Wilma L. Suarez-Pinzon

Cytokines and Their Roles in Pancreatic Islet β-Cell Destruction and Insulin-Dependent Diabetes Mellitus

Insulin-dependent diabetes mellitus (IDDM) is a disease that results from autoimmune destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. The autoimmune response against islet beta-cells is believed to result from a disorder of immunoregulation. According to this concept, a T helper 1 (Th1) subset of T cells and their cytokine products, i.e. Type 1 cytokines--interleukin 2 (IL-2), interferon gamma (IFNgamma), and tumor necrosis factor beta (TNFbeta), dominate over an immunoregulatory (suppressor) Th2 subset of T cells and their cytokine products, i.e. Type 2 cytokines--IL-4 and IL-10. This allows Type 1 cytokines to initiate a cascade of immune/inflammatory processes in the islet (insulitis), culminating in beta-cell destruction. Type 1 cytokines activate (1) cytotoxic T cells that interact specifically with beta-cells and destroy them, and (2) macrophages to produce proinflammatory cytokines (IL-1 and TNFalpha), and oxygen and nitrogen free radicals that are highly toxic to islet beta-cells. Furthermore, the cytokines IL-1, TNFalpha, and IFNgamma are cytotoxic to beta-cells, in large part by inducing the formation of oxygen free radicals, nitric oxide, and peroxynitrite in the beta-cells themselves. Therefore, it would appear that prevention of islet beta-cell destruction and IDDM should be aimed at stimulating the production and/or action of Type 2 cytokines, inhibiting the production and/or action of Type 1 cytokines, and inhibiting the production and/or action of oxygen and nitrogen free radicals in the pancreatic islets.

Intracellular Action of Matrix Metalloproteinase-2 Accounts for Acute Myocardial Ischemia and Reperfusion Injury

Background—Matrix metalloproteinases are best recognized for their ability to degrade the extracellular matrix in both physiological and pathological conditions. However, recent findings indicate that some of them are also involved in mediating acute processes such as platelet aggregation and vascular tone. The acute contractile defect of the heart after ischemia-reperfusion may involve the proteolytic degradation of the thin filament protein troponin I; however, the protease responsible for this remains obscure. Methods and Results—Here we report that matrix metalloproteinase-2 is colocalized with troponin I within the thin myofilaments of cardiomyocytes in ischemic-reperfused hearts and that troponin I is a novel intracellular target for proteolytic cleavage by matrix metalloproteinase-2. Inhibition of matrix metalloproteinase-2 activity prevented ischemia-reperfusion-induced troponin I degradation and improved the recovery of mechanical function of the heart. Conclusions—These data reveal for the first time a novel molecular mechanism by which matrix metalloproteinase-2 causes acute myocardial dysfunction after ischemia-reperfusion-injury and that matrix metalloproteinase-2 has a biological action within the cell.

Th1 to Th2 Cytokine Shifts in Nonobese Diabetic Mice: Sometimes an Outcome, Rather Than the Cause, of Diabetes Resistance Elicited by Immunostimulation

Numerous immunostimulatory protocols inhibit the development of T cell-mediated autoimmune insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse model. Many of these protocols, including treatment with the nonspecific immunostimulatory agents CFA or bacillus Calmette-Guérin (BCG) vaccine, have been reported to mediate protection by skewing the pattern of cytokines produced by pancreatic β-cell autoreactive T cells from a Th1 (IFN-γ) to a Th2 (IL-4 and IL-10) profile. However, most of these studies have documented associations between such cytokine shifts and disease protection rather than a cause/effect relationship. To partially address this issue we produced NOD mice genetically deficient in IFN-γ, IL-4, or IL-10. Elimination of any of these cytokines did not significantly alter the rate of spontaneous IDDM development. Additional experiments using these mice confirmed that CFA- or BCG-elicited diabetes protection is associated with a decreased IFN-γ to IL-4 mRNA ratio within T cell-infiltrated pancreatic islets, but this is a secondary consequence rather than the cause of disease resistance. Unexpectedly, we also found that the ability of BCG and, to a lesser extent, CFA to inhibit IDDM development in standard NOD mice is actually dependent upon the presence of the Th1 cytokine, IFN-γ. Collectively, our studies demonstrate that while Th1 and Th2 cytokine shifts may occur among β-cell autoreactive T cells of NOD mice protected from overt IDDM by various immunomodulatory therapies, it cannot automatically be assumed that this is the cause of their disease resistance.

