Immunomodulation: Principles and Mechanisms

1. Introduction

Equine clinicians frequently seek to increase normal immune responses as well as restore deficient and temper over-exuberant host immune responses. For these reasons, modulation of the immune system has become an area of intense interest in clinical medicine. Both immunostimulants and immunosuppressants are considered immunomodulators. There are a variety of ways to classify immunomodulators beyond this distinction, but for practical purposes, they may be best classified based on their origin (i.e., physiologic products [normal components of the immune response], microbial products, and chemically defined agents). Immune responses depend on complex and carefully integrated networks of regulatory events. Our current diagnostic methods do not allow us to precisely identify in vivo defects, deficiencies, or excesses of substances or regulators present within the immunoregulatory network. Consequently, our attempts to intervene with immunomodulators are often relatively crude and non-specific. Some of the information on which we base our use of immunomodulators has come from controlled, experimental studies; some of these studies were performed in vitro, and others were performed in vivo. In clinical patients, expectations based on these types of studies are frequently not realized. Reasons for this include the timing of the administration of the immunomodulator during the course of the disease and the fact that observation of a single immunological phenomenon resulting from use of an immunomodulator (e.g., an increase in lymphocyte count or lymphoproliferative responses) does not necessarily translate into improved clinical performance in the face of an infectious disease.

Immunomodulators have been embraced by clinicians with great enthusiasm, and the concept of their use remains appealing. However, although the clinical value of immunosuppressive drugs like corticosteroids and immunostimulant adjuvants in vaccines is clearly established, evidence for the clinical value of many immunostimulants is sparse. It is important to critically evaluate the immunomodulatory drugs on the market and to objectively evaluate the effects of therapy.

2. Immunosuppressors: Corticosteroids, Cytotoxic Drugs, and Fungal Derivatives

Corticosteroids are classic examples of immunosuppressive agents. Corticosteroids exhibit an extensive range of effects on elements of both the innate (inflammatory) and adaptive immune response. The mechanism of action of corticosteroids is illustrated in Figure 1. Corticosteroids, acting through their cytoplasmic steroid receptors, directly regulate as many as 1% of the genes in the genome, usually resulting in induction of transcription [1]. The effect on the adaptive immune response is complex [2]. For example, in cases of autoimmune disease (e.g., autoimmune hemolytic anemia), corticosteroids may act by reducing phagocytosis of antibody-coated cells by the reticuloendothelial system rather than decreasing antibody production. Nevertheless, corticosteroids have been shown to have effects on antibody production. In the horse, they can suppress de novo antigen-specific IgGa and IgGb responses and spare IgG(T) responses [3]. This can help explain the anti-inflammatory activity of corticosteroids, because IgGa and IgGb are the equine immunoglobulin subclasses that are most efficient at mediating inflammatory responses by complement fixation and recruitment of polymorphonuclear leukocytes. Cell migration is significantly affected by corticosteroids. In the horse, corticosteroids have been shown to suppress neutrophil migration as well as phagocytic and bactericidal activity [4].

Figure 1. Lipid-soluble corticosteroid molecules diffuse across the plasma membrane into the cytosol and bind to steroid receptors. This displaces a heat-shock protein (Hsp90) that is normally bound to the nascent steroid receptor, and it exposes a DNA-binding region. The new complex enters the nucleus and binds to specific regulatory DNA sequences, which results in modulation of transcription of a wide variety of genes.

Although corticosteroids are powerful immunosuppressive agents, they may also pre-dispose horses to life-threatening opportunistic infections [5] or recrudescence of viral infection [6-8]. Additional undesirable side effects include fluid retention and decreased wound healing. For this reason, prolonged or high-dose corticosteroid therapy must be used judiciously. Overall, corticosteroids remain the most potent and widely used immunosuppressive agents available to equine veterinarians, particularly for the treatment of non-infectious inflammatory diseases.

The two commonly used immunosuppressive cytotoxic drugs, azathioprine and cyclophosphamide, both interfere with DNA synthesis and act primarily on dividing cells [1]. This activity is useful for treatment of cancer and for suppression of dividing lymphocytes. Toxicity limits the use of these drugs. However, lower doses can be used in combination with corticosteroids. Two case reports describe the use of these drugs. They had some success in horses suffering from immune-mediated hemolytic anemia or thrombocytopenia after corticosteroid therapy alone had failed [9,10].

