Proceedings of the National Academy of Sciences of the United States of America, Jan 6, 2000
We previously produced three congenic strains carrying lupus susceptibility genes (Sle1-Sle3) fro... more We previously produced three congenic strains carrying lupus susceptibility genes (Sle1-Sle3) from the lupus-prone NZM2410 mouse on the C57BL/6 background and characterized their component phenotypes. Sle1 mediates the loss of tolerance to nuclear antigens; Sle2 lowers the activation threshold of B cells; and Sle3 mediates a dysregulation of CD4(+) T cells. We have now created a collection of bi- and tricongenic strains with these intervals and assessed the autoimmune phenotypes they elicit in various combinations. Our results indicate that Sle1 is key for the development of fatal lupus. The combination of Sle1 with Sle2, Sle3, or the BXSB-derived autoimmune accelerating gene yaa results in the development of systemic autoimmunity with variably penetrant severe glomerulonephritis culminating in kidney failure. In contrast, two locus combinations of Sle2, Sle3, and yaa failed to mediate fatal disease. These results indicate that the loss of tolerance to chromatin mediated by Sle1 is ...
Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive CD4(+) T cells ... more Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive CD4(+) T cells play an essential role. CD4(+) T cells rely on glycolysis for inflammatory effector functions, but recent studies have shown that mitochondrial metabolism supports their chronic activation. How these processes contribute to lupus is unclear. We show that both glycolysis and mitochondrial oxidative metabolism are elevated in CD4(+) T cells from lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice as compared to non-autoimmune controls. In vitro, both the mitochondrial metabolism inhibitor metformin and the glucose metabolism inhibitor 2-deoxy-d-glucose (2DG) reduced interferon-γ (IFN-γ) production, although at different stages of activation. Metformin also restored the defective interleukin-2 (IL-2) production by TC CD4(+) T cells. In vivo, treatment of TC mice and other lupus models with a combination of metformin and 2DG normalized T cell metabolism and reversed disease biomarkers. Further, CD4(+) T...
Journal of immunology (Baltimore, Md. : 1950), Jan 21, 2014
Pre-B cell leukemia homeobox 1 (Pbx1)-d is a dominant-negative splice isoform of the gene Pbx1 th... more Pre-B cell leukemia homeobox 1 (Pbx1)-d is a dominant-negative splice isoform of the gene Pbx1 that corresponds to the NZM2410 lupus susceptibility locus Sle1a1. Pbx1 is required to maintain stem cell self-renewal, including that of mesenchymal stem cells (MSCs). MSCs have immunosuppressive functions that require stem cell maintenance. We tested the hypothesis that the expression of Pbx1-d favors MSC differentiation and impairs their immunosuppressive functions. We demonstrate that Sle1a1 MSCs express high levels of Pbx1-d as compared with congenic C57BL/6J (B6) MSCs. Sle1a1 MSCs grew faster and differentiated significantly more rapidly into osteoblasts than did B6 MSCs. This corresponded to a significant decrease in the expression of genes associated with stemness and an increase in the expression of genes associated with differentiation. Additionally, Sle1a1 MSCs express a gene expression profile associated with an enhanced innate immunity and inflammation. Suppression of Ig produ...
Transurethral electrovaporization of the prostate is a new, minimally invasive technique being us... more Transurethral electrovaporization of the prostate is a new, minimally invasive technique being used by urologists for surgical ablation of prostatic tissue. There are insufficient data concerning factors affecting the vaporization and coagulation lesions produced by this technique. The aim of this study was to determine the role of various parameters for adequate tissue evaporation. This study compared bovine liver and human prostatic lesions made by the Vaportrode instrument with those produced by standard electrocautery loops, roller balls, and laser fibers. Additionally, two electrosurgical instruments with differing technical capabilities were compared for their ability to cause vaporization of tissue. Results revealed that the Vaportrode lesions were maximal with a new electrode when used with a Force 40S electrosurgical generator set at 300 W and a drag speed of 25 to 30 seconds per 10 mm of tissue. The lesions produced by this technique had a 74% greater coagulation volume compared to a standard cautery loop. The evaporation defect was comparable to a laser lesion produced in contact at 60 W. We conclude that electrovaporization under optimal conditions causes a vaporization lesion comparable to that produced by high power density laser prostatectomy. Additionally, the coagulation volume of a vaportrode lesion is considerably greater than that produced by standard electrocautery resection.
