Chapter 32 Biologic Response Modifiers
Interferons, Interleukins, Chemokines, and Hematopoietic Growth Factors∗
Natural or synthetic preparations given with the intent of altering the response of the host to a pathogen, neoplasm, or inflammatory process have been termed biological response modifiers. The goal of therapy may be to increase the effectiveness of the immune response directed toward a pathogen or neoplasm, to stimulate the proliferation of hematopoietic progenitor cells (e.g., in the treatment of chemotherapy-associated neutropenia), or to decrease a chronic inflammatory response (e.g., chronic inflammatory bowel disease). Biological response modifiers are not usually specific in an immunologic sense; rather, they alter physiologic systems through changes in regulatory pathways. They range from bacterial cell wall extracts to molecularly cloned cytokines. As mediators of physiologic processes, interferons, interleukins, and chemokines are candidates for therapeutic manipulation or even for use as drugs. Several recombinant human, feline, and canine cytokines are commercially available in pharmacologic quantities and are available for veterinary use (Box 32-1). Some generalizations about cytokine biology follow. See the reviews by Oppenheim and Feldman,1 Nicola,2 and Oppenheim and coworkers3 for general discussions of cytokine, growth factor, and chemokine biology and the article by Gangur and coworkers4 for a review of chemokines of veterinary relevance.
Box 32-1 Commercially Available Recombinant Canine and Feline Cytokines, Chemokines, and Growth Factors
Cytokines are polypeptides that regulate cell growth and differentiation, apoptosis, inflammation, immunity, and repair. They are of fundamental importance in the pathogenesis and treatment of disease. They transmit information to target cells regarding the physiologic status of the animal, resulting in a biologic response in the target tissue. More than 50 distinct human cytokines and their receptors have been described. Cytokine nomenclature can be bewildering, with several historical names attached to the same molecule (e.g., stem cell factor, c-kit ligand, and mast cell growth factor are all the same cytokine). Cytokines Online Pathfinder Encyclopaedia (COPE) is a useful Internet resource with a large amount of information concerning cytokine, chemokine, and growth factor biology (www.copewithcytokines.de/cope.cgi). Cytokines can be categorized into families on the basis of homology in primary amino acid sequence, three-dimensional structure, induction mechanisms, chromosomal location, similarities in receptor type, and functional homology. Families that are currently clinically relevant in companion animal medicine include interferons (IFNs), which have antiviral and immunoregulatory functions; interleukins (ILs), which have a wide variety of functions; chemokines, which have to do with chemotaxis and organ development; and hematopoietic growth factors.
Most cytokines are not constitutively produced but are secreted after activation of cells by viral, bacterial, and parasitic infections. The cellular sources of most cytokines are diverse, with production occurring in many types of cells. Production is normally short lived, usually for hours to a few days. Normally, they have autocrine and paracrine (rather than endocrine) effects, with little to no detectable circulating levels. Because of high cytokine–receptor affinity, cytokines are effective in the picogram to nanogram per mL range.
Cytokines typically have pleiotropic and redundant actions. The same activity may be induced by several structurally distinct cytokines acting at unrelated cell surface receptors. For example, IL-1, tumor necrosis factor-alpha (TNF-alpha),∗ and IL-6 are all mediators of the acute inflammatory response; induce fever and the synthesis of acute-phase proteins in the liver; increase vascular permeability; and induce adhesion molecules, fibroblast proliferation, platelet production, IL-6 and IL-8, and T and B cell activation. Gene deletion experiments reveal that few individual cytokines are absolutely essential to life or even to individual cell function, with notable exceptions such as TNF-alpha and transforming growth factor-β (TGF-β). Also, the cellular response of most cytokines is modulated by other cytokines, with synergistic, additive, and antagonistic interactions described. Cytokines form a complex feedback network by either inducing or suppressing the expression of other cytokines, forming cascades similar to the blood coagulation cascades.
Structurally, most cytokines consist of a single glycosylated polypeptide chain. They interact with cells by binding to specific high affinity cell surface receptors. The intracellular signal transduction cascade initiated by binding of a cytokine to its receptor eventually results in the production of DNA-binding proteins that influence transcription of various genes. In addition to cell surface receptors, soluble receptors have been described for many cytokines. Soluble receptors may be involved in cytokine transport or inhibit cytokine activity.
A number of proteins that inhibit the activity of cytokines have been reported. One of the best characterized is the IL-1 receptor antagonist (IL-1RA), which binds to receptor but fails to activate the cell. IL-1RA is currently being investigated as a therapy for rheumatoid arthritis in humans and may have a role in the treatment of other inflammatory disorders if appropriate delivery systems can be developed. Peptide antagonists of chemokines have shown promise in breaking the cycle of inflammation in experimental models of disease. A second way in which a cytokine can be inhibited is by binding of soluble cytokine receptors that are able to bind the cytokine and neutralize its activity, as discussed earlier.
Recombinant DNA technology has been used to produce cytokines, many of which are now commercially available. The recombinant process involves cloning the cDNA encoding the protein of interest and placing it into an expression system (bacterial, yeast, insect, mammalian cell culture) under conditions that permit large amounts of the protein to be produced. By convention, these products are designated by an “r” preceding the name of the cytokine and a designation of the species of origin (e.g., rhIL indicates recombinant interleukin of human origin, and rfeIFN indicates a recombinant interferon of feline origin). Many cytokines are conserved in the evolutionary sense, and biological effects occur when some human recombinant products are administered to companion animals.
It is tempting to administer cytokines in pharmacologic doses; however, several points should be considered. Perhaps most important, systemic levels of a given cytokine will perturb many cytokine cascades, with resultant side effects and toxicity. The use of IL-12 as a therapeutic agent, as discussed later, has been limited by potentially severe hematopoietic and hepatic toxicities in animals and man and was associated with two deaths in a phase 1 trial in humans.5 Dosing may be critical; for example, low doses of IFN appear to be immunostimulatory, whereas higher doses are immunosuppressive, and the specific type of IFN influences these effects. This concept of increasing doses of immunostimulatory cytokines leading to depression of the immune response has been demonstrated with IL-12 as well and appears to be due to the magnitiude of downstream IFN-γ and nitric oxide that is produced.6 Currently, recommended cytokine doses are empirically derived. With the exceptions of rfeIFN-α (in cats) and rhIL-1 (in dogs), the pharmacokinetics of cytokines in dogs and cats have not been well characterized.
The safety and efficacy of cytokines as biological response modifiers may be improved if they can be delivered directly to target cells. Delivery systems, such as liposomes and replication-defective adenoviruses, have been reported in the literature, with encouraging results. In some cases the cDNA for the cytokine of interest can be delivered (using a variety of methods) directly to the tissue of concern (usually a neoplasm), and controlled expression of the gene may confer clinical benefits. Both agonistic and antagonistic peptides have been sought for various cytokines in attempts to achieve systemic oral administration or suppress harmful effects.
KEY POINT 32-1
Natural or synthetic biological response modifiers are being developed as therapeutic mediators to increase the effectiveness of the immune response directed toward a pathogen or neoplasm, to stimulate the proliferation of hematopoietic progenitor cells (e.g., in the treatment of chemotherapy-associated neutropenia), or to decrease a chronic inflammatory response (e.g., chronic inflammatory bowel disease).
Interferons are cytokines secreted by virus-infected cells and were originally characterized by their nonspecific antiviral activity. Interferons bind to receptors on other cells and induce antiviral proteins, protecting those cells from infection. It is now known that IFNs induce a wide range of pleiotropic effects, including antiviral, antitumor, antiparasitic, and immunomodulatory effects.
