Autoimmune disease treatment in dogs currently relies on the use of broad-spectrum immunosuppressive drugs, which are associated with unacceptable adverse effects in some patients. Immune Mediated Hemolytic Anemia (IMHA) requires high doses and long-term administration of these medications, often resulting in a terrible choice for the pet owner between the disease, the medications or euthanasia (1) (2) . Stem cell therapy offers an alternative option for these pets.
Immune Mediated Hemolytic Anemia (IMHA) is caused by antibody deployment against red blood cells. The presence of these autoantibodies initiate destruction of the red blood cells through one of two ways: complement activation resulting in hemolysis (rupture) of the red blood cells within the blood stream, or macrophages in the spleen and liver gradually destroy the red blood cells(3). Immunosuppressive drugs dampen the immune response but do not address the underlying cause – the failure of immune tolerance. Stem Cell Therapy addresses both the immune tolerance issue of anti-red blood cell antibody production and protects the antibody tagged red blood cells from destruction. Stem Cells, unlike conventional therapeutics are alive and dynamically self-modulate their response to immune disease thereby preventing over-reaction or overdoses in therapy(4) .
The red blood cells of normal pets are produced by the bone marrow, they live for about 100 days and are then consumed by the macrophages in the spleen and liver in a process called eryptosis (red cell suicide)[ CITATION Flo12 \l 1033 ]. An average sized 25 lb. dog has about 60 x 1012 (60 trillion) total red blood cells at any one time and each day 600 billion red blood cells die and are replaced. Anemia is caused when the rate of destruction of cells is greater than the rate of production of new cells.
Immune reactions against red blood cells happen when antibodies are made against the proteins on the surface of these cells. These antibodies signal that these “tagged” cells should be destroyed. This red cell destruction happens either in the bloodstream (intravascular hemolysis) or within the liver and spleen (extravascular hemolysis).
The by-products of red blood cell destruction affect the liver and kidney and the lungs causing blood clots and multiple organ failure. Death occurs in approximately 25% of extravascular cases and 75% of intravascular cases. Thrombosis in the lungs is a very common cause of death in many of these dogs (5).
Immune Mediated Hemolytic Anemia has primary and secondary types. Primary IMHA occurs about 75% of the time where the antibodies are produced against “normal” red blood cell proteins. Secondary IMHA occurs when there is another disease affecting the red blood cells that initiates the IMHA. Factors such as infection, antibiotic administration, exposure to toxins, tick borne diseases,lymphoma, hemangiosarcoma, allergic reactions or anything that causes oxidative stress on the red
blood cells can cause the B-cells to attack the red blood cells with antibodies resulting in secondary IMHA. Secondary IMHA is usually a temporary disease that resolves when the offending substance or infection is removed, stopped, or treated. Primary IMHA occurs when normal red blood cell proteins are attacked abnormally. Primary IMHA has two forms – Intravascular and Extravascular.
Intravascular hemolysis (figure 1) occurs as a result of antibody production against the red blood cell surface antigens usually by the IgM class of antibody which activates complement. Complement causes the red cell to swell and burst within the blood stream. Once red blood cells are destroyed, the clinical signs are mostly attributable to loss of oxygen to the tissues. The by-products of red blood cell destruction affect the liver and kidney and the lungs causing blood clots and multiple organ failure.
Intravascular IMHA has a much higher mortality rate (75%) and occurs when the antibodies activate Complement.
Extravascular IMHA occurs when “nibbles” are bitten off the surface of the red blood cells by macrophages as the red blood cells pass through the spleen and liver (figure 2). This slower process is still very serious but has only a 25% mortality rate.
Immune Mediated Hemolytic Anemia is caused by a malfunction of immune tolerance. Immune tolerance is the discrimination of normal or “self” proteins versus abnormal or “non-self” proteins. Immune tolerance is mediated by lymphocytes. Lymphocytes are white blood cells produced in the bone marrow from the mother cell called a lymphoblast. B-Lymphocytes (B-cells) produce the abnormal antibodies that cause IMHA. Another group of cells called T-Lymphocytes (T-cells) get specific orders in the Thymus gland about determining “self” tissues vs. “non-self” and control “who” the B-cells produce antibodies against. IMHA arises when the T-cells and B-cells are out of balance.