Thromboxane synthase (TS) catalyzes the formation of thromboxane (TxA2) in monocytes/macrophages,... more Thromboxane synthase (TS) catalyzes the formation of thromboxane (TxA2) in monocytes/macrophages, platelets, and various tissues. TxA2 is likely to play a role in graft dysfunction due to its vasoconstrictive and platelet aggregatory properties. We studied the expression of TS in 7 normal native kidneys, 29 consecutive renal allograft biopsies (performed for rising serum creatinine, n = 23, and delayed graft function, n = 6), and one transplant nephrectomy specimen with severe acute rejection. TS expression was determined by immunocytochemistry using a monoclonal antibody against human TS, Kon-7. Histologic grading of the transplant biopsy specimens was based on the Banff classification. The degree of TS staining was graded in the glomeruli, interstitium, tubules and vessels from 0 to 3+. Of 29 biopsies, 13 had chronic nephropathy (CN), 6 had acute rejection (AR) with chronic nephropathy (AR/CN), 4 had acute rejection (AR), and 6 had acute tubular necrosis (ATN). TS staining of native kidneys showed sporadic interstitial cells. The biopsy and transplant nephrectomy specimens showed significant staining, predominantly in the glomeruli and interstitium. Positively staining cells appeared to be of macrophage/monocyte lineage by morphology. The mean glomerular staining grade was significantly increased in specimens with AR (2.3 +/- 0.9) and the mean interstitial staining was increased in specimens with AR/CN (2.2 +/- 0.9). Follow-up renal function 6 months post-biopsy showed that patients with higher TS staining grades had a faster decline in graft function. In conclusion, TS expression is increased in patients with acute rejection with or without chronic nephropathy and is associated with more rapid deterioration in function.
The ability to predict renal allograft dysfunction in the short term and predict graft survival b... more The ability to predict renal allograft dysfunction in the short term and predict graft survival by quantifying the macrophage infiltrate in allograft renal biopsies is described. Renal allograft biopsies performed for cause in 41 consecutive patients were scored for macrophages (macrophage index, MI) by use of a modified Banff score of inflammation (BSI), and the impact of the MI on serum creatinine (SCr) levels 3 months post-biopsy (post-Bx) and on graft survival was quantified. Biopsies were stained for macrophages, individual lesions semiquantified and MI, BSI and chronic allograft damage index (CADI) obtained. The effects of pathologic indices on 3 month post-Bx SCr and graft survival were quantified by multivariate analysis and Cox regression. Glomerular and interstitial macrophage scores correlated inversely with graft survival. MI predicted an increase in SCr 3 months post-Bx (P=0.02). MI >3 (hazard ratio 23.13, P=0.003) also had a powerful negative predictive value on graft survival.
The NZM2410 and NZM TAN (TAN) are two of 27 inbred strains derived from an intercross between the... more The NZM2410 and NZM TAN (TAN) are two of 27 inbred strains derived from an intercross between the NZW and NZB strains. NZM2410 mice develop a highly penetrant lupus nephritis mediated by three susceptibility loci, Sle1, Sle2 and Sle3. These three loci have been combined on a C57BL/6 background in a triple congenic strain that reconstitutes the NZM2410 autoimmune phenotype. Remarkably, inspite of the presence of Sle1, Sle2 and Sle3, TAN mice display a mild autoimmune phenotype reminiscent of NZW. Contrary to the lupus-prone strains, the majority of TAN CD4(+) T cells are in a naïve-inactivated stage. TAN mice show B-cell developmental abnormalities similar to lupus-prone mice, such an accumulation of transitional T1 cells and peritoneal B-1a cells. TAN mice show, however, a unique expansion of the splenic marginal zone, in which B cells express high levels of CD5 and CD9, fail to migrate to the follicles in response to LPS, and show sub-optimal binding of T-independent type 2 antigens. Therefore, TAN mice present a functional silencing of marginal zone B cells, which have been previously implicated with autoimmune process. The TAN strain thus provides a novel model for the analysis of the genetic determinants of B-cell autoreactivity.
