. This active form of caspase-8 was predominantly seen in the large pyramidal neurons of lamina V. Active caspase-3 was evident only in neurons located within lamina II/III starting at 24 hr after injury and in microglia throughout the core infarct at all times examined. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, gel electrophoresis of DNA, and neuronal cell quantitation indicated that there was an early nonapoptotic loss of cortical neurons followed by a progressive elimination of neurons with features of apoptosis. These data indicate that the pattern of caspase expression occurring during delayed neuronal cell death after focal stroke will vary depending on the neuronal phenotype.

Cells differ in the time required to execute cell death after receipt of a death signal. One reason may be the requirement for de novo synthesis of components of the death pathway. TSU-Pr1 prostate cancer cells treated with okadaic acid demonstrated activation of caspase-3, PARP cleavage, and nuclear fragmentation by 24 h and apoptosis by 72 h. Levels of procaspase-3 and procaspase-7, the precursor molecules of two effector caspases, were not depleted during apoptosis. Levels of procaspase-3 and -7 mRNA increased steadily in TSU-Pr1 cells up to 72 h after exposure to okadaic acid. Nuclear run-off experiments showed that the increase in mRNA was not due to transcriptional activation of caspase-3 and -7 mRNA. Antisense caspase-3 and caspase-7 oligodeoxynucleotides caused a depletion of procaspases-3 and -7 and a delay in apoptosis of TSU-Pr1 cells. Caspase antisense oligodeoxynucleotides inhibited apoptosis to a similar extent as peptide inhibitors of cysteine proteases. Synthesis of procaspases-3 and -7 was necessary to sustain programmed cell death in TSU-Pr1 prostate cancer cells.

Cardiomyocyte apoptosis has been demonstrated in animal models of cardiac injury as well as in patients with congestive heart failure or acute myocardial infarction. Therefore, apoptosis has been proposed as an important process in cardiac remodeling and progression of myocardial dysfunction. However, the mechanisms underlying cardiac apoptosis are poorly understood. The present study was designed to determine whether the family of caspase proteases and stress-activated protein kinase (SAPK/JNK) are involved in cardiac apoptosis. Cultured rat neonatal cardiac myocytes were treated with staurosporine to induce apoptosis as evidenced by the morphological (including ultrastructural) characteristics of cell shrinkage, cytoplasmic and nuclear condensation, and fragmentation. Nucleosomal DNA fragmentation in myocytes was further identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end-labeling (TUNEL). Staurosporine-induced apoptosis in myocytes was a time- and concentration-(0.25-1 micro M)-dependent process. Staurosporine-induced apoptosis in myocytes was reduced by a cell-permeable, irreversible tripeptide inhibitor of caspases, ZVAD-fmk, but not by the ICE-specific inhibitor, Ac-YVAD-CHO. At 10, 50 and 100 muM of ZVAD-fmk, staurosporine-induced myocyte apoptosis was reduced by 5.8, 39.1 (P<0.01) and 53.8% (P<0.01), respectively. Staurosporine, at 0.25-1 micro M, increased caspase activity in cardiomyocytes by five- to eight-fold, peaking at 4-8 h after stimulation. Based on substrate specificity analysis, the major component of caspases activated in myocytes was consistent with caspase-3 (CPP32). Moreover, the appearance of the 17-kD subunit of active caspase-3 in staurosporine-treated myocytes was demonstrated by immunocytochemical analysis. In contrast, staurosporine induced a rapid and transient inhibition of SAPK/JNK in myocytes. The SAPK activity in myocytes was reduced by 68.3 and 58.3% (P<0.01 v basal) at 10 and 30 min after treatment with 1 micro M of staurosporine, respectively. Our results suggest that staurosporine-induced cardiac myocyte apoptosis involves activation of caspases, mainly caspase-3, but not activation of the SAPK signaling pathway., (Copyright 1998 Academic Press Limited)

Tumour necrosis factor (TNF) elicits multiple biological effects through two distinct cell surface receptors, TNF-R1 (p55) and TNF-R2 (p75). Most TNF-mediated biological responses, such as cell death, gene induction, antiviral activity and cytokine production, have been attributed to TNF-R1 (refs 1-5). Gene targeting of this receptor confirms its role in the lethality attributable to low doses of lipopolysaccharide after sensitization with D-galactosamine; surprisingly, the toxicity of high doses of lipopolysaccharide was unaffected. The function of TNF-R2 is less well understood, although there are data supporting a role in T-cell development and the proliferation of cytotoxic T lymphocytes. To clarify the physiological role of TNF-R2, we have generated mice deficient in this receptor by gene targeting. The TNF-R2-/- mice show normal T-cell development and activity, but we find that they have increased resistance to TNF-induced death. Additionally, such mice injected subcutaneously with TNF show a dramatic decrease in tissue necrosis, indicating that this receptor plays a role in the necrotic effects of TNF.

Interleukin-8 (IL-8) is a proinflammatory cytokine that specifically attracts and activates human neutrophils. A murine gene with a high degree of homology to the two known human IL-8 receptors was cloned and then deleted from the mouse genome by homologous recombination in embryonic stem (ES) cells. These mice, although outwardly healthy, had lymphadenopathy, resulting from an increase in B cells, and splenomegaly, resulting from an increase in metamyelocytes, band, and mature neutrophils. Thus, this receptor may participate in the expansion and development of neutrophils and B cells. This receptor was the major mediator of neutrophil migration to sites of inflammation and may provide a potential therapeutic target in inflammatory disease.