Development of autoimmune diabetes in NOD mice is associated with the formation of peroxynitrite in pancreatic islet β-cells

Peroxynitrite (ONOO−) is a highly reactive oxidant species produced by the reaction of the free radicals superoxide (O2·–) and nitric oxide (NO·). Here we report a marked increase in nitrotyrosine (NT), a marker of peroxynitrite, in islet cells from NOD mice developing spontaneous autoimmune diabetes. By using specific antibodies and immunohistochemical methods, we found that NT-positive cells were significantly more frequent in islets from acutely diabetic NOD mice (22 ± 6%) than in islets from normoglycemic NOD mice (7 ± 1%) and control BALB/c mice (2 ± 1%). The NT+ cells in islets were identified to be macrophages and also β-cells. Most of the β-cells in islets from acutely diabetic NOD mice were NT+ (73 ± 8%), whereas significantly fewer β-cells were NT+ in islets from normoglycemic NOD mice (18 ± 4%) and BALB/c mice (5 ± 1%). Also, the percentage of β-cells in islets from NOD mice (normoglycemic and diabetic) correlated inversely with the frequency of NT+ β-cells. This study demonstrates for the first time that peroxynitrite, a reaction product of superoxide and nitric oxide, is formed in pancreatic islet β-cells of NOD mice developing autoimmune diabetes. This suggests that both oxygen and nitrogen free radicals contribute to β-cell destruction in IDDM via peroxynitrite formation in the islet β-cells.

Combination Therapy With Glucagon-Like Peptide-1 and Gastrin Restores Normoglycemia in Diabetic NOD Mice

OBJECTIVE—Glucagon-like peptide-1 (GLP-1) and gastrin promote pancreatic β-cell function, survival, and growth. Here, we investigated whether GLP-1 and gastrin can restore the β-cell mass and reverse hyperglycemia in NOD mice with autoimmune diabetes. RESEARCH DESIGN AND METHODS—Acutely diabetic NOD mice were treated with GLP-1 and gastrin, separately or together, twice daily for 3 weeks. Blood glucose was measured weekly and for a further 5 weeks after treatments, after which pancreatic insulin content and β-cell mass, proliferation, neogenesis, and apoptosis were measured. Insulin autoantibodies were measured, and adoptive transfer of diabetes and syngeneic islet transplant studies were done to evaluate the effects of GLP-1 and gastrin treatment on autoimmunity. RESULTS—Combination therapy with GLP-1 and gastrin, but not with GLP-1 or gastrin alone, restored normoglycemia in diabetic NOD mice. The GLP-1 and gastrin combination increased pancreatic insulin content, β-cell mass, and insulin-positive cells in pancreatic ducts, and β-cell apoptosis was decreased. Insulin autoantibodies were reduced in GLP-1–and gastrin-treated NOD mice, and splenocytes from these mice delayed adoptive transfer of diabetes in NOD-scid mice. Syngeneic islet grafts in GLP-1–and gastrin-treated NOD mice were infiltrated by leukocytes with a shift in cytokine expression from interferon-γ to transforming growth factor-β1, and β-cells were protected from apoptosis. CONCLUSIONS—Combination therapy with GLP-1 and gastrin restores normoglycemia in diabetic NOD mice by increasing the pancreatic β-cell mass and downregulating the autoimmune response.

Combination Therapy With Sirolimus and Interleukin-2 Prevents Spontaneous and Recurrent Autoimmune Diabetes in NOD Mice

Sirolimus is an immunosuppressant that inhibits interleukin (IL)-2 signaling of T-cell proliferation but not IL-2-induced T-cell apoptosis. Therefore, we hypothesized that administration of IL-2, together with sirolimus, might shift T-cell proliferation to apoptosis and prevent autoimmune destruction of islet beta-cells. We found that sirolimus and IL-2 therapy of female NOD mice, beginning at age 10 weeks, was synergistic in preventing diabetes development, and disease prevention continued for 13 weeks after stopping sirolimus and IL-2 therapy. Similarly, sirolimus and IL-2 were synergistic in protecting syngeneic islet grafts from recurrent autoimmune destruction after transplantation in diabetic NOD mice, and diabetes did not recur after stopping sirolimus and IL-2 combination therapy. Immunocytochemical examination of islet grafts revealed significantly decreased numbers of leukocytes together with increased apoptosis of these cells in mice treated with sirolimus and IL-2, whereas beta-cells were more numerous, and significantly fewer were apoptotic. In addition, Th1-type cells (gamma-interferon-positive and IL-2(+)) were decreased the most, and Th2-type cells (IL-4(+) and IL-10(+)) and Th3-type cells (transforming growth factor-beta1(+)) were increased the most in islet grafts of sirolimus and IL-2-treated mice. We conclude that 1) combination therapy with sirolimus and IL-2 is synergistic in protecting islet beta-cells from autoimmune destruction; 2) diabetes prevention continues after withdrawal of therapy; and 3) the mechanism of protection involves a shift from Th1- to Th2- and Th3-type cytokine-producing cells, possibly due to deletion of autoreactive Th1 cells.

Matrix metalloproteinase-2 mediates cytokine-induced myocardial contractile dysfunction.