Marginal zone (MZ) B cells contain a large number of autoreactive clones and the expansion of thi... more Marginal zone (MZ) B cells contain a large number of autoreactive clones and the expansion of this compartment has been associated with autoimmunity. MZ B cells also efficiently transport blood-borne antigen to the follicles where they activate T cells and differentiate into plasma cells. Using the B6.NZM2410.Sle1.Sle2.Sle3 (B6.TC) model of lupus, we show that the IgM+ CD1d(hi)/MZ B-cell compartment is expanded, and a large number of them reside inside the follicles. Contrary to the peripheral B-cell subset distribution and their activation status, the intrafollicular location of B6.TC IgM+ CD1d(hi)/MZ B cells depends on both bone marrow- and stromal-derived factors. Among the factors responsible for this intrafollicular location, we have identified an increased response to CXCL13 by B6.TC MZ B cells and a decreased expression of VCAM-1 on stromal cells in the B6.TC MZ. However, the reduced number of MZ macrophages observed in B6.TC MZs was independent of the IgM+ CD1d(hi)/B-cell location. B7-2 but not B7-1 deficiency restored IgM+ CD1d(hi)/MZ B-cell follicular exclusion in B6.TC mice, and it correlated with tolerance to dsDNA and a significant reduction of autoimmune pathology. These results suggest that follicular exclusion of IgM+ CD1d(hi)/MZ B cells is an important B-cell tolerance mechanism, and that B7-2 signaling is involved in breaching this tolerance checkpoint.
A growing body of evidence implicates inflammation in the development of diabetic nephropathy. We... more A growing body of evidence implicates inflammation in the development of diabetic nephropathy. We recently reported that diabetic endothelial nitric oxide synthase knockout (eNOS KO) mice develop advanced glomerular lesions resembling human diabetic nephropathy. Vascular endothelial growth factor (VEGF) is a major factor in diabetic nephropathy, and is known to be chemotactic for macrophages. Herein, we examined the association of VEGF with macrophage infiltration in experimental diabetic nephropathy. Glomerular macrophage infiltration was markedly increased in diabetic eNOS KO mice compared to diabetic C57BL/6 mice, and correlated with glomerular injury, such as mesangiolysis, glomerular microaneurysm and nodular lesions of glomerular sclerosis. An elevation of podocyte VEGF expression correlated with infiltration of Flt-1-positive macrophage in injured glomeruli in diabetic eNOS KO mice, suggesting that VEGF could contribute to macrophage migration. Neither renal nNOS nor iNOS expression was altered in both C57BL/6 and eNOS KO mice. To determine if lack of NO could affect VEGF activation of macrophages, we examined if exogenous NO can block macrophage migration induced by VEGF in in vitro studies. Exogenous NO blocked macrophage migration and hypertrophy in response to VEGF. NO mediated these effects in part by downregulating Flt-1 expression on the macrophage. In summary, NO negatively regulates VEGF-induced macrophage migration by inhibiting Flt-1 expression. The VEGF-endothelial NO uncoupling pathway might partially explain how VEGF causes glomerular disease in diabetes.
Abstract Thromboxane synthase (TS) catalyzes the formation of the eicosanoid thromboxane (TxA 2),... more Abstract Thromboxane synthase (TS) catalyzes the formation of the eicosanoid thromboxane (TxA 2), which is a potent vasoconstrictor and activator of platelet aggregation. It has a widespread distribution, including hemopoietic cells (platelets, macrophages, monocytes) ...