Fungal derivatives are relatively non-toxic alternatives to immunosuppressive cytotoxic drugs. Cyclosporine is a fungal derivative and has emerged as a major immunosuppressive agent for allograft survival, because it selectively inhibits proliferation, cytotoxicity, and lymphokine production by T cells. Cyclosporine is efficacious in suppressing specific immune responses with minimal non-specific toxic effects on polymorphonuclear leukocytes, monocytes, and macrophages. Thus, immunosuppressed patients suffer fewer severe secondary infections. The drug is not hazard free. In addition to suppressing lymphocyte responses in general, it is also toxic to the kidneys and other organs. In horses, the use of cyclosporine has been limited to topical therapy for ocular inflammatory disease including keratitis [11] and uveitis [12].

3. Immunostimulants

Physiologic Products: Immunoglobulins and Cytokines

We use immunoglobulins in a variety of ways to affect the immune system, most obviously for the treatment of failure of passive transfer of immunity or for antigen-specific passive immunoglobulin therapy for prevention of diseases such as tetanus or R. equi infection. Another mode of immunoglobulin therapy is polyclonal IV immunoglobulin (PIVIG) therapy using pools of immunoglobulin derived from several thousand donors. It was observed that PIVIG administration was useful for treatment of autoimmunity [13,14], and many mechanisms have been proposed to explain this effect [15,16]. Although the explanation for the mechanisms underlying PIVIG’s effects are uncertain, there are good examples of therapeutic success in the case of immunothrombocytopenic purpura [17]. However, diseases such as autoimmune diabetes mellitus are resistant to therapy [18]. There are no publications describing PIVIG therapy in horses, but it is possible that equine plasma pooled from many donors could similarly modulate immune responses in recipients.

Given the central role of cytokines in immunoregulation, their potential as immunomodulators is enormous. To date, only two cytokines have found clinical application in horses, interferon-a and granulocyte-colony stimulating factor (G-CSF). These treatments have been extensively reviewed [19].

Because much of the discussion on immunostimulants will depend on some understanding of cytokine networks, a brief review of the regulatory interactions of the innate and adaptive immune responses will be helpful. Encounters with antigenic pathogens are depicted in Figure 2, which illustrates antigen-presenting cells (APC) such as macrophages or dendritic cells phagocytosing the invader and presenting antigenic material to T-helper cells.

Figure 2. See text for explanation.

These encounters can be influenced by the pathogen’s simultaneous interactions with components of the innate (non-adaptive) immune system (e.g., natural killer [NK] cells in the case of viral and bacterial invaders or mast cells and basophils in the case of helminths). These cells can also receive additional signals from the antigen-presenting cell. The regulatory cytokines released by the innate immune system can in turn influence the outcome of antigen presentation to the T-helper cell by contributing to the polarization of the T-helper response toward either a Th-1 or a Th-2 phenotype. This is a critical step, because the phenotype of the T-helper cell significantly influences the type of immune response that is generated toward the pathogen (this process is illustrated in Figure 3). Many immunomodulators, including both antigen-specific and non-specific stimulants or suppressors, mediate their activity through affecting this axis.

Figure 3. Th1 and Th2 regulation. The Th1 lymphocyte subsets provide help for macrophage activation, cytolytic activity, and production of a subset of IgG subclasses. The Th2 subset promotes antibody responses including IgA, IgE, and the remainder of the IgG subclasses. This is mediated by production of cytokines that have a regulatory effect on each other.

Bacterial, Viral, and Plant Products

A variety of bacterial and fungal microorganisms or microbial products have been identified that have an immunomodulating effect on the immune system. A common feature of many of these products is a non-specific immunostimulant effect, purportedly caused by macrophage activation and release of cytokines including interferons, interleukin (IL)-1, tumor necrosis factor (TNF), or IL-6 [19]. Consequently, mild fever and malaise may be associated with this form of treatment. In the horse, these treatments are most commonly used in cases of respiratory infection or sarcoids.