Complement is the name for a group of molecules present in the blood plasma that enhances (complements) the immune system. These molecules when combined cause intravascular hemolysis in IMHA. Complement activation is shown in Figures 4 and 5.
Factor H regulates complement. Factor H is a chemical produced by the liver. Factor H provides control by binding to the surfaces of cells to protect cell surfaces from the cutting and puncturing of the knife-like complement molecule. Like the Kevlar worn by law enforcement officials protects them from knives and bullets, Factor H protects the cells from complement attack. Factor H is produced by stem cells (6) (7) (8) .
We have known for some time that Mesenchymal stem cells (MSCs) possess potent immuno-suppressive properties (9) . But only recently did we understand that MSCs produce Factor H (10) . Mesenchymal stem cell therapy has been shown to stop the damage from many auto-immune diseases; and one of the mechanisms for this effect is due to the production of Factor H by the mesenchymal stem cells and subsequent protection against complement damage (11) . MSCs production of Factor H is constitutive (12) . This means that Factor H is produced in relatively constant amounts regardless of the environment or stimulation. This is an important justification for the use of allogenic mesenchymal stem cells for the treatment of IMHA.
The purpose of the immune system is to remove foreign invaders. To do this it has to recognize who is supposed to be in the body and who is alien and a potential threat to the body. Red blood cells that are being attacked by antibodies in IMHA have been mistakenly determined to be foreign. How did this happen and how can this problem be fixed?
There are natural balances in the controls of the immune system that we are just coming to understand. These balances involve the production of different types of T-Lymphocytes (T-cells) depending on the need for immunity vs. the need to tolerate the presence of a seemingly abnormal protein or substance. For example, our intestine houses many species of bacteria that are arguably foreign to our bodies, yet our immune system tolerates their presence. Mammals who maintain placental pregnancy within their bodies must tolerate the foreign tissue of the developing fetus with different DNA than the mother and not attack it as foreign. The surface of the skin and opening of the mucous membranes harbor “normal” bacterial and fungal flora that must be tolerated by the immune system. On the other hand, cells that are infected or damaged or that have turned cancerous should be attacked and removed from the body by the same immune system. It is this balance that is
defective in patients with IMHA.
Recent experiments involving the thymus gland have uncovered previously unknown elements of the T-cell populations that help control these systems. The thymus is a small gland in the chest between the left and right lungs at the level of the trachea that has been found to be essential in the control of the immune system. T-cells are named after the thymus because they are processed within this small organ. Mutant mice which do not have a thymus gland spontaneously develop fatal,
widespread, early-onset autoimmunity (13) . Removal of the thymus gland in three-day old mice led to the rapid development of autoimmunity that was corrected by the infusion of T-cells from adult mice. This research advanced our understanding of the creation and management of the T-cell lineage and how it controls the immune system.
Certain T-cell types regulate or keep in check the aberrant or overactive activity of other T-cells. When the immune system functions normally, the immune system produces a population of T-cells that have regulatory functions called regulatory T cells or Tregs that are specialized for immune suppression (14) . In addition, it is becoming apparent that every immune reaction not only creates specific B-Cells and T-Cells but also Tregs and the balance between the populations of these cells is critical for the proper control of the immune response. IMHA is an example of an excessive attack on self-antigens as a result of improper immune tolerance resulting from dysregulation or deficiency of the Tregs (1) (14) .