Two distinct classes of IFNs have been described. Class I interferons are subdivided into alpha, omega, and beta interferons (a subclass of omega interferons, the tau interferons, has been described in ruminant embryos and is important in maintaining pregnancy). Class II interferons are composed of a single protein, IFN-γ. IFN-α and IFN-ω were originally described as being secreted by leukocytes, but they are likely produced by all nucleated cells. Humans have at least 24 different alpha and omega genes, dogs have two alpha genes and no omega genes, and the genetic complement of cats has not been reported. Feline IFN-α cDNA has been cloned and the cytokine produced in a silkworm-recombinant baculovirus system, and its pharmacokinetic properties have been studied.7-10 The pharmacokinetic data suggest that rfeIFN has similar pharmacokinetic properties to human IFNs and that it is distributed primarily to the liver and kidneys, is rapidly catabolized mainly in the kidneys, and is excreted in the urine without residual accumulation in the body. Since feline IFN-α was initially cloned, 14 additional subtypes have been described.11,12 IFN-β is classically described as being produced by fibroblasts; however, many other cells can be stimulated to secrete the cytokine. Humans and dogs have one IFN-β gene. Synthesis of IFN-α and IFN-β can be induced by live or inactivated viruses, bacterial cell walls, synthetic oligonucleotides, IL-1, IL-2, and TNF-alpha. IFN-α and IFN-β compete for similar receptors. IFN-γ is produced by activated T and natural killer (NK) cells and is structurally and functionally distinct from IFN-α/β. All mammals investigated have only one IFN-γ gene. IFN-γ binds to a receptor distinct from the α/β receptor. Canine IFN-γ has been characterized, the cDNA and chromosomal gene have been cloned, and production and characterization of recombinant canine from Escherichia coli has been described.13-15 Feline IFN-γ cDNA has also been cloned.16,17 After receptor binding, IFNs induce the transcription of a set of genes called IFN-stimulated genes (ISGs). Nearly 30 different ISGs have been identified. Induction of ISGs by IFN-α/β is rapid and transient, lasting for 3 to 4 hours. IFN-γ requires several hours of exposure before gene induction occurs. After the initial induction, ISG transcription declines and returns to basal levels.
IFNs inhibit the growth of almost all known viruses by interfering with viral RNA and protein synthesis. IFNs are induced by viral nucleic acid and bind to the receptors of nearby cells. The development of resistance to virus infections occurs within a few minutes and peaks within a few hours. Several proteins are induced, including RNase L (which cleaves viral RNA); nitric oxide synthetase (nitric oxide has antiviral activity); a protein kinase that phosphorylates an initiation factor called elL-2 (which inhibits viral protein synthesis by preventing the elongation of viral double-stranded RNA); and the Mx protein, which inhibits translation of viral mRN.18 IFN-γ inhibits viral replication by stimulating the release of other IFNs. IFNs are also induced by bacteria, fungi, and some protozoa, and the activation of phagocytic cells is important in the host response to these pathogens.
IFNs induce increased expression of class I and class II major histocompatibility complex (MHC) molecules on antigen-presenting cells, leading to enhanced antigen presentation. They increase phagocytosis and intracellular and extracellular killing by macrophages and neutrophils. The IFNs also modulate T, B, and NK cell function, with IFN-γ having the most potent immunomodulating activity.
IFNs have several effects on neoplastic cells, including modulation of oncogene expression. Downregulation of c-myc, c-fos, c-Ha-ras, c-mos, and c-src have been described in various models. IFN-α augments NK cell cytotoxicity against neoplastic cells and acts synergistically with IL-2 to increase NK activity. Antiangiogenic activity has also been described.
Large-scale production of IFNs is accomplished by culturing stimulated cells, leading to the production of “natural” or “native” IFN (denoted by N) products. Alternatively, IFNs can be produced by recombinant methods. Human IFNs from both sources are commercially available, and recombinant feline and canine IFN-α and IFN-γ are commercially available (see Box 32-1). Natural IFNs are less concentrated and may contain a mixture of IFN types with other cytokines.
In humans IFN-α has been approved for the treatment of hairy cell leukemia, melanoma, chronic myelogenous leukemia, Kaposi sarcoma, basal cell carcinoma, renal cell carcinoma, and genital warts. Effects on metastatic melanoma, endocrine–pancreatic tumors, metastatic colorectal and ovarian carcinoma, and bladder cancer have also been reported. IFN-β is also licensed for the treatment of chronic hepatitis B and hepatitis C infections. IFN-γ bound to polyethylene glycol (peg-IFN) has an increased circulating half-life and has shown improved efficacy in treating hepatitis C infections.19 IFN-β has been approved for the treatment of multiple sclerosis and IFN-γ for the treatment of chronic granulomatous disease. Experience using IFN therapy has generally shown that these products are most effective when combined with other antiviral or cytotoxic treatment modalities.20
Using a feline in vitro system, Weiss and Oostrom-Ram21 showed that low levels of rhIFN-α had no effect on lymphocyte blastogenesis, whereas higher levels significantly suppressed blastogenic responses. In vivo, cats given 102 or 104 IU/kg had significantly enhanced blastogenesis, whereas cats given 1 × 106 IU/kg had depressed lymphocyte stimulation. In cats the immunomodulating effects of rhIFN-α appeared to be dose dependent.
Five feline IFN-α subtypes have been recently cloned, expressed, and characterized in feline cell lines.11,22 All subtypes were shown to have antiviral effects on feline calicivirus and vesicular stomatitis virus in vitro. Additionally, they showed a species-specific antiproliferative effect on a feline tumor cell line. The IFN-α inducible Mx gene was upregulated 24 hours after administration to cats, demonstrating in vivo efficacy. In vitro work has also demonstrated the antiviral activity of IFN-α against feline herpesvirus-1 in the absence of any cytopathic effect on the cultured corneal cells.23 Activity of rfeIFN-ω, a closely related cytokine to rfeIFN-α against rotavirus, feline panleukopenia virus, feline calicivirus, and feline infectious peritonitis coronavirus, was documented in cell cultures of feline origin. The antiviral effect was more pronounced when the cell cultures were treated continuously than when they were pretreated only before challenge. RfeIFN-ω did not have activity in canine cells challenged with vesicular stomatitis virus, implying species specificity of action,24 yet de Mari and coworkers25 described clinical benefits of another rfeIFN-ω preparation in dogs with parvoviral infection (discussed later). Recombinant feline IFN-ω is marketed in Japan as IntercatR (Toray Industries, Tokyo, Japan), and a second rfeIFN-ω product is licensed in Europe as Virbagen omega (Laboratory Virbac, Carros, France).
