The effects of neutrophil cathepsin G on the glycoprotein (GP) Ib-IX complex of washed platelets ... more The effects of neutrophil cathepsin G on the glycoprotein (GP) Ib-IX complex of washed platelets were examined. Cathepsin G resulted in a concentration- and time-dependent decrease in the platelet surface GPIb- IX complex, as determined by flow cytometry, binding of exogenous von Willebrand factor (vWF) in the presence of ristocetin, and ristocetin- induced platelet agglutination. Cathepsin G resulted in proteolysis of the vWF binding site on GPIb alpha (defined by monoclonal antibody [MoAb] 6D1), as determined by increased supernatant glycocalicin fragment (a proteolytic product of GPIb alpha); decreased total platelet content of GPIb; and lack of effect of either cytochalasin B (an inhibitor of actin polymerization), prostaglandin I2 (an inhibitor of platelet activation), or prior fixation of the platelets. However, cathepsin G resulted in minimal decreases in the binding to fixed platelets of MoAbs TM60 (directed against the thrombin binding site on GPIb alpha) and WM23 (directed...
Proceedings of the National Academy of Sciences, 1998
Thrombin cleaves its G-protein-linked seven-transmembrane domain receptor, thereby releasing a 41... more Thrombin cleaves its G-protein-linked seven-transmembrane domain receptor, thereby releasing a 41-aa peptide and generating a new amino terminus that acts as a tethered ligand for the receptor. Peptides corresponding to the new amino terminal end of the proteolyzed seven-transmembrane domain thrombin receptor [TR 42–55, SFLLRNPNDKYEPF, also known as TRAP (thrombin receptor-activating peptide)], previously have been demonstrated to activate the receptor. In this study, we demonstrate that the 41-aa cleaved peptide, TR 1–41 (MGPRRLLLVAACFSLCGPLLSARTRARRPESKATNATLDPR) is a strong platelet agonist. TR 1–41 induces platelet aggregation. In whole-blood flow cytometric studies, TR 1–41 was shown to be more potent than TR 42–55 and almost as potent as thrombin, as determined by the degree of increase in: ( i ) platelet surface expression of P-selectin (reflecting α granule secretion); ( ii ) exposure of the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex; and ( iii ) fibri...
Mice lacking Tbk1 activity exhibit immune cell infiltrates in multiple tissues, altered circulati... more Mice lacking Tbk1 activity exhibit immune cell infiltrates in multiple tissues, altered circulating immune cell compartments, and increased susceptibility to LPS-induced lethality. TBK1 is critical for immunity against microbial pathogens that activate TLR4- and TLR3-dependent signaling pathways. To address the role of TBK1 in inflammation, mice were generated that harbor two copies of a mutant Tbk1 allele. This Tbk1Δ allele encodes a truncated Tbk1Δ protein that is catalytically inactive and expressed at very low levels. Upon LPS stimulation, macrophages from Tbk1Δ/Δ mice produce normal levels of proinflammatory cytokines (e.g., TNF-α), but IFN-β and RANTES expression and IRF3 DNA-binding activity are ablated. Three-month-old Tbk1Δ/Δ mice exhibit mononuclear and granulomatous cell infiltrates in multiple organs and inflammatory cell infiltrates in their skin, and they harbor a 2-fold greater amount of circulating monocytes than their Tbk1+/+ and Tbk1+/Δ littermates. Skin from 2-wee...
Autoantibody production and immune complex deposition within the kidney promote renal disease in ... more Autoantibody production and immune complex deposition within the kidney promote renal disease in patients with lupus nephritis. Thus, therapeutics that inhibit these pathways may be efficacious in the treatment of systemic lupus erythematosus. Bruton’s tyrosine kinase (BTK) is a critical signaling component of both BCR and FcR signaling. We sought to assess the efficacy of inhibiting BTK in the development of lupus-like disease, and in this article describe (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxy]phenyl)-1H-pyrazole-4-carboxamide (PF-06250112), a novel highly selective and potent BTK inhibitor. We demonstrate in vitro that PF-06250112 inhibits both BCR-mediated signaling and proliferation, as well as FcR-mediated activation. To assess the therapeutic impact of BTK inhibition, we treated aged NZBxW_F1 mice with PF-06250112 and demonstrate that PF-06250112 significantly limits the spontaneous accumulation of splenic germinal center B cells and plasma cells. Cor...