Mesenchymal stem cells (MSC’s) exert powerful immunomodulatory effects by both cell-to-cell contacts and by secreting powerful chemicals called cytokines and chemokines (15) . MSC’s affect both T- cells and B-cells while promoting the generation of regulatory T-cells. Mesenchymal stem cells release the cytokine Transforming Growth Factor Beta (TGF-β) which induces the development of Treg cells. MSC, also secrete the Interleukin-10 (IL-10) and Interleukin-6 (IL-6) (4) (13) (12) (15) (16) . The interplay between these interleukins and TGF-β has been well documented and is important in understanding the clinical effects noted using MSC’s as a therapeutic means for the treatment of autoimmune diseases (15) . This is an important justification for the use of allogenic mesenchymal stem cells for the treatment of IMHA.
To understand the implications of treatment choices made for the benefit of pets with IMHA it is best to understand how the immune system works normally when fighting disease and how it is deranged in the case of IMHA. Figure 8 explains in detail how different types of T-cells are produced in the thymus.
Lymphocytes are on constant surveillance; their job is to detect and remove invaders and abnormal cells. All lymphocytes are formed in the bone marrow, but, where they mature determines their function. B-cells mature in the bone marrow. T-cells mature in the thymus. The thymus is like “Boot Camp” for T-cells. The T-cells must pass two levels of test: “worthiness” and “loyalty”. The T-cells first prove worthiness by “memorizing” a list of all the body’s “self” proteins. This list is called the Major Histocompatibility Complex I (MCH I). The thymus tests each T-cell to make sure they have memorized the list. T-cells that pass the MHC I test are “worthy” of being a “soldier” and given a “badge” called CD8+. T-cells are also tested for the Major Histocompatibility Complex Type II (MCH II). This list has additional information about white blood cells that process antigens. Antigen processing is the identification of the “bad guys” viruses, bacteria, and other disease. The cells that pass this test are labeled with the CD4+ “badge”. T-cells that do not pass “Boot Camp” are “unworthy” and killed. This first level of “worthiness” testing creates T-cells with CD4+ badges and other T-cells with CD8+ badges.
T-cells that pass “Boot Camp” are termed Naïve T-cells. Only about 2% survive “Boot Camp”. CD8+ T-cells become “Killer T-Cells”. Their job is to check all cells of the body for the MCH I list. All cells in the body should have this MCH I “fingerprint”. If body cells do not have the MCH I then the job of the CD8+ cells are to kill these “not-self” cells on the spot. The second test for T-cells in “Boot Camp” is the “loyalty” test. During this test, the thymus cells encourage or “trick” the T-cells to defect and kill normal “self” body cells. T-cells fail this test by killing “self” body cells. T-cells that fall for this ploy are
deemed “not loyal” and are killed. There is a third group of T cells. There are some CD4+ T cells that do not pass or fail the “self”, “not-self” test. They survive by walking a fine line between protecting “self” cells and killing “self” cells. They are called “goldilocks” cells; “not-too-hot”, “not-too-cold”, “just
right!” These “goldilocks” cells become the most important Regulatory T-cells or Treg-cells. They get the Foxp3+ badge. See Figure 8. These cells are the ones that can call a halt to immune system battles. They are just now being recognized as an essential part of treating immune mediated diseases like IMHA.
The CD4+ T-cells that passed “Boot Camp” are ready for battle but do not know who the enemy is or how to fight them. The process of activation of a naïve T-cell is a two-stage process. Stage one occurs when the naïve T-cells are shown who their enemy is by an Antigen Presenting Cell (APC). This is like being shown a photo of the enemy. Once the enemy is known, the cells are then fully activated by the second stage of the activation process. This stage results in determining the type of “soldier” that the Boot Camp graduate will become. This second stage activation and programming determines the branch of the armed forces, the type of soldier and the method for killing the enemy that will be employed once he is activated.
The “soldier” first receives an antigen (enemy photo). Next, his “orders” come from chemical signals called cytokines. These cytokines affect the cell’s DNA and make actual transformations in the “soldier” cell type. The cell will then take on a different role and will, in turn, secrete different cytokines that will allow him to perform his mission against the enemy in the photo. This mission usually involves recruiting and directing B-cells to produce antibodies against the enemy. The new T-cell will also direct other parts of the innate immune system (neutrophils, natural kill cells etc..) with newly formed cytokines that have been unlocked in his DNA.