Parenterally administered rhIFN-α and IFN-β, with or without Propionibacterium acnes (which enhances IFN responses and augments T and NK cell activities) given prophylactically or therapeutically, did not significantly reduce the mortality rate of experimentally induced feline infectious peritonitis virus (FIPV) infection. Cats treated with high-dose IFN, 10 U/kg daily for 8 days and then on alternate days for an additional 2 to 3 weeks, however, had temporary suppression of clinical signs and decreased serum antibody responses to FIPV, and the mean survival time of cats treated with high-dose rhIFN-α was increased by a few weeks over that of untreated cats. IFN-related toxicities were not reported.26 It is possible that the benefits of the high-dose protocol were due to the dose-dependent immunosuppressive effects of IFN. The increase in survival times in this study using experimental challenge was only 2 to 3 weeks, and there are few data documenting the response of cats with naturally occurring disease. There are anecdotal reports of orally administered low-dose rhIFN-α therapy (as described later) inducing remissions from clinical FIP, but there is currently no published data. A study of subcutaneous rfeIFN-ω administered to 12 cats clinically diagnosed with FIP demonstrated long-term survival in 4 animals.27 As the authors acknowledge, this was not a case-controlled study and histologic confirmation of the disease was available only for those animals that died during the study. A recent placebo-controlled, double-blind trial in which rfeIFN-ω was administered subcutaneously to cats naturally infected with FIPV did not demonstrate any clinical, hematopoietic, or survival benefits to the therapy; conventional therapy with cytotoxic drugs and corticosteroids remains the treatment of choice.28,29
Recombinant hIFN-α has been shown to inhibit the production or release (or both) of feline leukemia virus (FeLV) in tissue culture systems.30 The parenteral administration of rhIFN-α alone or in combination with zidovudine (AZT) beginning 12 weeks after exposure to FeLV resulted in significant and sustained decreases in circulating virus levels. The anti-FeLV effect was limited by the production of anti–rhIFN-α antibodies detected 7 weeks after the start of therapy. IFN-associated toxicity was not observed.31 In a subsequent study, treatment of FeLV-infected cats with AZT, IFN-α, and adoptive transfer of lectin/IL-2–activated lymphocytes resulted in clearance of circulating virus in four of nine cats despite the appearance of anti-rhIFN-α antibodies. Combination therapy appeared to reconstitute antiviral humoral immunity, counteracted immunosuppression, and induced the reversal of retroviremia.32 Unfortunately, because of the problem of antibody induction, parenteral rhIFN-α appears to have little clinical utility as a monotherapy for FeLV infections. In vitro work supports the antiviral effects of rfeIFN-α and rhIFN-α but not rfeIFN-γ on feline immunodeficiency virus (FIV) replication.33 Ovine IFN-τ has demonstrable effects on the in vitro replication of both FIV and human immunodeficiency virus (HIV).34 A study of subcutaneous rfeIFN-α therapy in cats naturally infected with FeLV or co-infected with FeLV and FIV showed significant decreases in clinical scores and mortality rate during the 12 months of follow-up in the treated cats.35 Recently, low-dose oral IFN-α (as described later) was shown to improve clinical condition and survival in cats naturally infected with FIV.36
Two studies, one with experimental infection and one with naturally occurring infection, have examined the efficacy on rfeIFN-ω (Virbagen, Laboratory Virbac, Carros, France) on the outcome of canine parvoviral enteritis. Ten 8- to 9-week-old dogs inoculated with parvovirus were divided into rfeIFN-ω or placebo treatment groups and received three daily injections. All five of the placebo-treated dogs succumbed to fulminant enteritis, whereas four of five rfeIFN-ω–treated dogs survived and eventually recovered fully.37 In the second study of naturally occurring disease, 43 dogs were assigned to receive three daily intravenous injections of rfeIFN-ω, and 49 dogs received placebo treatment. There was a significant improvement in clinical signs and a significant decrease in mortality in the dogs receiving IFN therapy.25
In 1988 Cummins and coworkers 38 reported that after experimental infection with the Rickard strain of FeLV, the administration of 0.5 or 5 U of natural hIFN-α orally once daily for 7 consecutive days on alternate weeks for 1 month was associated with survival in 70% of IFN-treated cats, whereas 100% of placebo-treated control cats died. However, 12 of 13 cats treated with IFN did develop persistent viremia. In another report, four cats with FeLV-associated nonregenerative anemia were treated with 100,000 U bovine IFN-β orally for 5 consecutive days on alternate weeks. General clinical improvement, reduction in circulating antigen levels, and normalization of hematocrit levels were reported in all cats,39 and one cat cleared its viremia. Similar findings were reported by Steed,40 who treated four FeLV-infected cats with low-dose natural hIFN-α or bovine IFN-β. Weiss and coworkers41 reported on 69 FeLV-infected cats with clinical signs treated orally with either low-dose rhIFN-α or bovine IFN-β. Cats treated with rhIFN had significantly higher survival rates than did cats given bovine IFN; both groups had increased survival rates compared with historical controls. In general, clinical responses were observed within the first or second week of oral IFN administration, with increased appetite, greater activity, weight gain, resolution of fever, improved hemogram and leukocyte counts, and quicker recovery from secondary bacterial infections when antibiotics were administered. Most cats remained viremic.
In contrast to the aforementioned reports, Kociba and coworkers42 reported that low-dose oral hIFN-α had no significant effects on viremia, course of the disease, or differential leukocyte counts in experimental FeLV infection. In their system the Kawakami–Theilen strain (A, B, and C subgroups, which consistently induce fatal erythroid aplasia) was administered to 12-week-old kittens. Methylprednisolone acetate was also given the day of inoculation. Neither rhIFN-α nor human natural IFN-α induced any significant benefit compared with placebo. A second study administering low-dose oral rhIFN-α showed a similar lack of benefit clinically or biochemically in naturally infected, clinically ill FeLV-positive cats.43
Pedretti and coworkers36 recently reported that FIV-infected cats given a daily oral dose of 10 IU/kg rhIFN-α had a significantly prolonged survival rate compared with placebo-treated cats. In addition, clinical remission of many of the typical immunopathologic lesions occurred despite no significant reduction in circulating viral levels. This study selected FIV-infected cats that presented with evidence of immunosuppression and clinical signs of acquired immunodeficiency syndrome (AIDS) and utilized a mixture of multiple human IFN-α subtypes. Two large, double-blind, placebo-controlled trials of low-dose oral IFN-α failed to show any efficacy in ameliorating clinical signs, increasing CD4+ T cell counts, or decreasing mortality rates in HIV-infected humans.44,45
If orally administered low-dose human IFN-α has any effect in cats with retroviral infections, a direct systemic antiviral effect is unlikely. It may be possible that IFN may be acting as a biological response modifier after binding to cellular receptors in the oral cavity/pharynx, triggering cytokine cascades that have systemic immunomodulatory effects. Appetite stimulation may be due to direct central nervous system effects. When it was used orally in cats, adverse effects have not been reported, and anti-rhIFN-α neutralizing antibodies do not appear to develop.
IFNs inhibit cell proliferation of both normal and malignant cells and have numerous immunomodulating effects. Several human cancers respond to IFN therapy. At present, there are only preclinical data suggesting that IFNs may have some utility in the treatment of cancer in companion animals. When canine mammary tumor and melanoma cell lines were incubated with canine IFN-γ, significantly increased expression of MHC antigen class I and II antigens and tumor-associated antigens were observed.46 Increased expression of these antigens may be of benefit in tumor cell recognition and rejection by the immune system. Pretreatment of canine macrophages with rcIFN-γ resulted in increased in vitro tumor cell killing in canine models of melanoma and osteosarcoma.47,48 In another study growth of canine and feline tumors was inhibited by rhIFN-α and rhIFN-γ in vitro. Sensitivity to IFN varied according to the type of neoplasm, with round cell tumors being most sensitive.49 Recombinant rfeIFN was found to have a dose-dependent inhibitory effect on cell growth and colony formation on cell lines derived from canine acanthomatous epulis, benign mixed mammary tumor, squamous cell carcinoma, and malignant melanoma.50 Recent work has demonstrated that intratumoral injection of rfeIFN-ω in cats with fibrosarcomas is safe and feasible; subsequent studies will be necessary to determine efficacy.51
There are currently 35 defined ILs, named in order of discovery and classified into groups according to their structure, function, or both. ILs are a diverse group of cytokines, with functions including enhancement or suppression of various cells of the immune system (e.g., IL-1, IL-2, IL-4, IL-9, IL-10, IL-12, IL-13, IL-17, IL-23, IL-27), hematopoietic growth factor activity (e.g., IL-3, IL-11, IL-17, IL-32, IL-34), and the regulation of leukocyte function (e.g., IL-5 modulates eosinophil function; IL-8 is chemotactic for neutrophils). Some are growth factors for cells of the immune system (e.g., IL-7, IL-11, IL-14, IL-15), whereas others enhance the acute phase response (e.g., IL-1, IL-6). Most ILs have multiple effects on various cells and are part of complex regulatory cascades. Depending on the specific IL, they are produced by T and B lymphocytes, macrophages, dendritic cells (DCs), fibroblasts, and other stromal cells.
