Cytokines in Allergic Disease
The immune system is unique in that it can very selectively discriminate between self and nonself, leaving self alone while rapidly processing and destroying nonself (foreign) antigens in a primary immune response. In addition, a functioning immune system remembers previous encounters with these foreign antigens, resulting in a more vigorous and rapid secondary response (called immunologic memory). These responses depend on both T cells (dependent on the thymus gland for function) and, when specific antibody is made, B cells (which ultimately differentiate into antibody secreting plasma cells). These cells are present in bone marrow, lymphoid organs (i.e. thymus, lymph nodes, tonsils, spleen, etc) and peripheral blood. Many of these lymphoid cells look alike when viewed under a microscope. They can be categorized by the presence of specific cell surface markers called clusters of differentiation (CD). Each CD marker is given a specific number and is found on certain cell types. Thus the function of a cell can often be predicted by the specific CD marker present. For example, all mature human T cells have CD3 on their cell surface (thus they are CD3+). In contrast, human B cells are CD19+ but CD3-. CD3+ T cells can further be divided by function. T cells capable of killing target cells (called cytotoxic T cells- CTL) and/or downregulating immune responses (called suppressor T cells) are CD8+. T cells that help B cells make antibody or other T cells (such as CTL) become active are called helper T cells (TH) and are CD4+.
While it has been appreciated for many years that TH cells existed, it was unclear how helper cells worked until recent studies showed that soluble peptides secreted by activated TH could turn on selective portions of the immune response. Initially, these glycoproteins produced by lymphocytes were called lymphokines. It is now known that many different cell types can produce these immune mediators. The current , more accurate term now used is cytokine.
All cytokines have certain properties in common. They are all small molecular weight peptides or glycopeptides. Many are produced by multiple cell types such as lymphocytes, monocytes/macrophages, mast cells, eosinophils, even endothelial cells lining blood vessels. Each individual cytokine can have multiple functions depending upon the cell that produces it and the target cell(s) upon which it acts (called pleiotropism). Also, several different cytokines can have the same biologic function (called redundancy). Cytokines can exert their effect through the bloodstream on distant target cells (endocrine), on target cells adjacent to those that produce them (paracrine) or on the same cell that produces the cytokine (autocrine). Physiologically it appears that most cytokines exert their most important effects in a paracrine and/or autocrine fashion. Their major functions appear to involve host defense or maintenance and repair of the blood elements (Table 1).
Cytokines are categorized by their major specific function(s). There are four major categories of cytokines (Table 2). Interferons are so named because they interfere with virus replication. There are three major types based upon the source of the interferon. Interferon alpha (IFNa) is produced by the buffy coat layer from white blood cells and is used in treatment of a variety of malignant and immune disorders. Interferon beta (IFNb) is produced by fibroblasts and is currently being evaluated in the treatment of multiple sclerosis. Interferon gamma (IFNg) is produced by activated T cells and is an important immunoregulatory molecule, particularly in allergic diseases.
The colony stimulating factors are so named because they support the growth and differentiation of various elements of the bone marrow. Many are named by the specific element they support such as granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF). Other CSFs include Interleukin (IL) -3, which can stimulate a variety of hematopoietic precursors and is being evaluated as a therapy in aplastic anemia and bone marrow transplantation; and c-Kit ligand (stem cell factor) which has recently been demonstrated as a cytokine necessary to cause the differentiation of bone marrow stem cells into their various precursor elements for eventual differentiation into RBC, WBC and megakaryocytes (platelets).
The tumor necrosis factors (TNF) are so called because injecting them into animals causes a hemorrhagic necrosis of their tumors. TNFa is produced by activated macrophages and TNFb is produced by activated T cells (both TH and CTL). These molecules appear to be involved in the pathogenesis of septic shock and much research is aimed at trying to inhibit their activity in septic patients. Attempts have also been made to use the TNFs clinically to treat human tumors. Because of their extremely narrow therapeutic window (efficacy vs toxicity), few view this as a useful stand-alone cancer therapy.
