Identification of a Cytotoxic Form of Dimeric Interleukin-2 in Murine Tissues

Interleukin-2 (IL-2) is a multi-faceted cytokine, known for promoting proliferation, survival, and cell death depending on the cell type and state. For example, IL-2 facilitates cell death only in activated T cells when antigen and IL-2 are abundant. The availability of IL-2 clearly impacts this process. Our laboratory recently demonstrated that IL-2 is retained in blood vessels by heparan sulfate, and that biologically active IL-2 is released from vessel tissue by heparanase. We now demonstrate that heparanase digestion also releases a dimeric form of IL-2 that is highly cytotoxic to cells expressing the IL-2 receptor. These cells include “traditional” IL-2 receptor-bearing cells such as lymphocytes, as well as those less well known for IL-2 receptor expression, such as epithelial and smooth muscle cells. The morphologic changes and rapid cell death induced by dimeric IL-2 imply that cell death is mediated by disruption of membrane permeability and subsequent necrosis. These findings suggest that IL-2 has a direct and unexpectedly broad influence on cellular homeostatic mechanisms in both immune and non-immune systems

Role of the Microenvironment in Immune Responses to Transplantation

Through the constant interplay of cellular and extracellular components, the microenvironment of tissues directs immune responses. In solid organ transplantation, one factor that significantly alters the microenvironment of tissues is reperfusion injury, which occurs to a certain extent in essentially all cadaver organs. The damage that results from reperfusion injury initiates a cascade of signals to surveillance cells such as macrophages, mast cells, and dendritic cells, augmenting both innate and allo-immune responses. Chemokines, released from surveillance cells and others, orchestrate an influx of cells into the allograft, and subsequently drive the migration of dendritic cells and lymphocytes to proper areas within lymph nodes for the efficient generation of allo-immune responses. Heparan sulfate, a component of the extracellular matrix, binds chemokines and thus regulates their localization within tissues. This association is one of a multitude of examples of the interplay between cells and their extracellular surroundings. In addition to the association with chemokines, heparan sulfate binds cytokines such as IFN-gamma and IL-2. In the spleen, heparan sulfate localizes IL-2 to the marginal zone, red pulp, and interdigitating dendritic cells of the T cell zone. Our laboratory recently determined that the contribution of heparan sulfate-bound IL-2 to immune responses is substantial, finding that bound, rather than free, IL-2 drives immune responses. This finding reiterates the critical nature of the interaction between cells and the extracellular matrix. Disruptions in these interactions may lead to dysregulation of immune responses and, in turn, pathologies such as tissue fibrosis or autoimmunity. Further studies into the exchange between cells and the extracellular matrix will likely lead to new lines of therapies aimed at correcting these abnormalities before irreversible damage occurs

Role of the Microenvironment in Immune Responses to Transplantation

Through the constant interplay of cellular and extracellular components, the microenvironment of tissues directs immune responses. In solid organ transplantation, one factor that significantly alters the microenvironment of tissues is reperfusion injury, which occurs to a certain extent in essentially all cadaver organs. The damage that results from reperfusion injury initiates a cascade of signals to surveillance cells such as macrophages, mast cells, and dendritic cells, augmenting both innate and allo-immune responses. Chemokines, released from surveillance cells and others, orchestrate an influx of cells into the allograft, and subsequently drive the migration of dendritic cells and lymphocytes to proper areas within lymph nodes for the efficient generation of allo-immune responses. Heparan sulfate, a component of the extracellular matrix, binds chemokines and thus regulates their localization within tissues. This association is one of a multitude of examples of the interplay between cells and their extracellular surroundings. In addition to the association with chemokines, heparan sulfate binds cytokines such as IFN-gamma and IL-2. In the spleen, heparan sulfate localizes IL-2 to the marginal zone, red pulp, and interdigitating dendritic cells of the T cell zone. Our laboratory recently determined that the contribution of heparan sulfate-bound IL-2 to immune responses is substantial, finding that bound, rather than free, IL-2 drives immune responses. This finding reiterates the critical nature of the interaction between cells and the extracellular matrix. Disruptions in these interactions may lead to dysregulation of immune responses and, in turn, pathologies such as tissue fibrosis or autoimmunity. Further studies into the exchange between cells and the extracellular matrix will likely lead to new lines of therapies aimed at correcting these abnormalities before irreversible damage occurs

Regulation of T Cell Homeostasis by Heparan Sulfate-Bound IL-2

Although IL-2 is commonly thought to promote proliferation of T lymphocytes, mice deficient in IL-2 exhibit splenomegaly, lymphocytosis, and autoimmunity, suggesting this cytokine may have a prominent role in T cell homeostasis. Since the number of T cells in the bloodstream and lymphoid organs is tightly controlled, it is likely that the availability of IL-2 must also be closely regulated. One mechanism altering the local availability of cytokines is association with heparan sulfate, a glycosaminoglycan found on cell surfaces and within extracellular matrices. Here we show that an association between IL-2 and heparan sulfate localizes IL-2 to lymphoid organs such as the spleen. We also show that IL-2, sequestered in this way, contributes to the activation of T lymphocytes and primes T lymphocytes for activation-induced cell death

Regulation of T Cell Homeostasis by Heparan Sulfate-Bound IL-2

Although IL-2 is commonly thought to promote proliferation of T lymphocytes, mice deficient in IL-2 exhibit splenomegaly, lymphocytosis, and autoimmunity, suggesting this cytokine may have a prominent role in T cell homeostasis. Since the number of T cells in the bloodstream and lymphoid organs is tightly controlled, it is likely that the availability of IL-2 must also be closely regulated. One mechanism altering the local availability of cytokines is association with heparan sulfate, a glycosaminoglycan found on cell surfaces and within extracellular matrices. Here we show that an association between IL-2 and heparan sulfate localizes IL-2 to lymphoid organs such as the spleen. We also show that IL-2, sequestered in this way, contributes to the activation of T lymphocytes and primes T lymphocytes for activation-induced cell death

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2’ deoxyuridine (BrdU) and 5-ethynyl-2′-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2’ deoxyuridine (BrdU) and 5-ethynyl-2′-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites

Regional Manifestations and Control of the Immune System

Although immune responses are generally considered to be systemic, local events such as interaction of complement products with blood vessels and with inflammatory cells play a pivotal role in determining the nature and manifestations of immune responses. This paper will discuss how blood vessel physiology and immunity influence one another to reach homeostasis upon exposure to an infectious agent. We review new insights into the mechanisms by which the microenvironment of tissues protects against microbial invasion yet facilitates migration of leukocytes and ‘decides’ whether immunity or tolerance ensues and whether, in the face of immunity, protective responses or tissue injury ensues. These ‘decisions’ are made based on interaction of components of normal tissues such as proteoglycans and injured tissues such as cell-associated cytokines with receptors on immune cells and blood vessels