Human rIL-1α was shown to be chemokinetic and chemotactic for canine neutrophils in vitro and to cause dose-dependent and selective neutrophil infiltration after intradermal administration.52 The pharmacokinetics of a single dose of human IL-1 has been studied in the dog. IL-1 was rapidly distributed, with a volume of distribution approximately twice that of the total body water of a lean dog. The terminal half-life was less than 30 minutes. Within approximately 1 hour after dosing,53 IL levels were below the quantifiable limit of the enzyme-linked immunosorbent assay (ELISA). As IL-1 is a central mediator of inflammation, these data are useful for studying the physiology of IL-1; however, pharmacologic efforts will focus on inhibition of IL-1 activity, with applications in the therapy of acute and chronic inflammatory disorders and septic shock. The canine IL-1 receptor antagonist, which functions as a competitive inhibitor of IL-1, has been cloned.54
IL-2 synthesis is triggered by antigen-induced activation of T lymphocytes, and its most important activity is the promotion of clonal expansion of antigen-specific T cells. In NK cells and macrophages, IL-2 also promotes proliferation, production of IFN-γ, and cytolytic activity. It also induces growth of B cells as well as immunoglobulin secretion. An important clinical consideration is the observation that NK cells cultured in the presence of IL-2 have enhanced cytotoxic activity, with increased capability for lysis of neoplastic cells. These activated cells are called lymphokine-activated killer (LAK) cells. IL-2 is thus an attractive agent for the therapy of neoplastic disorders; however, parenteral administration of IL-2 is associated with significant hepatic, hematopoietic, and vascular toxicity, which appears to be largely due to the secondary release of TNF-alpha and nitric oxide.55-57
IL-2 was licensed in the United States in 1992 for the treatment of metastatic renal cell cancer, becoming the first biological agent approved for treatment of any cancer in humans. The availability of recombinant IL-2 spurred the development of adoptive immunotherapy, which refers to the transfer to the tumor-bearing patient immune cells that mediate antitumor effects. Adoptive immunotherapy has been performed with LAK cells and tumor-infiltrating lymphocytes. IL-2 is necessary for the generation of these cells in vitro and in vivo and is also administered systemically along with these cells in an effort to keep them functioning in the patient. More recently, IL-2 gene therapy for various cancers has been developed.
Several in vitro studies have demonstrated that rhIL-2 has activity in companion animals similar to the activity recognized in humans. Feline lymphocytes responded appropriately to the cytokinetic action of systemically administered rhIL-2,58 and adoptive immunotherapy of FeLV-infected cats with lectin/rhIL-2–activated lymphocytes, IFN-α, and AZT led to reconstitution of antiviral humoral immunity, counteracted immunosuppression, and induced the reversal of retroviremia in a subset of treated cats.32 Feline IL-2 cDNA was cloned, and the recombinant protein was shown to promote proliferation of feline, but not human, cells.59,60 Helfand and coworkers61 demonstrated that the immunobiology of IL-2 in the dog is similar to that of humans. Tumor cytotoxicity was induced in vitro in canine lymphocytes with rhIL-2, demonstrating that functional and morphologic changes compatible with LAK cells could be obtained in dogs.62-64
Infusion of rhIL-2 into normal dogs resulted in lymphocytosis and enhanced in vitro lysis of a canine tumor cell line. Side effects included vomiting, diarrhea, and inactivity.65 Recombinant human TNF and rhIL-2 were administered in a sequential schedule to 30 dogs with a variety of spontaneous neoplasms. Objective tumor responses were seen in dogs with oral melanomas and cutaneous mast cell tumors. Dose-limiting toxicities were primarily gastrointestinal.66
In an effort to develop a delivery system that would be associated with less toxicity, Khanna and coworkers67 nebulized free rhIL-2 and rhIL-2–containing liposomes into normal dogs. Free IL-2 resulted in increased peripheral blood mononuclear cell activation compared with saline-treated control dogs. IL-2 liposomes resulted in significantly increased bronchoalveolar lavage (BAL) effector leukocyte numbers and activation compared with empty liposomes. In dogs with primary and metastatic lung cancer, nebulized IL-2 increased the total number of BAL macrophages, eosinophils, and lymphocytes compared with pretreatment levels, and there was increased expression of CD3 on BAL lymphocytes. Mean BAL cytolytic activity increased compared with pretreatment activity during therapy to a maximum at day 15, and then it decreased (despite continued aerosol therapy) to pretreatment levels at day 30. Antibodies reacting with hIL-2 developed in the serum of all treated dogs, possibly accounting for the decrease in BAL cytolytic activity at day 30 compared with day 15. Toxicity was not recognized with either IL-2 preparation. Complete regression of pulmonary metastases in two of five dogs with metastatic osteosarcoma was maintained at greater than 370 and 700 at day 68. A phase I trial of liposome-complexed canine IL-2 administered parenterally to dogs with chemotherapy-resistant, metastatic osteosarcoma was recently completed.69 The liposome formulation resulted in preferential expression of the IL-2 within the lung. The weekly injections were well tolerated, and 3 of the 14 dogs completing the 12-week therapy had partial or complete radiographic regression of metastases, with an additional four dogs showing stable disease. Despite the limitations in interpreting efficacy in a phase I pilot study, there was a significant increase in overall survival times in the treated dogs compared with historic controls with similar tumor staging.
Quintin-Colonna and coworkers70 reported that dogs with oral melanoma treated with local resection, 45-Gy radiation therapy, and repeated local injections of xenogeneic Vero cells transfected with an hIL-2–expressing plasmid had longer median survival times than did dogs treated with resection and radiation therapy alone; dogs treated with surgery, radiation therapy, and nonengineered Vero cells; or dogs treated with surgery, radiation therapy, and injection of rhIL-2 into the tumor bed. Similar results were seen in cats with fibrosarcomas. An unexpected observation was the development of metastatic fibrosarcoma in three of five cats that relapsed in the group treated with engineered cells. Complications seen in some dogs and cats included anaphylaxis associated with injection of Vero cells and local inflammatory reactions.70 Canarypox virus and attenuated vaccinia virus vectors have been used to deliver rfIL-2 and rhIL-2, respectively, to cats with soft tissue sarcomas.71 The injections were well tolerated, and there was a significant reduction in tumor recurrence rate with either of the virus delivery systems compared with controls 12 months after treatment.
IL-2 therapy appears to have a place in the immunotherapy of feline and canine cancers, particularly when administered in a manner that concentrates expression within the tumor, thereby limiting systemic toxicity. Adenoviral delivery and incorporation into liposomes appear to be particularly promising modes of therapy. Canine and feline IL-2 cDNA have been cloned59,72,73 and are commercially available (see Box 32-1).