The largest group is the interleukins, so named because their fundamental function appears to be communication between (inter-) various populations of white blood cells (leucocytes -leukin). Interleukins (IL) are given numbers. They are produced by a variety of cell types such as monocytes/macrophages, T cells, B cells and even non-leucocytes. The major interleukins currently of greatest interest to allergists are IL-4, IL-5, IL-10 and IFNg. IL-4 causes a switch to IgE production by differentiating B cells. IFNg can inhibit that switch and prevent the production of specific IgE. IL-10 can actually inhibit the activity of IFNg, allowing the original IL-4 to proceed in the IgE cascade. Thus, an allergic response can be viewed as an allergen-specific production of excess IL-4 and/or IL-10 , lack of adequate IFNg production or both. Eosinophilic inflammation, a major component of allergic reactions, is under control of IL-5 and TNFa
The Allergic Response
There are three fundamental components of allergic reactions:
formation of allergen-specific IgE ;
activity of mast cells caused by allergen reexposure, which crosslinks IgE on the surface of mast cells, activating them to cause the signs and symptoms of an immediate hypersensitivity reaction ; and
allergic inflammation, mediated primarily by recruitment and activation of eosinophils.
Each of these events involve cellular recruitment to the reaction site (called chemotaxis) along with activation of these cells to produce their products and altered cellular traffic to gather the cells together in an optimal fashion to promote the allergic reaction. Remember, the host mistakenly believes this is a protective reaction. A group of proteins called adhesion molecules can be stimulated on both inflammatory cell surfaces as well as target cells (i.e. endothelial lining of blood vessels, lung tissue, etc.). These molecules function to “localize” the inflammatory reaction at the site of tissue injury and/or antigen deposition. In addition, certain adhesion molecules likely have a role in inflammatory cell activation, further enhancing allergic inflammation.
IgE is one of five isotypes of antibody formed in humans. The cell responsible (B cell) starts with an IgM molecule on its surface that is specific for the antigen (or in the case on an allergic response, an allergen). T helper (TH2) cells assist B cells in making antibody by producing cytokines. One particular cytokine (IL-4) is responsible for causing the isotype switch from IgM to IgE. Although necessary, IL-4 is not in and of itself sufficient to cause a switch to IgE. A second signal, which can come from a variety of sources, is needed to complete the switch. Once formed, IgE seeks to bind to either the inciting allergen or to IgE receptors located on a variety of cell types. Mast cells have high affinity IgE receptors.
Of note, other cytokines are active in the regulation of IgE production. IFNg, produced by TH1 helper cells, can antagonize the ability of IL-4 to induce IgE production Recent studies have shown that T cells from nonatopic patients, when stimulated in vitro by specific allergen, produces primarily IFNg while T cells from atopic patients produce allergen-induced IL-4. Further, TH2 cells can produce IL-10, which can inhibit the production of cytokines such as IFNg. Thus IgE could be the prevalent antibody if TH2 rather than TH1 helper cells are stimulated in an atopic individual.
Once allergen-specific IgE is generated and bound to mast cells, subsequent allergen exposure causes crosslinking of mast cell-bound IgE resulting in degranulation. This process takes only a matter of minutes and releases a variety of mediators, including histamine. Histamine binds to target receptors in the nose, lung, skin, gastrointestinal tract and near blood vessels via specific histamine receptors, especially H1 receptors. This activates a series of events leading to increased vascular permeability and dilation, stimulation of nerve fibers and initiation of inflammatory cascades that are collectively responsible for the signs and symptoms of immediate hypersensitivity – itching, sneezing, increased mucus secretion (i.e. rhinorrhea, etc.), bronchospasm and, if enough vascular tissue is involved, hypotension.
Mast cells themselves both respond to and produce cytokines. IL-3 is a mast cell growth factor. Mast cells make IL-4 when stimulated. This may be particularly important in the propagation of IgE- producing B cells as well as the differentiation of T helper cells to the TH2 pathway (both necessary for IgE production). In addition, IL-4 appears to be a secondary but important growth factor for mast cells. Mast cells also make and secrete TNFa. This cytokine has important inflammatory properties that are consistent with the known proinflammatory activities of mast cells.