Experimental autoimmune encephalomyelitis (EAE), a T cell-mediated inflammatory disease of the CN... more Experimental autoimmune encephalomyelitis (EAE), a T cell-mediated inflammatory disease of the CNS, is a rodent model of human multiple sclerosis. IL-23 is one of the critical cytokines in EAE development and is currently believed to be involved in the maintenance of encephalitogenic responses during the tissue damage effector phase of the disease. In this study, we show that encephalitogenic T cells from myelin oligodendrocyte glycopeptide (MOG)-immunized wild-type (WT) mice caused indistinguishable disease when adoptively transferred to WT or IL-23-deficient (p19 knockout (KO)) recipient mice, demonstrating that once encephalitogenic cells have been generated, EAE can develop in the complete absence of IL-23. Furthermore, IL-12/23 double-deficient (p35/p19 double KO) recipient mice developed EAE that was indistinguishable from WT recipients, indicating that IL-12 did not compensate for IL-23 deficiency during the effector phase of EAE. In contrast, MOG-specific T cells from p19KO ...
Highly reactive oxygen species rapidly inactivate nitric oxide (NO), an endothelial product which... more Highly reactive oxygen species rapidly inactivate nitric oxide (NO), an endothelial product which inhibits platelet activation. We studied platelet inhibition by NO in two brothers with a cerebral thrombotic disorder. Both children had hyperreactive platelets, as determined by whole blood platelet aggregometry and flow cytometric analysis of the platelet surface expression of P-selectin. Mixing experiments showed that the patients' platelets behaved normally in control plasma; however, control platelets suspended in patient plasma were not inhibited by NO. As determined by flow cytometry, in the presence of plasma from either patient there was normal inhibition of the thrombin-induced expression of platelet surface P-selectin by prostacyclin, but not NO. Using a scopoletin assay, we measured a 2.7-fold increase in plasma H 2 O 2 generation in one patient and a 3.4-fold increase in the second patient, both compared with control plasma. Glutathione peroxidase (GSH-Px) activity was decreased in the patients' plasmas compared with control plasma. The addition of exogenous GSH-Px led to restoration of platelet inhibition by NO. These data show that, in these patients' plasmas, impaired metabolism of reactive oxygen species reduces the bioavailability of NO and impairs normal platelet inhibitory mechanisms. These findings suggest that attenuated NO-mediated platelet inhibition produced by increased reactive oxygen species or impaired antioxidant defense may cause a thrombotic disorder in humans.
Previously we have shown that adoptive transfer of CD4+CD45RBhiCD25- T cells (naïve CD4 cells), ... more Previously we have shown that adoptive transfer of CD4+CD45RBhiCD25- T cells (naïve CD4 cells), into scid/scid mice can induce psoriasis-like disease. The affected mice develop scaly and raised skin plaques and immune features in this model mimic those of human psoriasis. Data from others suggests that skin infiltrating CD8+ T cells contribute to disease exacerbations in human psoriasis. To understand the role of CD8 cells in the mouse model of psoriasis, we adoptively transferred either naïve CD4 T cells, or CD8+ CD62LhiCD25- cells (naïve CD8 T cells) or combined naïve CD4 and CD8 cells into scid/scid mice and monitored disease progression. We found that adoptive transfer of naïve CD8+ T cells alone resulted in slightly less severe skin lesions when compared to either naïve CD4 T cell transfer or combined CD4 and CD8 T cell transfer. We also found that mice receiving CD8+ T cells experienced slightly reduced epidermal hyperplasia and inflammation than mice given other combina...