IL-12 is a proinflammatory cytokine that has shown great promise as a therapeutic agent in experimental models of infectious disease and cancer. Multiple lines of evidence indicated that IL-12, a heterodimer produced in response to endotoxins, intracellular parasites, and CD40 ligation, is pivotal in initiating a cell-mediated immune response.74-76 Direct contact with microbial products or activated T cells can prompt antigen-presenting cells (e.g., macrophages, DCs) to release IL-12. In turn, IL-12 induces the synthesis of IFN-γ by T and NK cells. This early IL-12 generation is critical to early host containment of many intracellular pathogens, including Toxoplasma gondii,77 Listeria monocytogenes,78 Leishmania major,79 and Mycobacterium tuberculosis. 80 To date, most veterinary studies using IL-12 have been designed to characterize the adjuvant effect of IL-12 in boosting the cell-mediated immune response to FeLV, FIV, and FIPV vaccination. Two studies administering intradermal FIV DNA showed protection from FIV infection in three of four cats when IL-12 was included in the vaccination, whereas all four cats that received the DNA vaccine alone became infected.81,82 Similar DNA vaccinations protocols against FeLV challenge showed increased protection only when IL-12 was co-administered with IL-18, another cytokine known to enhance cell-mediated immunity.83 Vaccination of cats against FIPV in combination with co-delivery of feline IL-12 encoding plasmids did not enhance protection and may have actually potentiated the infection.84 There is precedence for the inhibition of cell-mediated immunity when IL-12 is administered at high doses,6,85 and this may have played a role in the outcome of this study. Lasarte and coworkers6 demonstrated that the depression of cell-mediated immunity at high IL-12 doses was related to the generation of large amounts of nitric oxide.
IL-12 is one of the most widely studied cytokines in cancer immunotherapy and has been found to eradicate experimental tumors, elicit long-term antitumor immunity,86-88 and possess antiangiogenic properties.89,90 The role of IL-12 as chemotherapeutic agent has been limited because of its toxicities. In mice, daily administration of 0.1 to 10 μg of recombinant murine IL-12 for up to 2 weeks resulted in liver function abnormalities; gastrointestinal toxicity; and hematopoietic changes, including anemia, neutropenia, lymphopenia, and thrombocytopenia.91-93 In humans the toxic effects include fever, anemia, neutropenia, lymphocytopenia, thrombocytopenia, liver function test abnormalities, rhinitis, stomatitis, and colitis.94 In a phase II clinical trial in renal carcinoma using rhIL-12, two deaths were reported.5 The authors have seen dose-dependent hematopoietic toxicities (anemia, neutropenia, and thrombocytopenia) in preclinical trials of systemic administration of a replication-defective adenovirus encoding murine IL-12 in cats.94a
One approach that can be used to avoid the systemic toxicity of IL-12 is to use local intratumoral IL-12 gene therapy. A replication-defective adenovirus containing feline IL-12 under the control of the heat-shock promoter has been developed and characterized in vitro.95 An added advantage of employing the heat-shock promoter is that IL-12 expression can be temporally induced by providing external hyperthermia to the tumor. A phase I dose escalation trial with feline soft tissue sarcomas was recently completed. A maximum tolerable dose was reached, although all cats completed the treatment regimen. At the highest dose of adenovirus, fever, anemia, and thrombocytopenia were reported.96 Further follow-up and expanded studies will be required to determine clinical efficacy of this cytokine treatment. Intramuscular injection of a plasmid DNA expression vector of canine IL-12 induced enhanced peripheral blood mononuclear cell production of IFN-γ in treated dogs.97 Recombinant mIL-12 has been shown to significantly inhibit the growth of a canine hemangiosarcoma cell line transplanted into mice.98 It is unclear whether the growth inhibition was mediated mainly through antiangiogenic mechanisms, NK cell activation, or a combination of both.
IL-12 is a potent immunostimulant that holds tremendous promise as a vaccine adjuvant or cancer immunotherapeutic. The very potency of this cytokine contributes to its limitations, and continued studies designed to diminish systemic toxicities will be necessary before it can be commonly employed. Both feline and canine IL-12 have been cloned and expressed,99,100 and the recombinant proteins are currently commercially available (see Box 32-1).
IL-6 is a proinflammatory cytokine (along with IL-1 and TNF-α) with pleiotropic activity, including effects on B and T cells and induction of acute phase proteins. Elevated levels of IL-6 have been reported in Chinese Shar-Peis with recurrent febrile illnesses, and IL-6 dysregulation has been postulated to play an etiologic role in the syndrome.101 Systemic inflammatory response syndrome (SIRS) and sepsis are associated with increased IL-6 production, and high plasma IL-6 levels are negatively correlated with outcome in canine SIRS patients.102 Cranial cruciate ligament rupture in dogs results in increased levels of synovial fluid IL-6.103 Dogs with juvenile polyarteritis syndrome were also found to have increased levels of serum IL-6 activity during acute illness but undetectable levels during convalescence. Treatment of acutely ill dogs with prednisone resulted in rapid clinical improvement accompanied by a decrease in IL-6 activity; withdrawal of prednisone resulted in reappearance of signs and high serum IL-6 activity. Clinically, the most important inhibitors of IL-6 expression are glucocorticoids.104 Specific blockade of IL-6 or the IL-6 receptor (or both) is a potentially promising means of controlling clinical disease in chronic inflammatory conditions.105 IL-6 also has marked effects on megakaryocyte and platelet physiology. In dogs 80 μg/kg per day of rhIL-6 increased platelet counts modestly and enhanced the sensitivity of platelets to activation in response to thrombin and platelet-activating factor.106 Other investigators have shown that IL-6 promoted increases in plasma fibrinogen and von Willebrand factor (vWF) and a decrease in free protein S concentrations.107 These effects on the clotting mechanism may result in an overall prohemostatic tendency, which may prove beneficial for the amelioration of bleeding associated with a variety of conditions. Additional investigation is required to determine if IL-6–mediated alterations of hemostasis may lead to pathologic thrombosis. The cDNAs for feline and canine IL-6 have been cloned, the recombinant proteins have been described,108-110 and they are commercially available (see Box 32-1).