OBJECTIVE Pro-inflammatory cytokines depress myocardial contractile function by enhancing peroxynitrite production, yet the mechanism by which peroxynitrite does this is unknown. As matrix metalloproteinases (MMPs) can be activated by peroxynitrite and can proteolytically cleave troponin I in hearts, we determined whether this occurs in cytokine-induced myocardial dysfunction. METHODS Isolated working rat hearts were perfused with buffer containing interleukin-1 beta, interferon-gamma, and tumor necrosis factor-alpha. RESULTS Cytokines induced a marked decline in mechanical function during 60-120 min of perfusion. This decline was accompanied by increased myocardial inducible NO synthase activity and perfusate dityrosine (a marker of peroxynitrite), compared to control hearts. Before the decline in mechanical function there was enhanced MMP-2 activity in the perfusate. This was accompanied by decreased tissue levels of MMP-2, tissue inhibitor of matrix metalloproteinases-4 and troponin I in cytokine-treated hearts. The collagen content of the heart was not affected by cytokine treatment. A neutralizing anti-MMP-2 antibody or the MMP inhibitors Ro31-9790 or PD166793 attenuated the decline in myocardial function. Moreover, the MMP-2 antibody prevented the decline in myocardial MMP-2 and troponin I levels. CONCLUSIONS Myocardial contractile dysfunction caused by pro-inflammatory cytokines results in MMP-2 activation and a decline in tissue inhibitor of matrix metalloproteinases-4 in the heart. Troponin I is also a target for the proteolytic action of MMP-2 during acute heart failure triggered by pro-inflammatory cytokines. Inhibition of MMPs may be a novel pharmacological strategy for the treatment of acute inflammatory heart disease.

Imbalance Between Tissue Inhibitor of Metalloproteinase-4 and Matrix Metalloproteinases During Acute Myoctardial Ischemia-Reperfusion Injury

Background—We have previously reported that matrix metalloproteinase-2 (MMP-2) contributes to myocardial ischemia-reperfusion injury by degradation of troponin I, a regulatory element of the contractile proteins. MMP activities are also tightly regulated by tissue inhibitors of metalloproteinase (TIMPs). The change in TIMPs during acute myocardial ischemia-reperfusion injury is not clear. Methods and Results—Isolated rat hearts were perfused either aerobically for 75 minutes or subjected to 15, 20, or 25 minutes of global, no-flow ischemia followed by 30 minutes of aerobic reperfusion. During reperfusion after ischemia, there was a rapid, enhanced release of TIMP-4, the most abundant TIMP in the heart, into the coronary effluent, as shown both by reverse zymography and Western blot. There was a negative correlation between the recovery of cardiac mechanical function and the release of TIMP-4 during reperfusion in hearts subjected to different durations of ischemia. Immunogold electron microscopy revealed a close association of TIMP-4 with the sarcomeres in aerobically perfused hearts. Moreover, TIMP-4 was present only in thin myofilaments prepared from aerobically perfused hearts but not in ischemic-reperfused hearts. An enhanced MMP activity was shown in ischemic-reperfused hearts by in situ zymography. Conclusions—Loss of TIMP-4 from the cardiac myocyte leads to an increase in net myocardial MMP activity that contributes to acute myocardial stunning injury.

Role of Cytokines in the Pathogenesis of Autoimmune Diabetes Mellitus

Studies over the last 20 years have examined the possible involvement of cytokines in the pathogenesis of type 1 diabetes (T1DM) through a variety of approaches: (a) correlation studies of cytokines expressed in islets in relation to T1DM development, (b) cytokine augmentation studies, and (c) cytokine deficiency studies. Cytokine augmentations have been created by (a) adding cytokines to islets in vitro, (b) expressing cytokine genes transgenically in β-cells, and (c) administering cytokines and cytokine-producing cells. Cytokine deficiencies have been created by (a) disrupting genes encoding cytokines or their receptors, (b) neutralizing cytokines by anticytokine antibodies or soluble cytokine receptors, (c) blocking cytokine receptors by receptor antagonists or antibodies, and (d) deleting cytokine receptor-positive cells. Information obtained from these studies is summarized in Tables 1 to 3.

DNA fragmentation is an early event in cytokine-induced islet beta-cell destruction

SummaryThe cytokines, interleukin 1, tumour necrosis factor, and interferon gamma are cytotoxic to islet beta cells, however, their mechanisms of beta-cell killing are not fully characterized. Since DNA damage is a mechanism of cytokine-induced cell death in some cell types, we sought evidence for cytotoxic effects of cytokines at a nuclear level in islet beta cells by measuring DNA fragmentation in rat islets and islet beta-cell lines. The individual cytokines, interleukin 1 (10 U/ml), tumour recrosis factor (103 U/ml) and interferon gamma (103 U/ml) inhibited insulin release from rat islets, but did not cause DNA fragmentation or destroy islet cells; by contrast, combination of the three cytokines induced DNA fragmentation and islet-cell death. Cytokine-induced DNA fragmentation preceded cell lysis in islet beta-cell lines (RINm5F, rat insulinoma cells; and NIT-1, NOD/Lt mouse transgenic beta cells), whereas in non-islet cell lines (GH-3, rat pituitary; and PC-12, rat adrenal) the cytokines induced cell lysis and no or late DNA fragmentation. Nicotinamide prevented both DNA fragmentation and destruction of RINm5F islet cells by the cytokines. These findings identify DNA as an early target of cytokine action in islet beta cells, and implicate DNA fragmentation as a mechanism of cytokine-induced beta-cell destruction.