Journal of immunology (Baltimore, Md. : 1950), 2014
Inhibitors of Bruton's tyrosine kinase (BTK) possess much promise for the treatment of oncolo... more Inhibitors of Bruton's tyrosine kinase (BTK) possess much promise for the treatment of oncologic and autoimmune indications. However, our current knowledge of the role of BTK in immune competence has been gathered in the context of genetic inactivation of btk in both mice and man. Using the novel BTK inhibitor PF-303, we model the clinical phenotype of BTK inhibition by systematically examining the impact of PF-303 on the mature immune system in mice. We implicate BTK in tonic BCR signaling, demonstrate dependence of the T3 B cell subset and IgM surface expression on BTK activity, and find that B1 cells survive and function independently of BTK. Although BTK inhibition does not impact humoral memory survival, Ag-driven clonal expansion of memory B cells and Ab-secreting cell generation are inhibited. These data define the role of BTK in the mature immune system and mechanistically predict the clinical phenotype of chronic BTK inhibition.
partial DiGeorge syndrome is severely disturbed starting at infancy and may be more skewed in pat... more partial DiGeorge syndrome is severely disturbed starting at infancy and may be more skewed in patients with upper respiratory infections than in those without infections.
Objective. Patients with psoriasis and psoriatic arthritis respond well to tumor necrosis factor ... more Objective. Patients with psoriasis and psoriatic arthritis respond well to tumor necrosis factor ␣ (TNF␣) blockers in general; however, there is now mounting evidence that a small cohort of patients with rheumatoid arthritis who receive TNF␣ blockers develop psoriasis. This study was undertaken to explore the mechanisms underlying TNF␣ blockade-induced exacerbation of skin inflammation in murine psoriasislike skin disease. Methods. Skin inflammation was induced in BALB/c scid/scid mice after they received CD4؉ CD45RB high CD25؊ (naive CD4) T cells from donor mice. These mice were treated with either antiinterleukin-12 (anti-IL-12)/23p40 antibody or murine TNFRII-Fc fusion protein and were examined for signs of disease, including histologic features, various cytokine levels in the serum, and cytokine or FoxP3 transcripts in the affected skin and draining lymph node (LN) cells. In a separate study, naive CD4؉ T cells were differentiated into Th1 or Th17 lineages with anti-CD3/28 magnetic beads and appropriate cytokines in the presence or absence of TNF␣. Cytokine gene expression from these differentiated cells was also determined. Results. Neutralization of TNF␣ exacerbated skin inflammation and markedly enhanced the expression of the proinflammatory cytokines IL-1, IL-6, IL-17, IL-21, and IL-22 but suppressed FoxP3 expression in the skin and reduced the number of FoxP3-positive Treg cells in the draining LNs. TNF␣ also demonstrated a divergent role during priming and reactivation of naive T cells. Conclusion. These results reveal a novel immunoregulatory role of TNF␣ on Th17 and Treg cells in some individuals, which may account for the exacerbation of skin inflammation in some patients who receive anti-TNF treatments.
Psoriasis is a chronic skin disease resulting from the dysregulated interplay between keratinocyt... more Psoriasis is a chronic skin disease resulting from the dysregulated interplay between keratinocytes and infiltrating immune cells. We report on a psoriasis-like disease model, which is induced by the transfer of CD4 + CD45RB hi CD25cells to pathogen-free scid/scid mice. Psoriasis-like lesions had elevated levels of antimicrobial peptide and proinflammatory cytokine mRNA. Also, similar to psoriasis, disease progression in this model was dependent on the p40 common to IL-12 and IL-23. To investigate the role of IL-22, a Th17 cytokine, in disease progression, mice were treated with IL-22-neutralizing antibodies. Neutralization of IL-22 prevented the development of disease, reducing acanthosis (thickening of the skin), inflammatory infiltrates, and expression of Th17 cytokines. Direct administration of IL-22 into the skin of normal mice induced both antimicrobial peptide and proinflammatory cytokine gene expression. Our data suggest that IL-22, which acts on keratinocytes and other nonhematopoietic cells, is required for development of the autoreactive Th17 cell-dependent disease in this model of skin inflammation. We propose that IL-22 antagonism might be a promising therapy for the treatment of human psoriasis.