IL-11 is a pleiotropic cytokine that enhances the activity of primitive, erythroid, and megakaryocyte progenitor cells and the production of hepatic acute phase proteins, and it supports growth of the intestinal epithelium. In animal models rhIL-11 has been shown to be effective in reconstituting platelet levels after the administration of chemotherapy or radiation therapy and in protection against radiation- or drug-induced damage to the intestinal epithelium. IL-11 has also been shown to increase circulating levels of vWF and rhIL-11 increases vWF in dogs with von Willebrand disease (vWD) with kinetics that are more gradual and sustained than desmopressin.111 Unlike the release of preformed vWF from platelets induced by desmopressin, administration of IL-11 results in the increased production of vWF RNA. Phase II trials of IL-11 in human patients with vWD have recently been completed.112 Clinically, rhIL-11 has also been used to support platelet levels in human cancer patients and in the management of Crohn’s disease and ulcerative colitis. A side effect that limits the administration of rhIL-11 is plasma volume expansion, resulting in edema, a fall in hematocrit levels, and cardiac arrhythmias. In dogs rhIL-11 increased platelet counts, platelet size, ploidy, and the number of megakaryocytes in marrow and peripheral blood. Pneumonitis may be a dose-limiting side effect in dogs.113
Chemokines are largely responsible for leukocyte chemotaxis, mediating inflammation and coordinating the host response to infection. The chemokines can be divided into two large and two small subfamilies on the basis of the pattern of cysteine residues found at the amino terminal end. The two large groups consist of the CXC subfamily, in which one amino acid is interspersed between the two cysteines, and the CC subfamily, in which the two cysteines are directly apposed. The two minor subfamilies consist of chemokines in which a single cysteine is found at the amino terminal end (XC) and ones in which the cysteines are separated by three amino acids (CX3C). Chemokines bind to one or more seven transmembrane, G protein–coupled receptors that, again, fall into two major and two minor subfamilies. In an attempt to standardize the nomenclature, the chemokines are grouped as CCL, CXCL, XCL, and CX3CL, and the receptors as CCR, CXCR, XCR, and CX3CR, where L denotes ligand and R denotes receptor and numbers are added in the order in which they have been described.114 In this chapter chemokines are referred to according to the standard nomenclature with their former names provided in brackets if they have been commonly used in the literature. Currently 46 chemokines and 19 chemokine receptors have been identified.115 Canine and feline CXCR4 and CCR5 have been isolated and demonstrate significant sequence homology and tissue distribution with their murine and human counterparts.116-118
Chemokines play a central role in inflammatory and allergic diseases. In response to signals such as lipopolysaccharide (LPS) from gram-negative bacteria or some of the inflammatory cytokines described earlier, such as IL-1 or TNF-α, chemokines are released at the site of injury, setting up a chemotactic gradient. Fibroblasts, smooth muscle cells, and epithelial cells have all been shown to be significant sources of chemokines. Neutrophils will move toward high levels of the chemokines CXCL1-3, CXCL6,7 and CXCL8 (IL-8); eosinophils respond to CCL5 (RANTES), CCL11,24,26 (eotaxin-1,2,3), CCL13 (MCP-4), and CCL28; and monocytes, DCs, and lymphocytes are attracted to CCL6 (C10), CCL2 (MCP-1), CCL3 (MIP1-α), and CCL4 (MIP1-β). There is a high level of overlap and promiscuity in the types of cells recruited by a particular chemokine based, in part, on the ability of some chemokines to bind multiple receptors and some receptors to bind multiple chemokines.
In addition to directing leukocytes to the site of inflammation or injury, chemokines provide the necessary gradients for DC trafficking to lymph nodes. Immature DCs circulate and act as sentinels for pathogens. In the immature state, they express a chemokine receptor repertoire that is poised to respond to many of the inflammatory chemokines. After recruitment to the site of inflammation, DCs encounter antigen and associated inflammatory cytokines and undergo maturational changes, including the expression of the chemokine receptor CCR7. The ligands for CCR7 are CCL19 (MIP3β) and CCL21 (6Ckine), which are expressed within the T cell–rich areas of lymphoid follicles. The end result is that the antigen-loaded, mature DC is preferentially drawn to the draining lymph node, where it will encounter T lymphocytes, thereby activating a specific cell-mediated response to the pathogen.
To describe chemokines in terms of leukocyte recruitment only would be to ignore their other important physiologic roles. The normal development of lymphoid organs depends on signals derived from chemokines, as does structural development of the fetal neurologic system119,120 and intestinal vascular networks.121 Chemokines have been proposed to play a role in embryo implantation and menstruation.122 Many tumors express chemokine receptors, and they have been shown to play a role in tumor growth and in directing organ-specific metastasis.123,124 The discovery that HIV uses the chemokine receptors CCR5 and CXCR4 as co-receptors for viral entry into target cells has helped highlight the way in which some pathogens exploit chemokine pathways.125,126
Elevated chemokine levels have been demonstrated in a variety of inflammatory and autoimmune diseases in humans, including asthma, inflammatory bowel disease, rheumatoid arthritis, and sepsis. Because much of the pathology associated with these diseases is due to the cascade of effects caused by the ongoing chemotactic stimulus, chemokine antagonists have emerged as potential therapeutics.127 The CC chemokine receptor CCR1 and its ligands CCL3 (MIP1-α) and CCL5 (RANTES) have been shown to be important in the pathology of models of rheumatoid arthritis, respiratory syncytial virus infection and sepsis. Serum levels of both CCL3 and CCL5 are elevated in septic humans,128 and mice lacking CCR1, the receptor for these chemokines, were significantly more likely to survive experimental sepsis.129 Limiting viral replication is clearly important in virally mediated pneumonia, but blocking chemokine-mediated recruitment of inflammatory cells has also shown dramatic benefits in experimental systems. In a murine model of respiratory syncytial virus infection, either blocking CCR1 signaling or deleting CCR1 expression in conjunction with antiviral treatment dramatically enhances survival compared with antiviral treatment alone.130 Antagonists of CCR1 have been developed, and despite the species specificity of some antagonists,131 cross-species efficacy has been demonstrated for other compounds. Liang and coworkers132 have developed a selective peptide antagonist of CCR1 that has efficacy in both human and rat systems, although the binding affinity of the peptide is significantly lower in rats. Additionally, this peptide was shown to have good oral bioavailability in dogs. Relatively minor modifications of antagonist structure have been shown to improve activity across divergent species.133
CXCL8 (IL-8)–induced neutrophil influx into tissues by way of CXCR1 and CXCR2 is clearly beneficial in controlling the early stages of bacterial infection. There are other instances in which CXCL8 expression potentiates the pathology of diseases such as sepsis/peritonitis, acute respiratory distress syndrome, exacerbations of human chronic obstructive pulmonary disease and bovine pneumonic pasteurellosis.134-137 CXCL8 is expressed in increased levels in several bacterial diseases of ruminants and in equine chronic obstructive pulmonary disease.4 Li and coworkers138 generated a potent CXCL8 antagonist that blocks signaling through both CXCR1 and CXCR2 in bovine neutrophils. They have shown efficacy in vitro against the neutrophil chemotactic activities of fluids from bovine pasteurella pneumonia and endotoxin- induced mastitis and in vivo against intradermal neutrophil influx in response to endotoxin.139 Because this antagonist also effectively blocks human CXCL8 activities on human neutrophils, it may have uses in feline and canine inflammatory diseases.
Introducing chemokines into tumors could be advantageous in recruiting immune effector cells to help destroy the tumor, and this has been shown to be true in murine cancer models. CCL16, CCL20, MIP3α, CCL21 (6Ckine) expression within murine tumors all resulted in increased infiltration of DCs, increased tumor immunogenicity, and decreased tumor growth.140-143 Bringing DCs and T lymphocytes into the tumor is not always sufficient, and providing a simultaneous activation stimulus helps overcome the otherwise immunosuppressive tumor microenvironment. This has been accomplished by delivering the chemokines in adenoviral vectors where the virus itself provides an immune stimulus140 or by simultaneously blocking immunosuppressive cytokines or providing innate immune system stimuli.143,141 Because there is significant cross-species sequence homology in many of the chemokines144 and precedence for cross-species efficacy between mice and humans with chemokines such as CCL20,140 some of the currently available murine and human chemokines may prove useful in veterinary cancer applications.