Proceedings of the National Academy of Sciences of the United States of America, Jan 6, 2000
We previously produced three congenic strains carrying lupus susceptibility genes (Sle1-Sle3) fro... more We previously produced three congenic strains carrying lupus susceptibility genes (Sle1-Sle3) from the lupus-prone NZM2410 mouse on the C57BL/6 background and characterized their component phenotypes. Sle1 mediates the loss of tolerance to nuclear antigens; Sle2 lowers the activation threshold of B cells; and Sle3 mediates a dysregulation of CD4(+) T cells. We have now created a collection of bi- and tricongenic strains with these intervals and assessed the autoimmune phenotypes they elicit in various combinations. Our results indicate that Sle1 is key for the development of fatal lupus. The combination of Sle1 with Sle2, Sle3, or the BXSB-derived autoimmune accelerating gene yaa results in the development of systemic autoimmunity with variably penetrant severe glomerulonephritis culminating in kidney failure. In contrast, two locus combinations of Sle2, Sle3, and yaa failed to mediate fatal disease. These results indicate that the loss of tolerance to chromatin mediated by Sle1 is ...
Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive CD4(+) T cells ... more Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive CD4(+) T cells play an essential role. CD4(+) T cells rely on glycolysis for inflammatory effector functions, but recent studies have shown that mitochondrial metabolism supports their chronic activation. How these processes contribute to lupus is unclear. We show that both glycolysis and mitochondrial oxidative metabolism are elevated in CD4(+) T cells from lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice as compared to non-autoimmune controls. In vitro, both the mitochondrial metabolism inhibitor metformin and the glucose metabolism inhibitor 2-deoxy-d-glucose (2DG) reduced interferon-γ (IFN-γ) production, although at different stages of activation. Metformin also restored the defective interleukin-2 (IL-2) production by TC CD4(+) T cells. In vivo, treatment of TC mice and other lupus models with a combination of metformin and 2DG normalized T cell metabolism and reversed disease biomarkers. Further, CD4(+) T...
Journal of immunology (Baltimore, Md. : 1950), Jan 21, 2014
Pre-B cell leukemia homeobox 1 (Pbx1)-d is a dominant-negative splice isoform of the gene Pbx1 th... more Pre-B cell leukemia homeobox 1 (Pbx1)-d is a dominant-negative splice isoform of the gene Pbx1 that corresponds to the NZM2410 lupus susceptibility locus Sle1a1. Pbx1 is required to maintain stem cell self-renewal, including that of mesenchymal stem cells (MSCs). MSCs have immunosuppressive functions that require stem cell maintenance. We tested the hypothesis that the expression of Pbx1-d favors MSC differentiation and impairs their immunosuppressive functions. We demonstrate that Sle1a1 MSCs express high levels of Pbx1-d as compared with congenic C57BL/6J (B6) MSCs. Sle1a1 MSCs grew faster and differentiated significantly more rapidly into osteoblasts than did B6 MSCs. This corresponded to a significant decrease in the expression of genes associated with stemness and an increase in the expression of genes associated with differentiation. Additionally, Sle1a1 MSCs express a gene expression profile associated with an enhanced innate immunity and inflammation. Suppression of Ig produ...
Transurethral electrovaporization of the prostate is a new, minimally invasive technique being us... more Transurethral electrovaporization of the prostate is a new, minimally invasive technique being used by urologists for surgical ablation of prostatic tissue. There are insufficient data concerning factors affecting the vaporization and coagulation lesions produced by this technique. The aim of this study was to determine the role of various parameters for adequate tissue evaporation. This study compared bovine liver and human prostatic lesions made by the Vaportrode instrument with those produced by standard electrocautery loops, roller balls, and laser fibers. Additionally, two electrosurgical instruments with differing technical capabilities were compared for their ability to cause vaporization of tissue. Results revealed that the Vaportrode lesions were maximal with a new electrode when used with a Force 40S electrosurgical generator set at 300 W and a drag speed of 25 to 30 seconds per 10 mm of tissue. The lesions produced by this technique had a 74% greater coagulation volume compared to a standard cautery loop. The evaporation defect was comparable to a laser lesion produced in contact at 60 W. We conclude that electrovaporization under optimal conditions causes a vaporization lesion comparable to that produced by high power density laser prostatectomy. Additionally, the coagulation volume of a vaportrode lesion is considerably greater than that produced by standard electrocautery resection.