Parapoxvirus Ovis

Parapoxvirus ovis is the etiologic viral agent of contagious ecthyma or "orf" in sheep. The immunostimulant properties of poxvirus were first noted after routine smallpox vaccination. Some patients receiving the vaccine experienced spontaneous tumor regression and resolution of chronic viral and bacterial infections. Marketed under the trade name Baypamune [a], inactivated parapoxvirus ovis has been used extensively in Europe for prophylaxis and treatment of infectious disease in companion animals (including horses) and pigs [19]. The immunostimulant properties (increased NK activity and macrophage activation) are independent of viral replication and depend on a component of the viral envelope. Efficacy has been proven against viral and bacterial disease in several species [19], and there is some evidence in horses that prophylactic administration before weaning reduces signs of respiratory disease after weaning [20]. The recommended dosage schedule is two to four doses at 48-h intervals, and the immunostimulant activity is reported to occur within hours of administration. The maximum duration of immunostimulatory activity after parapoxvirus ovis administration is 8 days. In studies of treatment of sarcoid, there was no evidence of efficacy [21].

Recently, we conducted a study to test the value of Baypamune when used prophylactically in horses that we subjected to transport stress and then exposed to influenza virus-infected and contagious horses in a group-housing environment. Influenza-naïve mixed-breed yearling horses were used for this trial. A control group of 10 horses was established in Wisconsin in a group-housing environment for 1 wk, and all horses were subsequently infected with equine influenza virus by nebulization using a face mask on day 0 of the experiment. Two groups of 20 horses each were shipped from Montana to Wisconsin. They departed on day 0 and arrived on day 2. One of these groups was treated with Baypamune on days -2, 0, and 2 (treatment group). The other group received a placebo injection (untreated group). Personnel did not know which horses were receiving the placebo and which horses were receiving Baypamune.

Clinical signs of disease (rectal temperature, respiratory rate, nasal discharge, cough, and body weight) and clinico-pathologic date (complete blood cell counts and nasal viral shedding) were measured daily from day 2 to day 16. After day 16, the horses were periodically checked until the experiment was terminated on day 28. Serum antibodies to influenza virus were measured by single radial hemolysis (SRH) [22] every week throughout the experiment. Viral shedding was determined by egg inoculation and determination of egg infectious dose 50% (EID50). The Wilcoxian rank-sum test was used to test for differences between the treated and untreated groups, and p ≤ 0.05 was considered significant.

All horses in the control group developed clinical signs of the influenza virus infection on day 2, including pyrexia, tachypnea, mucopurulent nasal discharge, paroxysmal coughing, and body weight loss. Both the treated and untreated group horses developed similar signs of disease by day 5 (i.e., 3 days after exposure to the influenza virus-infected control group horses). Elevations in respiratory rate and rectal temperature and the onset of coughing were slightly reduced in the treated group, although this was only statistically significant for respiratory rate. Both the treated and untreated group horses developed leucopenia by day 9 with a rebound leucocytosis evident from day 14 to 22. All horses in all groups shed influenza virus, and EID50s were similar in the treated and untreated groups.

Results of the SRH analyses of the serum anti-influenza virus antibody responses showed that all horses were seronegative on day 0 and again on day 7 (Fig. 4). All horses showed evidence of a primary antibody response on days 14, 21, and 28. This response was significantly lower in the untreated group on each of these days.

Figure 4. Mean influenza virus-specific antibody levels determined by SRH assay. Control horses (n = 10) were experimentally infected with the influenza virus on day 0. Treated horses (n = 20) received an immunostimulant on days -2, 0, and 2. Untreated horses received a placebo. Treated and untreated horses were infected by exposure to the control horses.

The implications of this study for our understanding of the spread of the influenza virus are considerable, but with regard to the efficacy of the immunostimulant Baypamune, the findings are more equivocal. Although the use of an immunostimulant did not substantively reduce clinical or virological signs of infection in the face of this severe challenge infection, it did result in significantly increased antibody responses in treated horses. The reduction in antibody responses in the untreated group horses compared with the control group horses seems to be associated with the stress of transport in the untreated group. The restoration of antibody responses in the treated horses to levels equal to those in the control group is consistent with the fact that immunostimulant treatment reduces the immunosuppressive effect of transport. Although clinical disease was not affected in this model, this result does indicate the potential value of this immunostimulant therapy for improving immune responses in immunosuppressed horses.

Acemannan

Acemannan is an extract of the aloe vera plant predominately used for treatment of fibrosarcomas in dogs and cats [19]. It can also be used as a vaccine adjuvant, and in addition, it has antiviral activity. Anecdotal reports describe efficacy for treatment of equine respiratory disease (IV) and sarcoids (intralesional), although both forms of treatment are associated with side effects including syncope, tachycardia, tachypnea, and sweating. Controlled studies of efficacy in the horse are lacking.