Eosinophils both respond to and manufacture certain cytokines. IL-5 appears to be a major growth factor for eosinophils. IL-5 is also produced by TH2 cells, further supporting the developing allergic cascade. Eosinophils can secrete many cytokines such as IL-3,GM-CSF, TNFa and IL-1 when activated. Any or all of these cytokines serve to enhance and sustain the allergic inflammatory process by mast cell activation (IL-3), further eosinophil recruitment (TNFa), altering the target tissue (IL-1) and even direct tissue damage. The activated eosinophils also produce and secrete multiple basic proteins and lipid mediators associated with allergic inflammation.
Inflammation has three major components: recruitment , where the inflammatory cells are drawn from the circulation under direct a chemical influence called chemotaxis; altered traffic, where the inflammatory cells are held at the site of developing inflammation; and activation, where the inflammatory cells exert their influence , e.g. producing cytokines, lytic enzymes, phagocytosis, etc. In allergic inflammation, a combination of TH2 cell and mast cell activity appear to be most responsible for the initiation of the eosinophilic activities.
A major source of chemotactic molecules is the activated mast cell. TNFa is a major proinflammatory cytokine whose activities include chemotaxis. Activated mast cell secrete TNFa and therefore may directly influence recruitment of eosinophils. Once activated, eosinophils are themselves a source of secreted TNFa which may serve to continue the recruitment of new eosinophils to the site of inflammation.
Altered traffic also involves changing how the inflammatory cells migrate through tissue. Most leucocytes migrate into tissue from the circulation. This process involves sticking to the endothelial lining of the blood vessel and movement (called diapedesis) between adjacent cells of the blood capillary endothelium to the site of developing inflammation in the tissue. Fundamental to this process is. the expression of adhesion molecules on both the leucocytes and endothelial target tissue. These adhesion molecules are necessary to keep the inflammatory cell at the target tissue site as well as , in certain cases, participate in the cellular activation process.
Cytokines play a fundamental role in adhesion molecule expression. IL-1 can act on the endothelial cell to increase the expression of several adhesion molecules such as ELAM-1(endothelial leucocyte adhesion molecule) , ICAM-1 (intercellular adhesion molecule) and VCAM-1 (vascular cell adhesion molecule). The specific biochemical properties of these molecules is a subject for future discussion. It appears that VCAM-1 expression may be most important in allergic (eosinophilic) inflammation . Another adhesion molecule of special importance in allergic reactions is VLA-4. This molecule is expressed on activated lymphocytes, mast cells and eosinophils. Thus , expression of VCAM-1 on endothelium (of , say, the nose or lung) and VLA-4 on activated mast cells and eosinophils are necessary steps for eosinophilic infiltration of these organs in allergic late phase reactions. This has become particularly significant with the discovery that IL-4 induces the expression of both VCAM-1 on endothelial surfaces and VLA-4 on eosinophils. TNFa further enhances IL-4-induced VCAM-1 expression.
Thus, the cytokine influences on allergic inflammatory reactions can be summarized into three major components:
- Induction of allergen-specific IgE
- IL-4 upregulates IgE production
- Gamma IFN downregulates IgE production
- IL-10 inhibits the production/activity of gamma IFN
- Mast cell activation
- IL-3, IL-4 – mast cell growth factors,
- TNF alpha – proinflammatory, chemotactic
- Th2 differentiation
- VCAM-1, VLA-4 induction
- Eosinophil inflammation
- IL-5 – eosinophilic growth factor
- IL-3 – supports mast cell growth
- GM-CSF – proinflammatory effects
It follows that excess, allergen-specific TH2 activity could produce activation of each component of the allergic cascade. It is reasonable to hypothesize that allergic disease is characterized by an imbalance between allergen- specific TH1 and TH2 activities.