Thromboxane synthase (TS) catalyzes the formation of thromboxane (TxA2) in monocytes/macrophages,... more Thromboxane synthase (TS) catalyzes the formation of thromboxane (TxA2) in monocytes/macrophages, platelets, and various tissues. TxA2 is likely to play a role in graft dysfunction due to its vasoconstrictive and platelet aggregatory properties. We studied the expression of TS in 7 normal native kidneys, 29 consecutive renal allograft biopsies (performed for rising serum creatinine, n = 23, and delayed graft function, n = 6), and one transplant nephrectomy specimen with severe acute rejection. TS expression was determined by immunocytochemistry using a monoclonal antibody against human TS, Kon-7. Histologic grading of the transplant biopsy specimens was based on the Banff classification. The degree of TS staining was graded in the glomeruli, interstitium, tubules and vessels from 0 to 3+. Of 29 biopsies, 13 had chronic nephropathy (CN), 6 had acute rejection (AR) with chronic nephropathy (AR/CN), 4 had acute rejection (AR), and 6 had acute tubular necrosis (ATN). TS staining of native kidneys showed sporadic interstitial cells. The biopsy and transplant nephrectomy specimens showed significant staining, predominantly in the glomeruli and interstitium. Positively staining cells appeared to be of macrophage/monocyte lineage by morphology. The mean glomerular staining grade was significantly increased in specimens with AR (2.3 +/- 0.9) and the mean interstitial staining was increased in specimens with AR/CN (2.2 +/- 0.9). Follow-up renal function 6 months post-biopsy showed that patients with higher TS staining grades had a faster decline in graft function. In conclusion, TS expression is increased in patients with acute rejection with or without chronic nephropathy and is associated with more rapid deterioration in function.
The ability to predict renal allograft dysfunction in the short term and predict graft survival b... more The ability to predict renal allograft dysfunction in the short term and predict graft survival by quantifying the macrophage infiltrate in allograft renal biopsies is described. Renal allograft biopsies performed for cause in 41 consecutive patients were scored for macrophages (macrophage index, MI) by use of a modified Banff score of inflammation (BSI), and the impact of the MI on serum creatinine (SCr) levels 3 months post-biopsy (post-Bx) and on graft survival was quantified. Biopsies were stained for macrophages, individual lesions semiquantified and MI, BSI and chronic allograft damage index (CADI) obtained. The effects of pathologic indices on 3 month post-Bx SCr and graft survival were quantified by multivariate analysis and Cox regression. Glomerular and interstitial macrophage scores correlated inversely with graft survival. MI predicted an increase in SCr 3 months post-Bx (P=0.02). MI >3 (hazard ratio 23.13, P=0.003) also had a powerful negative predictive value on graft survival.
The NZM2410 and NZM TAN (TAN) are two of 27 inbred strains derived from an intercross between the... more The NZM2410 and NZM TAN (TAN) are two of 27 inbred strains derived from an intercross between the NZW and NZB strains. NZM2410 mice develop a highly penetrant lupus nephritis mediated by three susceptibility loci, Sle1, Sle2 and Sle3. These three loci have been combined on a C57BL/6 background in a triple congenic strain that reconstitutes the NZM2410 autoimmune phenotype. Remarkably, inspite of the presence of Sle1, Sle2 and Sle3, TAN mice display a mild autoimmune phenotype reminiscent of NZW. Contrary to the lupus-prone strains, the majority of TAN CD4(+) T cells are in a naïve-inactivated stage. TAN mice show B-cell developmental abnormalities similar to lupus-prone mice, such an accumulation of transitional T1 cells and peritoneal B-1a cells. TAN mice show, however, a unique expansion of the splenic marginal zone, in which B cells express high levels of CD5 and CD9, fail to migrate to the follicles in response to LPS, and show sub-optimal binding of T-independent type 2 antigens. Therefore, TAN mice present a functional silencing of marginal zone B cells, which have been previously implicated with autoimmune process. The TAN strain thus provides a novel model for the analysis of the genetic determinants of B-cell autoreactivity.