The converse to the potentially beneficial aspects of tumor chemokine expression is the documented role that chemokines and tumor chemokine receptor expression play in the natural course of tumor progression. Chemokines can act as tumor growth factors either directly or through the recruitment of leukocytes. Some tumor cells both express the chemokine receptor and produce its ligand, allowing autocrine enhancement of proliferation and survival. Melanoma cells expressing CXCR2 and producing CXCL1 and CXCL8 (IL8) are one such example.143 Macrophages recruited to tumors can elaborate tumor cell growth factors and increase angiogenesis within the tumor.144 Chemokine receptor expression by tumor cells has also been shown to play a substantial role in metastasis. Tumor cells expressing chemokine receptors will move toward chemokine gradients, resulting in metastasis to draining lymph nodes or distant sites. CCR7 expression by melanoma cells is correlated with metastasis to draining lymph nodes.145,146 It appears that the tumor cells are taking advantage of the normal chemokine gradient for activated, CCR7-expressing DC homing to draining lymph nodes. CXCR4 is expressed by a wide variety of tumors, including mesenchymal, epithelial, and hematopoietically derived cancers.147 Malignant mammary tumors have been shown to express high levels of CXCR4, and its ligand, CXCL12 (SDF1), is preferentially expressed in common target tissues for mammary tumor metastasis such as lung, liver, bone marrow, and lymph nodes.148 Oonuma and coworkers149 have demonstrated increased expression of feline CXCR4 in metastatic mammary tumors of cats and high levels of CXCL12 production in feline lymph node, lung, and liver. Additionally, they demonstrated increased in vitro tumor cell migration toward CXCL12 gradients and inhibition of this migration when CXCR4 peptide antagonists were added. The same CXCR4 antagonist has shown efficacy in inhibiting the number of lung metastases in a mouse model of mammary carcinoma.132 Some cases of canine osteosarcoma have been shown to express CXCR4 and migrate toward CXCL12 in vitro.150 Selective chemokine receptor antagonists hold promise as potential in vivo therapeutics to minimize cancer metastasis, and feline mammary tumors appear to be an appropriate system to apply these compounds. The documented role of chemokine receptor expression in human squamous cell carcinoma,151 melanoma,152,153 and prostate carcinoma154 awaits exploration in dogs and cats.
The discovery that HIV uses the chemokine receptors CXCR4 and CCR5 as co-receptors for viral entry125,126,155 has prompted the search for and development of drugs to inhibit virus binding. Subsequent work has shown that FIV uses CXCR4, but apparently not CCR5, as a co-receptor as well.156 Bicyclams are potent CXCR4 antagonists, and one such compound, AMD3100, has been shown to inhibit in vitro replication of FIV.157,158 Antagonists of feline CXCR4 have been developed and tested successfully in vitro.159 The number of CXCR4 antagonists is continually expanding,160 and FIV-infected cats may well benefit from these compounds in the future.
Rapid advances in the understanding of chemokine biology have allowed the exploration of ways to manipulate these factors in a wide variety of diseases. Whether they are used to recruit immune effector cells to tumors or their actions are blocked in an attempt to minimize inflammation or viral infection, manipulation of chemokines has the potential to be a powerful therapeutic tool.
In humans the hematopoietic system produces in the order of 1011 cells daily and is able to rapidly increase production even further when stimulated by hematopoietic growth factors. Hematopoietic growth factors include erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), monocyte colony-stimulating factor (M-CSF), thrombopoietin, stem cell factor (SCF), and most of the ILs. Hematopoietic growth factors act synergistically at various levels in the hematopoietic developmental system, and their actions are rarely restricted to a given lineage. The cDNAs for all of the known human factors, and some of the canine and feline factors, have been cloned. Recombinant hG-CSF (filgrastim), rhGM-CSF (sargramostim), and rhEPO (epoetin) are commercially available. For humans this has led to significant advances in the management of a variety of hematologic and neoplastic disorders. Canine and feline G-CSF, GM-CSF, and SCF have all been cloned and expressed,161-165 and canine and feline GM-CSF and SCF are commercially available (see Box 32-1).
In dogs EPO is secreted by cells adjacent to the proximal convoluted tubules in response to renal hypoxia. EPO stimulates the proliferation and maturation of erythroid progenitor cells, primarily colony-forming unit erythroid cells. Megakaryocytes are also stimulated by EPO. Recombinant human EPO was the first commercially available hematopoietic growth factor released for clinical use in humans and is indicated for the treatment of anemia secondary to chronic renal failure, anemia secondary to the treatment of HIV with AZT, and anemia secondary to cancer chemotherapy. The use of recombinant human, feline, and canine EPO in small animals is addressed elsewhere in this text (see Chapters 15 and 18).
G-CSF is produced by fibroblasts and endothelial cells stimulated by IL-1 or TNF and by macrophages stimulated by bacterial endotoxins. Its major effects are on neutrophils and neutrophil progenitors. Recombinant hG-CSF is commercially available and for humans is indicated to decrease the incidence of infection in patients with nonmyeloid malignancies receiving myelosuppressive chemotherapy with or without bone marrow transplantation, congenital neutropenia, and cyclic neutropenia.
In normal dogs rhG-CSF induces rapid and marked neutrophilia but, similar to the situation with rhEPO, also induces antibodies that react with both rhG-CSF and endogenous canine G-CSF, leading to chronic but reversible neutropenia.166,167 Whether dogs with cancer receiving immunosuppressive chemotherapy are able to mount an immune response to rhG-CSF has not yet been determined. In dogs treated with total body irradiation, rhG-CSF therapy was associated with earlier recovery of neutrophils and platelets and reduced the lethality of the hematopoietic insult compared with untreated irradiated controls.16 In gray Collies with cyclic hematopoiesis, rhG-CSF eliminated neutropenic episodes but did not correct abnormalities in platelet aggregation or serotonin content or decreased neutrophil myeloperoxidase activity.167,169 Recombinant hG-CSF has also been used to treat drug-induced pancytopenia in a dog.170
Canine G-CSF has been cloned but is not commercially available. In normal dogs rcG-CSF was shown to induce rapid and marked increases in neutrophils, moderate increases in lymphocyte and monocyte counts, and bone marrow hyperplasia. For example, five normal dogs were given 5 μg/kg rcG-CSF per day subcutaneously for 4 weeks. The mean neutrophil counts increased from 6537/μL to 26,330/μL within 24 hours after the first injection to a maximum of 72,125/μL by day 19. Blood counts returned to normal within 5 days after discontinuation of rcG-CSF, and clinically significant toxicoses were not associated with rcG-CSF administration. The induction of neutrophilia was induced again on repeated administration.171-173 Recombinant cG-CSF prevented neutropenia and associated clinical signs in cyclic hematopoietic dogs but did not completely eliminate the cycling of neutrophils in cyclic hematopoietic dogs. Also, the time to bone marrow reconstitution was not decreased in dogs treated with rcG-CSF after autologous bone marrow transplantation, emphasizing that rcG-CSF action depends on the presence of progenitor cells in the bone marrow.174
To evaluate the utility of rcG-CSF in the management of chemotherapy-induced neutropenia, myelosuppression was induced with mitoxantrone in normal dogs and then treated with daily rcG-CSF for 20 days. None of the dogs receiving rcG-CSF developed serious neutropenia, whereas four of five untreated dogs did. These findings demonstrate that rcG-CSF is capable of reducing the duration and severity of mitoxantrone-induced myelosuppression.175 The optimal cost-effective timing and duration of treatment for the management of therapy-induced myelosuppression has not been determined for canine or human origin cytokines, and it may only be necessary to treat when neutrophil counts fall below 1000 cells/μL, and only a few days of therapy may be necessary.176 In addition to cancer treatment–induced neutropenia, rcG-CSF has been used to accelerate the rate of recovery from neutropenia in dogs with parvovirus. It has not been useful in dogs without neutrophil progenitors, such as those with aplastic anemia secondary to ehrlichia infections or estrogen toxicity.177 Historical and bone marrow evaluations are important in determining which animals are likely to respond to therapy with any hematopoietic growth factor.