Different soldier types are required because the body must defend different fronts (the gut, the skin, mucous membranes, respiratory tract etc.) from different invaders: viruses, fungi, worms, bacteria and cancer. In addition, once the war is over, there must be peacekeepers (Tregs) who stop the battle and prepare the body for repair and regeneration. For example: T H 1 lymphocytes are a common soldier type. They are given an enemy photo (antigen) by an antigen presenting cell then given the interleukin 12 (IL- 12) “orders”. In response to these chemical orders they secrete interferon and interleukin 2 (IL-2) which chemically controls the less trained cells of the immune system. The interferon and IL-2 produced by the T H 1 cells act on macrophages, cytotoxic T cells and IgG antibody producing B-cells to attack the enemy in the photo. On the other side of the battle, Tregs secrete other chemicals that keep the T H 1 cells in check. When the Treg cells malfunction, we get autoimmune disease. T H 1 malfunction is the cause of Type 4 hypersensitivity, flea allergy in dogs and Type I diabetes. Appendix 1 lists the different T cell types or soldiers, their chemical signals and the diseases that result when they malfunction.
Our newest understanding is that upon the issuance of “orders” to attack a certain enemy, countervailing orders are also issued simultaneously to create Treg cells directed to protect the same enemy. In addition, these Treg cells have certain advantages over the regular army version of T-cells. Treg cells could be onsidered Seal Team 6 type cells as they are faster, multiply quicker, have a higher affinity for the target and can outmaneuver the typical T-cell (14) . They can
even kill the T-cells or B-cells if necessary. They are there to regulate the war to prevent the soldiers from going too far in their mission. They are an essential component of the balance of the immune system and when the immune system goes awry, they bring it back into order. Treg cells come in two types one type is created by the “goldilocks” decision in the thymus and is designated the “Thymus” Treg cell or tTreg. nother Treg cell can be generated in the heat of battle as a type of battlefield promotion and is called an “Induced” Treg cell or iTreg. Mesenchymal Stem Cells produce iTreg cells by releasing TGF-β and IL-6. Treg cells stop the immune reaction by producing cytokines that shut down antibody production by the B-cells, inhibit the APCs, and they can produce Granzyme B which kills activated T-cells (13) .
Tregs function to suppress the immune responses to beneficial microbes in the gut and act to protect the developing fetus from harm. Tregs also have been found to protect cancer cells from being destroyed and stopping excessive Treg activity is being actively researched as a method for cancer therapy.
Most of the medications used against IMHA are considered anti-metabolites. That is, they inhibit cellular metabolism as their mode of action. They turn off DNA synthesis, or synthesis of one of the essential elements of DNA function or inhibit the communication between the outer parts of the cell and the DNA. These medications do this to all cells of the body equally without regard for the knock-on effects to other tissues they might cause (liver damage, blood clot formation loss of immune function etc.).
Let’s consider the common therapies for IMHA and IMTP, their mode of action, side effects and the results of their use.
Glucocorticoids (prednisolone, prednisone, methylprednisolone and dexamethasone) bind receptors in all cells of the body that turn off parts of the DNA (17) . This results in reduced release of the chemicals that cause cell death, reduced release of inflammatory chemicals, reduction in the number of receptors for antibodies which blocks cell destruction (18) . The part of the DNA that processes foreign antigens is blocked resulting in reduced immune function (19) .
The side effects of glucocorticoids are well documented; steroid resistance occurs in up to 30% of patients (20) . We also see thrombosis (blood clots) (21) , gastrointestinal ulceration, liver disease, and reduced resistance to disease. Signs of Cushing’s disease are seen with prolonged use of glucocorticoids.
The efficacy of glucocorticoids is not well documented. “To the best of our knowledge, glucocorticoids have never been subjected to rigorous evaluation by randomized double-blinded, placebo-controlled trials for treating canine immune-mediated disease (2) ”. Glucocorticoid therapy itself carries considerable morbidity, and treatment failure (2) . Nevertheless, withholding glucocorticoids to compare the effect of another immunosuppressive drug against placebo is cited as unethical (22) . 6-month survival from IMHA is reported to be 72% when used with azathioprine (23) (3) .