Marginal zone (MZ) B cells contain a large number of autoreactive clones and the expansion of thi... more Marginal zone (MZ) B cells contain a large number of autoreactive clones and the expansion of this compartment has been associated with autoimmunity. MZ B cells also efficiently transport blood-borne antigen to the follicles where they activate T cells and differentiate into plasma cells. Using the B6.NZM2410.Sle1.Sle2.Sle3 (B6.TC) model of lupus, we show that the IgM+ CD1d(hi)/MZ B-cell compartment is expanded, and a large number of them reside inside the follicles. Contrary to the peripheral B-cell subset distribution and their activation status, the intrafollicular location of B6.TC IgM+ CD1d(hi)/MZ B cells depends on both bone marrow- and stromal-derived factors. Among the factors responsible for this intrafollicular location, we have identified an increased response to CXCL13 by B6.TC MZ B cells and a decreased expression of VCAM-1 on stromal cells in the B6.TC MZ. However, the reduced number of MZ macrophages observed in B6.TC MZs was independent of the IgM+ CD1d(hi)/B-cell location. B7-2 but not B7-1 deficiency restored IgM+ CD1d(hi)/MZ B-cell follicular exclusion in B6.TC mice, and it correlated with tolerance to dsDNA and a significant reduction of autoimmune pathology. These results suggest that follicular exclusion of IgM+ CD1d(hi)/MZ B cells is an important B-cell tolerance mechanism, and that B7-2 signaling is involved in breaching this tolerance checkpoint.
A growing body of evidence implicates inflammation in the development of diabetic nephropathy. We... more A growing body of evidence implicates inflammation in the development of diabetic nephropathy. We recently reported that diabetic endothelial nitric oxide synthase knockout (eNOS KO) mice develop advanced glomerular lesions resembling human diabetic nephropathy. Vascular endothelial growth factor (VEGF) is a major factor in diabetic nephropathy, and is known to be chemotactic for macrophages. Herein, we examined the association of VEGF with macrophage infiltration in experimental diabetic nephropathy. Glomerular macrophage infiltration was markedly increased in diabetic eNOS KO mice compared to diabetic C57BL/6 mice, and correlated with glomerular injury, such as mesangiolysis, glomerular microaneurysm and nodular lesions of glomerular sclerosis. An elevation of podocyte VEGF expression correlated with infiltration of Flt-1-positive macrophage in injured glomeruli in diabetic eNOS KO mice, suggesting that VEGF could contribute to macrophage migration. Neither renal nNOS nor iNOS expression was altered in both C57BL/6 and eNOS KO mice. To determine if lack of NO could affect VEGF activation of macrophages, we examined if exogenous NO can block macrophage migration induced by VEGF in in vitro studies. Exogenous NO blocked macrophage migration and hypertrophy in response to VEGF. NO mediated these effects in part by downregulating Flt-1 expression on the macrophage. In summary, NO negatively regulates VEGF-induced macrophage migration by inhibiting Flt-1 expression. The VEGF-endothelial NO uncoupling pathway might partially explain how VEGF causes glomerular disease in diabetes.
Abstract Thromboxane synthase (TS) catalyzes the formation of the eicosanoid thromboxane (TxA 2),... more Abstract Thromboxane synthase (TS) catalyzes the formation of the eicosanoid thromboxane (TxA 2), which is a potent vasoconstrictor and activator of platelet aggregation. It has a widespread distribution, including hemopoietic cells (platelets, macrophages, monocytes) ...
Uploads
Papers by Byron Croker