The effects of neutrophil cathepsin G on the glycoprotein (GP) Ib-IX complex of washed platelets ... more The effects of neutrophil cathepsin G on the glycoprotein (GP) Ib-IX complex of washed platelets were examined. Cathepsin G resulted in a concentration- and time-dependent decrease in the platelet surface GPIb- IX complex, as determined by flow cytometry, binding of exogenous von Willebrand factor (vWF) in the presence of ristocetin, and ristocetin- induced platelet agglutination. Cathepsin G resulted in proteolysis of the vWF binding site on GPIb alpha (defined by monoclonal antibody [MoAb] 6D1), as determined by increased supernatant glycocalicin fragment (a proteolytic product of GPIb alpha); decreased total platelet content of GPIb; and lack of effect of either cytochalasin B (an inhibitor of actin polymerization), prostaglandin I2 (an inhibitor of platelet activation), or prior fixation of the platelets. However, cathepsin G resulted in minimal decreases in the binding to fixed platelets of MoAbs TM60 (directed against the thrombin binding site on GPIb alpha) and WM23 (directed...
Proceedings of the National Academy of Sciences, 1998
Thrombin cleaves its G-protein-linked seven-transmembrane domain receptor, thereby releasing a 41... more Thrombin cleaves its G-protein-linked seven-transmembrane domain receptor, thereby releasing a 41-aa peptide and generating a new amino terminus that acts as a tethered ligand for the receptor. Peptides corresponding to the new amino terminal end of the proteolyzed seven-transmembrane domain thrombin receptor [TR 42–55, SFLLRNPNDKYEPF, also known as TRAP (thrombin receptor-activating peptide)], previously have been demonstrated to activate the receptor. In this study, we demonstrate that the 41-aa cleaved peptide, TR 1–41 (MGPRRLLLVAACFSLCGPLLSARTRARRPESKATNATLDPR) is a strong platelet agonist. TR 1–41 induces platelet aggregation. In whole-blood flow cytometric studies, TR 1–41 was shown to be more potent than TR 42–55 and almost as potent as thrombin, as determined by the degree of increase in: ( i ) platelet surface expression of P-selectin (reflecting α granule secretion); ( ii ) exposure of the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex; and ( iii ) fibri...
Mice lacking Tbk1 activity exhibit immune cell infiltrates in multiple tissues, altered circulati... more Mice lacking Tbk1 activity exhibit immune cell infiltrates in multiple tissues, altered circulating immune cell compartments, and increased susceptibility to LPS-induced lethality. TBK1 is critical for immunity against microbial pathogens that activate TLR4- and TLR3-dependent signaling pathways. To address the role of TBK1 in inflammation, mice were generated that harbor two copies of a mutant Tbk1 allele. This Tbk1Δ allele encodes a truncated Tbk1Δ protein that is catalytically inactive and expressed at very low levels. Upon LPS stimulation, macrophages from Tbk1Δ/Δ mice produce normal levels of proinflammatory cytokines (e.g., TNF-α), but IFN-β and RANTES expression and IRF3 DNA-binding activity are ablated. Three-month-old Tbk1Δ/Δ mice exhibit mononuclear and granulomatous cell infiltrates in multiple organs and inflammatory cell infiltrates in their skin, and they harbor a 2-fold greater amount of circulating monocytes than their Tbk1+/+ and Tbk1+/Δ littermates. Skin from 2-wee...
Autoantibody production and immune complex deposition within the kidney promote renal disease in ... more Autoantibody production and immune complex deposition within the kidney promote renal disease in patients with lupus nephritis. Thus, therapeutics that inhibit these pathways may be efficacious in the treatment of systemic lupus erythematosus. Bruton’s tyrosine kinase (BTK) is a critical signaling component of both BCR and FcR signaling. We sought to assess the efficacy of inhibiting BTK in the development of lupus-like disease, and in this article describe (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxy]phenyl)-1H-pyrazole-4-carboxamide (PF-06250112), a novel highly selective and potent BTK inhibitor. We demonstrate in vitro that PF-06250112 inhibits both BCR-mediated signaling and proliferation, as well as FcR-mediated activation. To assess the therapeutic impact of BTK inhibition, we treated aged NZBxW_F1 mice with PF-06250112 and demonstrate that PF-06250112 significantly limits the spontaneous accumulation of splenic germinal center B cells and plasma cells. Cor...