Recombinant G-CSFs of both human and canine origin have been studied in normal cats. In one study, 5 μg/kg rcG-CSF per day was administered to healthy cats for 42 days. Mean neutrophil counts increased from 10,966 cells/μL to 30,688 cells/μL within 24 hours after the first dose. Neutrophil counts increased and remained elevated until cytokine administration was discontinued at 42 days. No adverse effects were reported.178 Normal cats given rhG-CSF developed neutropenia before the end of 3 weeks of therapy, presumably because antibodies developed against the growth factor.179 Neutropenia has not been observed in cats given rcG-CSF, likely because of greater homology between the cat and dog cytokines.178 It is not known if cats with immunosuppressive disorders or cats receiving antineoplastic chemotherapy are able to form antibodies that react with human-derived cytokines. Cats with Chédiak–Higashi syndrome treated with rcG-CSF had increased neutrophil counts and improvement in neutrophil function.180 Recombinant G-CSF (of any species origin) may also be useful in the management of feline panleukopenia, neutropenia associated with FeLV and FIV infections, and sepsis.
GM-CSF is produced by the bone marrow stroma and T and B cells and is a regulator of the intermediate stages of hematopoiesis. It supports the expansion and growth of both granulocytic and macrophage lineages and also enhance the function of mature macrophages and neutrophils.181 In humans rhGM-CSF is indicated for the acceleration of hematopoietic reconstitution after autologous bone marrow transplantation in lymphoproliferative disorders. Recombinant hGM-CSF induces leukocytosis (primarily neutrophils but also eosinophils and monocytes) in normal dogs. In dogs undergoing total body irradiation and supported with rhGM-CSF, there was decreased severity and shortened duration of neutropenia, indicating that rhGM-CSF can be effective monotherapy for radiation-induced bone marrow failure in dogs. Anti–rhGM-CSF antibodies developed in 1 to 2 weeks and persisted for at least 150 days. Another potential concern with rhGM-CSF is that platelet counts dropped to nadirs of 20% to 30% normal levels.182,183
Canine GM-CSF has been cloned and its activity investigated in normal dogs, where it induced significant increases in neutrophil and monocyte levels. As with the human recombinant product, mean platelet counts decreased significantly. Further investigation into the mechanism of thrombocytopenia suggested that GM-CSF activates hepatic macrophages, with resultant increases in phagocytosis of platelets.184,185 After otherwise lethal total body irradiation, rcGM-CSF was not effective in promoting hematopoietic recovery or improving survival.186 In studies of partial body irradiation in dogs, 7 days of treatment with rhG-CSF resulted in a more efficient and rapid reconstitution of neutrophil numbers than similar treatment with rhGM-CSF.187 It is thought that G-CSF results in an increase in the production of migratory hematopoietic progenitors from the protected marrow sites and an enhanced seeding of these progenitors into the regions of damaged marrow. These results suggest that, in situations of severely limited stem cell response, G-CSF may be more effective than GM-CSF in eliciting a rapid neutrophil recovery.
In an effort to avoid daily systemic administration of recombinant GM-CSF, direct intramarrow injection of adenoviral vector–cGM-CSF constructs (AdcGM-CSF) in normal dogs has been carried out.188 Replication-deficient adenoviral vectors efficiently transduce marrow stromal cells and induce high levels of cytokine production. In vivo, high levels of protein production are found in bone marrow aspirates 72 hours after direct intramarrow administration of AdcGM-CSF. Localized myeloid expansion of marrow and significant peripheral leukocytosis have been identified in all treated dogs, and peripheral blood changes last for up to 3 weeks after a single intramarrow injection. It appears that adenoviral-mediated cytokine expression from the marrow of a single large bone (ileum) leads to compartmentalized expression of GM-CSF and an increase in hematopoiesis. Recent studies have demonstrated that intravenous administration of canine GM-CSF ligated to silica nanoparticles results in detectable GM-CSF for up to 10 days and prevented or restored vinblastine-induced neutropenia in dogs.189,190 None of the dogs had detectable anti–GM-CSF antibodies 28 days after injection.
GM-CSF has been used to expand DC precursors from peripheral blood and bone marrow for potential therapeutic purposes. Ex vivo expanded DCs have been stimulated with antigen and reintroduced in experimental models of infectious and neoplastic diseases.191,192 Both human and feline GM-CSF have been used to culture myeloid DCs from feline peripheral blood and bone marrow,193,162,194 and canine GM-CSF has been used with canine peripheral blood and bone marrow.195,196 In vitro culture, antigen stimulation, and reintroduction of bone marrow–derived DCs from three dogs with melanoma have been performed.197 These cells were cultured with a combination of rhGM-CSF, SCF, and FLT3 ligand, matured with rhTNF-α and administered in conjunction with radiation therapy. One dog developed antigen-specific cytotoxic T cell activity and remained tumor free 48 months after therapy. It is likely that more studies employing DC vaccination will be carried out in canine and feline patients in the near future.
GM-CSF is a mediator of antibody-dependent cellular cytotoxicity and increases MHC expression, giving GM-CSF potential utility as a modulator of antitumor immunity. A vaccine consisting of irradiated hGM-CSF transfected canine melanoma cell line has been reported to produce GM-CSF at the site of intradermal injection for extended periods in normal dogs.198 A phase I clinical trail using autologous tumor cells in dogs with melanoma and soft tissue sarcomas showed significant tumor infiltration of neutrophils and macrophages in treated tumors.199
SCF (c-kit ligand, mast cell growth factor) is produced primarily by bone marrow stroma, with effects on a wide range of precursor cells at different stages of differentiation, including primitive hematopoietic stem cells. It has little activity as a single agent; however, it is a potent co-stimulatory molecule when administered in combination with other hematopoietic growth factors. Canine SCF cDNA has been cloned and studied in long-term bone marrow cultures. Alone, rcSCF was nonstimulatory for committed marrow precursors. Synergistic stimulation of granulocyte/macrophage colony-forming units was demonstrated between rcSCF, rhGM-CSF, and rhIL-6.200 In vivo, rcSCF induced neutrophilia in normal dogs and supported hematopoietic recovery in normal dogs undergoing total body irradiation without marrow transplant; however, results were similar to those obtained with rcG-CSF.201 Recombinant canine SCF also prevented neutropenic periods in gray Collies with cyclic hematopoiesis.202
Hematopoietic growth factors have proved quite useful in small animal veterinary medicine for managing cytopenias and anemias. The recent cloning and expression of canine- and feline-specific reagents will help alleviate the problem of antibody generation with repeated use. Additional uses of factors such as GM-CSF in cancer chemotherapy and in the in vitro generation of DCs will expand the clinical utility of this class of growth factors.
The concept of enhancing the normal immune response against infections and tumors has been considered for decades. The administration of various natural and synthetic products to simulate systemic infections has largely given way to the idea that specific cytokines can be used effectively when administered locally or systemically. IFNs, ILs, chemokines, and hematopoietic growth factors may offer substantial clinical benefit in chronic viral infections and in cancers. EPO has been shown to have great utility in the management of chronic renal failure. The recent increase in the commercial availability of canine and feline reagents allows for increased exploration of their efficacy in diseases of dogs and cats. These products may have significant clinical impact on several highly fatal disorders of dogs and cats. When administered systemically, cytokines perturb complex regulatory pathways, and serious side effects may occur. Innovative delivery methods, such as liposomes, adenoviral delivery, and even oral administration, may increase the therapeutic index of these molecules. Biological response modification, cytokine biology, and associated delivery systems are rapidly changing fields, and the small animal veterinarian will need to watch for significant advances in these areas over the next several years.
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∗ Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine with biological activities that include modulation of tumor growth, infections, septic shock/systemic inflammatory response syndrome, and autoimmunity. The TNF family of cytokines is distinct from the IL and IFN families. Canine and feline TNF-alpha have been cloned. They are not discussed in this chapter.
∗ From Kruth SA: Biological response modifiers: interferons, interleukins, recombinant products, liposomal products, Vet Clin North Am Small Anim Pract 28:269-295, 1998.