Azathioprine is a T-cell inhibitor. It has an 11-day lead time, this may be too long for severe or acute cases. The side effects include bone marrow suppression (24), acute pancreatitis, hepatopathy and gastrointestinal distress (25) . There are no prospective controlled studies. A few large retrospective studies lend limited support to its use (26)(2) .
Cyclophosphamide kills T-lymphocytes and regulatory T-cells. It is commonly used for cancer chemotherapy. Side-effects include hemorrhagic cystitis and bone marrow suppression (2) . Efficacy studies show increased morbidity (makes conditions worse) Does not justify its use as an immunosuppressive agent (2) .
Cyclophosphamide kills T-lymphocytes and regulatory T-cells. It is commonly used for cancer chemotherapy. Side-effects include hemorrhagic cystitis and bone marrow suppression (2) . Efficacy studies show increased morbidity (makes conditions worse) Does not justify its use as an immunosuppressive agent (2) .
Cyclosporin blocks T cell DNA transcription. It also blocks Interleukin 2 formation. The side-effects of cyclosporine are gastrointestinal upsets and anorexia. Cyclosporin has good studies showing that it is effective for atopy (27) and anal furunculosis (28) . Prospective controlled studies for other conditions are lacking (2) .
Human Intravenous immunoglobulin competitively inhibits the binding of canine immunoglobulin G to macrophages (29) It works from day 1 but only lasts 3 weeks (30) . The side effects include increased blood clotting and it increases inflammation in healthy dogs (31) . Although IVIG has been used for various conditions in dogs, efficacy data are limited to case reports and two small controlled studies (32)
Vincristine increases platelet numbers. The main side-effect is myelosuppression in higher doses, and it has been shown to cause abnormal platelet function in vitro (33) .
Leflunomide is only available in some countries. It inhibits T and B cell proliferation. It has profound anti-inflammatory effects. It may induce regulatory T cells (2) . Leflunomide is used when pets are refractory to glucocorticoids or the side-effects are intolerable (34) . Controlled studies in the dog are confined to
renal transplantation.
Mycophenolate mofetil blocks purine biosynthesis which blocks T cell and B cell proliferation. It may kill activated T- cells (35) . It has low toxicity and side-effects are limited to gastrointestinal effects. Is being used for refractory myasthenia gravis, IMHA and pemphigus vulgaris (2) .
Clodronate is used for management of hypercalcemia from vitamin D intoxication in dogs. It kills macrophages and dendritic cells that eat antibody tagged red blood cells. This drug is well-tolerated and has few side effects. Controlled trials of LC are in progress at Colorado State University (CSU) for management of IMHA in dogs (36) .
Plasmapheresis is the partial removal of autoantibodies, immune complexes and complement components from the blood. It can increase the chances of blood clots in the lungs. Most commonly used for IMHA as a rescue therapy. Has some utility in this regard in dogs. Plasmapheresis has been studied in Immune Mediated Thrombocytopenia Patients and no patients showed a response (37) . For this reason, it is no longer recommended in humans for chronic IMHA or IMTP.
Splenectomy is the removal of the spleen as the organ most responsible for the destruction of antibody coated red blood cells. Surgery is usually done when medical therapy has failed. Complications include bleeding, infection, thrombosis, prolonged hospitalization, readmission to the hospital and therapeutic failure. Results are extremely variable (38) . No prospective studies available.