Experimental autoimmune encephalomyelitis (EAE), a T cell-mediated inflammatory disease of the CN... more Experimental autoimmune encephalomyelitis (EAE), a T cell-mediated inflammatory disease of the CNS, is a rodent model of human multiple sclerosis. IL-23 is one of the critical cytokines in EAE development and is currently believed to be involved in the maintenance of encephalitogenic responses during the tissue damage effector phase of the disease. In this study, we show that encephalitogenic T cells from myelin oligodendrocyte glycopeptide (MOG)-immunized wild-type (WT) mice caused indistinguishable disease when adoptively transferred to WT or IL-23-deficient (p19 knockout (KO)) recipient mice, demonstrating that once encephalitogenic cells have been generated, EAE can develop in the complete absence of IL-23. Furthermore, IL-12/23 double-deficient (p35/p19 double KO) recipient mice developed EAE that was indistinguishable from WT recipients, indicating that IL-12 did not compensate for IL-23 deficiency during the effector phase of EAE. In contrast, MOG-specific T cells from p19KO ...
Highly reactive oxygen species rapidly inactivate nitric oxide (NO), an endothelial product which... more Highly reactive oxygen species rapidly inactivate nitric oxide (NO), an endothelial product which inhibits platelet activation. We studied platelet inhibition by NO in two brothers with a cerebral thrombotic disorder. Both children had hyperreactive platelets, as determined by whole blood platelet aggregometry and flow cytometric analysis of the platelet surface expression of P-selectin. Mixing experiments showed that the patients' platelets behaved normally in control plasma; however, control platelets suspended in patient plasma were not inhibited by NO. As determined by flow cytometry, in the presence of plasma from either patient there was normal inhibition of the thrombin-induced expression of platelet surface P-selectin by prostacyclin, but not NO. Using a scopoletin assay, we measured a 2.7-fold increase in plasma H 2 O 2 generation in one patient and a 3.4-fold increase in the second patient, both compared with control plasma. Glutathione peroxidase (GSH-Px) activity was decreased in the patients' plasmas compared with control plasma. The addition of exogenous GSH-Px led to restoration of platelet inhibition by NO. These data show that, in these patients' plasmas, impaired metabolism of reactive oxygen species reduces the bioavailability of NO and impairs normal platelet inhibitory mechanisms. These findings suggest that attenuated NO-mediated platelet inhibition produced by increased reactive oxygen species or impaired antioxidant defense may cause a thrombotic disorder in humans.
Previously we have shown that adoptive transfer of CD4+CD45RBhiCD25- T cells (naïve CD4 cells), ... more Previously we have shown that adoptive transfer of CD4+CD45RBhiCD25- T cells (naïve CD4 cells), into scid/scid mice can induce psoriasis-like disease. The affected mice develop scaly and raised skin plaques and immune features in this model mimic those of human psoriasis. Data from others suggests that skin infiltrating CD8+ T cells contribute to disease exacerbations in human psoriasis. To understand the role of CD8 cells in the mouse model of psoriasis, we adoptively transferred either naïve CD4 T cells, or CD8+ CD62LhiCD25- cells (naïve CD8 T cells) or combined naïve CD4 and CD8 cells into scid/scid mice and monitored disease progression. We found that adoptive transfer of naïve CD8+ T cells alone resulted in slightly less severe skin lesions when compared to either naïve CD4 T cell transfer or combined CD4 and CD8 T cell transfer. We also found that mice receiving CD8+ T cells experienced slightly reduced epidermal hyperplasia and inflammation than mice given other combina...