Echinacea

Extracts of Echinacea angustifolia has been reported to be an immunostimulant. One study in horses evaluated the effect of an experimental oral Echinacea extract on neutrophil number, phagocytosis, and lymphocyte count [23]. Findings provided limited support for changes in cell count and behavior consistent with immunostimulation; however, the evidence was very limited.

Chemically Defined Agents: Levamisole

Levamisole is a synthetic anthelmintic used for treatment of nematode infections that has also been reported to restore impaired host immune defenses [19]. Levamisole seems to have little effect on the normal immune system, but it seems to stimulate a subnormal response and suppress hyperactive responses. The effects are dose related. Low doses are reported to enhance responses, and higher doses are reported to suppress responses. In cattle, levamisole has been shown to enhance lymphoproliferative responses in vitro, although in vivo co-administration with vaccine had no immunostimulant effect [24]. Similarly, levamisole did not prevent corticosteroid-mediated immunosuppression in cattle [25] or enhance post-partum lymphoproliferative responses in pigs [24].

In humans, levamisole enhances lymphoproliferative responses in post-operative patients and reduces viremia in patients with chronic hepatitis-B infection [26,27]. Levamisole improves cell-mediated immune responses and lymphocyte cytotoxicity in children suffering from severe protein-calorie malnutrition and chronic respiratory infection [28]. It is also reported to be an effective adjunct treatment for rheumatoid arthritis and chronic bronchitis [29]. Controlled investigation of the immunostimulatory effects of levamisole in healthy or immunocompromised horses has not been reported. Levamisole has been recommended by some clinicians as adjunct treatment of equine protozoal myelitis.

4. Antigen Specific Immunomodulation

Vaccination and Adjuvants

Vaccination is a critically important tool in preventing infectious disease in humans and animals, and both passive and active vaccination are extensively employed in the horse [30]. Vaccination can be active where an antigen-specific immune response is provoked in the vaccinated animal by administration of an antigen in the form of a dead, live, or DNA vaccine. Success of all of these forms of vaccination is frequently dependent on the use of an effective adjuvant, which is a compound capable of amplifying and directing immune responses [31]. As such, adjuvants are one of the most important forms of immunomodulating agents in use in equine medicine.

Passive vaccination is accomplished by administering preformed antibodies either as a plasma transfusion or in a concentrated form, such as in commercially available tetanus antitoxin. This strategy can be highly effective in diseases for which there is no available form of active vaccination (e.g., R. equi) or in high-risk situations when there is inadequate time for protection to be generated by active vaccination.

Hyposensitization

Because horses suffer from a number of hyposensitivity diseases, attempts have been made to perform antigen-specific immunosuppression. Examples include Sweet Itch and Recurrent Airway Obstruction. The principle of this type of therapy is that the immune response to an allergen can be redirected to reduce hypersensitivity disease. For example, Type 1 hypersensitivity disease may depend on a Th2 immune response. Treatments that can change this to a Th1 immune response may eliminate or control the hypersensitivity disease by changing the immune response from one dominated by IgE to one dominated by IgG [32]. Typically, hyposensitization treatments use injections of the allergen itself, starting with very small doses and gradually increasing the dose over time. This form of treatment depends on correct identification of the allergen against which the hypersensitivity disease is directed, and the difficulty in identifying these allergens using available intradermal testing methodologies [33,34] may provide an explanation for the mixed success of hyposensitization treatment in horses [35]. Prospects for hyposensitization therapy may be improved by new techniques to produce large numbers of recombinant allergens [36,37]. These allergens are far better defined than conventionally prepared allergen extracts, and in initial experimental studies of recurrent airway obstruction (RAO), they were found to be far superior in terms of specificity and sensitivity for detection of allergen-specific IgE. Such developments, together with the developments of DNA vaccination strategies incorporating CpG immunomodulation for hyposensitization [38-40], mean that there is a good chance that new and effective therapies will be developed in the future.

5. Conclusions

The properties and efficacy of immunosuppressive agents are generally well documented as are the effects of antigen-specific immunostimulant adjuvants. These treatment modalities are well-established and important tools in equine clinical medicine. Our understanding of the practical value of non-specific equine immunostimulants is less developed and requires careful scrutiny of controlled clinical studies to select useful treatments.

Footnote

1. Baypamune, Bayer Corporation, Animal Health Division, PO Box 390, Shawnee Mission, KS 66201-0390.