Allogenic mesenchymal stem cell therapy is the intravenous use of adipose derived stem cells from the same species. In intravascular IMHA where red blood cells are destroyed by complement activation (3) Stem cells block Complement attacks by secreting Factor H (27)(28)(29) (30) . Regulatory T-cells increase red cell tolerance (2) . Lack of red cell tolerance causes IMHA. Stem Cells create Regulatory T-cells by production of TGF-β and Il-6 (15) (31) . Stem Cells down regulate the production of antibodies by B-cells and down regulate macrophage consumption of red blood cells. The stem cells response is modulated by the relative need and will not affect the rest of the immune system’s ability to fight disease. Allogenic Stem Cells are not allergenic or recognized by the immune system. They have been shown safe when given intravenously in numerous human Phase I and Phase II Clinical Trials (39), (40), (4) .
Randomized clinical trials for IMHA therapy in dogs with Stem Cells are lacking. It should be noted that neither are there randomized controlled trials for the use of Parachutes to prevent death from gravitational challenges. Gordon C S Smith’s article explores the need for common sense and observation as an alternative to this method of determination of effectiveness of a therapeutic protocol. Such is the case with stem cell therapy (41) . More general research has shown that stem cells possess anti-inflammatory and immunomodulatory properties; primarily by modulating the type and function of T-Lymphocytes (42), (43), (44), (45), (46), (47), (48), (49), (50) (51) . Unlike pharmaceutical treatments that deliver a single agent at a specific dose, MSCs are site regulated and secrete bioactive factors and signals at variable concentrations in response to local microenvironmental cues (4) .
The drugs listed above all interfere with DNA in some way. The reason these medications work for IMHA is that IMHA is caused by lymphocytes and lymphocytes are
among the most rapidly dividing cells of the body. Lymphocytes use their DNA more and are therefore, more affected by substances that damage DNA than other cells.
Tregs divide faster, have a higher metabolic rate and are more affected than other lymphocytes by these medications (14) . These medications do nothing to cure the
disease, or to stop the creation of the offending antibodies in fact they may inhibit immune tolerance by killing Treg cells. These medications only act to stop the
manufacture of the antibodies after they have been created. In some cases, the side-effects of the drugs are more damaging than the disease.
IMHA is a serious, fatal disease that has not yielded to the advanced immunosuppressive drugs and therapies just listed. Unlike these anti-metabolites, Mesenchymal Stem Cells are site regulated producing effects through chemokines, cytokines, soluble factors and direct cell to cell contact that adjusts to the local cellular environment (4) . Primary IMHA that is expressing IgM antibodies is causing intravascular hemolysis through the activation of complement. This form of IMHA is among the most lethal, killing most pets within the first 10 to 14 days (3) . Mesenchymal Stem Cells constitutively produce Factor H which blocks the rapid red blood cell destruction mediated by complement in this form of the disease. Extravascular hemolysis occurs in the spleen and liver and is the result of macrophage action on antibody tagged red blood cells. MSC’s produce the cytokine IL- 10 and the soluble factor Indoleamine 2,3 dioxygenase (IDO) which downregulates the phagocytosis of macrophages. These two actions of MSC’s effectively stop the red blood cell destruction in IMHA within the first 24 hours. These effects would be short-lived if the actual antibody production were not stopped. B-cells reproduce in a clonal fashion to produce plasma cells which deliver massive numbers of anti-red blood cell antibodies per minute. MSC’s significantly curtail this antibody production by affecting the T-cell mediated control of the plasma cells (52) . The development of the anti-red blood cell antibodies in the first place is a problem with the immune tolerance and balance of Inflammatory T-cells and Regulatory T cells. MSC’s produce the soluble factor Transforming Growth Factor Beta (TGFβ) which stimulates the formation of iTreg cells. These cells can stop the plasma cell production of the antibodies as well as to kill the Th2 cells that are stimulating the B- Cells to produce antibodies. In addition, Treg cells stop the Antigen Presenting Cells (APC’s) such as macrophages and dendritic cells from processing red blood cell antigens as foreign. These Treg cells then form memory Treg cells that remember who friend vs. foe is thereby promoting lasting Immune Tolerance of the red cell surface antigens in the future.
Understanding the recent advances in T-cell function shows that allogenic stem cells provide a safe alternative to immunosuppressive drugs for the treatment of IMHA. They also offer the potential of a cure rather than life-long therapy.
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