Journal of immunology (Baltimore, Md. : 1950), 2014
Inhibitors of Bruton's tyrosine kinase (BTK) possess much promise for the treatment of oncolo... more Inhibitors of Bruton's tyrosine kinase (BTK) possess much promise for the treatment of oncologic and autoimmune indications. However, our current knowledge of the role of BTK in immune competence has been gathered in the context of genetic inactivation of btk in both mice and man. Using the novel BTK inhibitor PF-303, we model the clinical phenotype of BTK inhibition by systematically examining the impact of PF-303 on the mature immune system in mice. We implicate BTK in tonic BCR signaling, demonstrate dependence of the T3 B cell subset and IgM surface expression on BTK activity, and find that B1 cells survive and function independently of BTK. Although BTK inhibition does not impact humoral memory survival, Ag-driven clonal expansion of memory B cells and Ab-secreting cell generation are inhibited. These data define the role of BTK in the mature immune system and mechanistically predict the clinical phenotype of chronic BTK inhibition.
partial DiGeorge syndrome is severely disturbed starting at infancy and may be more skewed in pat... more partial DiGeorge syndrome is severely disturbed starting at infancy and may be more skewed in patients with upper respiratory infections than in those without infections.
Objective. Patients with psoriasis and psoriatic arthritis respond well to tumor necrosis factor ... more Objective. Patients with psoriasis and psoriatic arthritis respond well to tumor necrosis factor ␣ (TNF␣) blockers in general; however, there is now mounting evidence that a small cohort of patients with rheumatoid arthritis who receive TNF␣ blockers develop psoriasis. This study was undertaken to explore the mechanisms underlying TNF␣ blockade-induced exacerbation of skin inflammation in murine psoriasislike skin disease. Methods. Skin inflammation was induced in BALB/c scid/scid mice after they received CD4؉ CD45RB high CD25؊ (naive CD4) T cells from donor mice. These mice were treated with either antiinterleukin-12 (anti-IL-12)/23p40 antibody or murine TNFRII-Fc fusion protein and were examined for signs of disease, including histologic features, various cytokine levels in the serum, and cytokine or FoxP3 transcripts in the affected skin and draining lymph node (LN) cells. In a separate study, naive CD4؉ T cells were differentiated into Th1 or Th17 lineages with anti-CD3/28 magnetic beads and appropriate cytokines in the presence or absence of TNF␣. Cytokine gene expression from these differentiated cells was also determined. Results. Neutralization of TNF␣ exacerbated skin inflammation and markedly enhanced the expression of the proinflammatory cytokines IL-1, IL-6, IL-17, IL-21, and IL-22 but suppressed FoxP3 expression in the skin and reduced the number of FoxP3-positive Treg cells in the draining LNs. TNF␣ also demonstrated a divergent role during priming and reactivation of naive T cells. Conclusion. These results reveal a novel immunoregulatory role of TNF␣ on Th17 and Treg cells in some individuals, which may account for the exacerbation of skin inflammation in some patients who receive anti-TNF treatments.
Psoriasis is a chronic skin disease resulting from the dysregulated interplay between keratinocyt... more Psoriasis is a chronic skin disease resulting from the dysregulated interplay between keratinocytes and infiltrating immune cells. We report on a psoriasis-like disease model, which is induced by the transfer of CD4 + CD45RB hi CD25cells to pathogen-free scid/scid mice. Psoriasis-like lesions had elevated levels of antimicrobial peptide and proinflammatory cytokine mRNA. Also, similar to psoriasis, disease progression in this model was dependent on the p40 common to IL-12 and IL-23. To investigate the role of IL-22, a Th17 cytokine, in disease progression, mice were treated with IL-22-neutralizing antibodies. Neutralization of IL-22 prevented the development of disease, reducing acanthosis (thickening of the skin), inflammatory infiltrates, and expression of Th17 cytokines. Direct administration of IL-22 into the skin of normal mice induced both antimicrobial peptide and proinflammatory cytokine gene expression. Our data suggest that IL-22, which acts on keratinocytes and other nonhematopoietic cells, is required for development of the autoreactive Th17 cell-dependent disease in this model of skin inflammation. We propose that IL-22 antagonism might be a promising therapy for the treatment of human psoriasis.
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Papers by Stephen Benoit