Therapeutic Potentials of Cytokines in Allergic Diseases
This knowledge provides possibilities of new targets for therapeutic activity. Many therapeutic modalities have been examined for their effects on these TH subpopulations . Several have been demonstrated to have significant activities on this arm of the immune response. Currently, the most extensively used antiinflammatory agents, in various topical and systemic formulations, are the corticosteroids. While steroids have direct activities on inflammatory cells such as mast cells and eosinophils, they also exert a regulatory effect on cytokine production.
Recall that the immune reaction that leads to IgE production involves antigen presenting cells(APC- usually macrophages), T cells and B cells. The APC produces IL-1, TNFa and IL-6, all proinflammatory cytokines. In addition, IL-1 activates helper T cells to produce IL-2 (and other cytokines) which activates the immune cascade. Corticosteroids have a fundamental effect on IL-1 secretion and, thus, the ultimate production of IL-2 and resulting T cell activity. Further, mast cells produce their own cytokines including proinflammatories such as TNFa. These are also downregulated in the presence of corticosteroids
Perhaps most exciting is recent data from several investigators that address the effects of allergen immunotherapy (AIT) on cytokine production. What has been appreciated for many years is the positive clinical effects of AIT on symptoms of allergic diseases such as rhinitis and asthma. Until recently, the mechanisms were largely unknown. Now we are beginning to understand that the process of AIT can selectively change allergen-specific cytokine profiles. That is, when an atopic patient’s peripheral blood lymphocytes (PBL) are stimulated in vitro with allergen, they produce mostly IL-4. In contrast, PBL from patients who were highly atopic but treated with AIT did not produce IL-4 but rather produced large amounts of IFNg. In other words, it appears that successful AIT is characterized by a switch from allergen-specific TH2 to TH1 expression. The effect appears to be durable and related to length of AIT. It correlates reasonably well with symptom improvement.
The fact that successful AIT takes several years to achieve and the still controversial positions about duration of therapy suggests that we are not yet at the definitive stage for allergy therapy that we desire. This raises the possibility of new therapeutic modalities that can achieve clinical control of allergic inflammation with the rapidity of corticosteroids and specificity of AIT. Such modalities could include cytokines themselves or pharmacologic agents that can modulate specific cytokine profiles.
Recombinant cytokines are being studied in a variety of clinical conditions such as malignant, infectious, autoimmune and allergic/asthmatic diseases. What is apparent is the limited use of these recombinant cytokines for three reasons: (a) they are extremely expensive, something not viewed kindly in the new age of managed health care; (b) they are extremely toxic, causing various systemic symptoms such as fever, chills, muscle aches and fatigue. In higher doses they are potentially life threatening by their effects on causing hypotension; and (c) since these immune-based diseases (particularly allergic/asthmatic) are most likely an imbalance of “good” and “bad” cytokines, giving large pharmacologic doses of recombinant cytokines may create other imbalances in the host that could create other disease entities.
With those concerns, several studies have provided promising results in diseases such as atopic dermatitis (AD). IFNg has been shown to clinically improve children with severe AD and can be steroid-sparing. It is still extremely expensive and can make the children ill. IFNg is also being el\valuated in topical form in asthma.
Finally, there are new immunomodulators being studied that can selectively activate cytokine production in a positive way at the inflammatory site. While not of themselves antigen-specific, they tend to be more effective in networks that are already antigen (or allergen)-activated. This may in part explain the long known observation that, in certain patients, treatment with AIT that does not involve a specific allergen (i.e. animal dander) provides relief of documented symptoms after successful therapy for pollens. Microenvironmental production of IFNg to pollens may act on the animal dander-specific TH2 clone to downregulate its activity. Such a hypothesis remains to be verified.
The understanding of the allergen-specific TH1/TH2 functional ration and its importance in the pathophysiology of allergic diseases may also have utility in clinical monitoring situations. It is provocative to consider clinically useful laboratory assays that could establish the TH2 nature of a specific response and then monitor response to therapy such as AIT by watching for the change to a normal balance. This could also be useful in evaluating therapeutic agents to determine dose and duration of therapy. Such concepts are currently being investigated as research related to allergic inflammation and cytokines continues to move steadily from bench to bedside.