Helper T-Cell Subsets
Helper T-cell (Th cell) subsets were originally reported by Mossman and colleagues 1 in 1986, who described two types of mouse CD4+ T-cell clones distinguished by their patterns of cytokine production (figure 1). Th1 cells are responsible for cellular immunity and mainly produce interleukin-2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor β (TNF-β). Th2 cells, in contrast, are responsible for humoral immunity, and mainly produce IL-4, IL-5, IL-6, and IL-13. Although IL-10 was originally described as a product of Th2 cells, 2 it is now clear that Th1 cells also produce IL-10. 3 Many cells other than CD4+ cells—such as CD8+ cells, monocytes, B cells, natural killer (NK) cells, mast cells, and eosinophils—also produce Th1 and Th2 cytokines. Some investigators suggest, therefore, that these cytokines should be called type 1- or Th1-like cytokine and type 2- or Th2-like cytokine, respectively.
Both Th1 and Th2 cells mature from a naive Th cell type (Thp; precursor Th cell) through Th0 cells, which produce both Th1 and Th2 cytokines. 4 The subset imbalances appear to contribute to the pathogenesis of several diseases. Rheumatoid arthritis, multiple sclerosis, and insulin-dependent diabetes mellitus, for example, are considered to be Th1-cell-mediated diseases. In contrast, allergic diseases and systemic lupus erythematosus are thought to be Th2-cell-mediated diseases.
Chen and associates 5 found a third T-cell type, Th3, which has a unique pattern of cytokine production in experimental allergic (autoimmune) encephalomyelitis (EAE; an animal model of multiple sclerosis). Th3 cells have also been investigated in an experimental autoimmune model of insulin-dependent diabetes mellitus 6 and in human autoimmune diseases such as multiple sclerosis and colitis. 7,8 This cell type is assumed to play a role in immunosuppression of the Th1 response via transforming growth factor-β, although the contribution of Th3 cells to cytokine balance is largely still unclear, especially with regard to ocular immunology.
Th-cell maturation consists of activation, differentiation, and proliferation. This maturation and Th1/Th2 balance are organized by several cytokines composing a so-called cytokine network with autocrine or paracrine functions of up-or downregulation. Naive Th cells need T-cell receptor stimulation to be activated by antigen-presenting cells (APCs), such as macrophages, B cells, or dendritic cells, as well as a costimulatory signal via B7/CD28 and CD40/CD40 ligand interaction. Activated Th cells mature into memory/effector Th cells, which have the ability to express several cytokines, receptors, and the other products necessary for carrying out their multiple functions.
Th cell differentiation appears to vary by cell type. Once naive CD4+ Th cells (Thp) are activated, they acquire the ability to produce both Th1 and Th2 cytokines (Th0 cells). Subsequently, however, IL-12 produced by APCs induces differentiation to Th1 cells, whereas IL-4 leads to differentiation toward a Th2 cell type. 9 IL-4, which induces differentiation to a Th2 cell type, is thought to be produced by NK1.1+ T cells. 10 Once Th2 cells are induced, however, IL-4 produced by Th2 cells can also promote Th2 differentiation in an autocrine fashion.
Proliferation of Th cells depends on the coexistence of IL-2 and other cytokines. Th1-cell proliferation requires coexpression of IL-2 and IL-12. T-cell receptor stimulation of murine Th1 cells can induce IL-2 production but not T-cell proliferation. 11 Addition of IL-12, however, can promote Th1-cell proliferation in an IL-2-dependent fashion. This function is due to the expression of IL-2 receptor on Th1 cells induced by the synergistic effect of IL-12 and IL-2. The proliferation of Th2 cells depends on IL-4 and also on IL-2. IL-1, which is produced mainly by macrophages, upregulates IL-2 receptor expression on Th2 cells but not on Th1 cells. 12 Hence, Th1 versus Th2 development depends on IL-12 and IL-1, respectively. Furthermore, IL-1 and IL-12 production by APCs requires the stimulation of CD40 by CD40L expressed on CD4+ T cells. 13–15
Importance of Th Cells in Uveitis
Over the last decade, numerous studies have attempted to clarify the pattern of Th cytokine production in various Th-cell-mediated diseases, including various forms of uveitis. CD8+ T cells, considered to be cytotoxic/suppressor T lymphocytes, also have the ability to produce Th1 or Th2 cytokines (Tc1, Tc2). However, activated CD4+ T cells predominate in inflammatory foci in most forms of uveitis, 16–19 although activated CD8+ cells have been shown to be abundant in the early stage of exogenous uveitis. 17 CD8+ cells also predominate in patients with Fuchs' uveitic syndrome; however, these cells do not appear to be activated. 20 It is not clear, therefore, whether CD8+ cells are pathogenic in this disease. Accordingly, Th cells (CD4+ T cells) are assumed to be more important in the pathogenesis of uveitis.
Experimental autoimmune uveoretinitis (EAU), the most common animal model of noninfectious uveitis, has been well investigated to clarify the role of Th cells and the pattern of Th cytokine production in uveitis. Ocular inflammation in this model can be induced in rats, mice, and other animals by immunization with a retinal antigen, usually interphotoreceptor retinoid-binding protein or S-antigen, together with adjuvant. Adoptive transfer of T cells from primarily immunized animals can also induce EAU. 21 Barton and associates 22 demonstrated the kinetics of cytokine mRNA expression in retinas of EAU rats. Th1 cytokine mRNA reached maximal levels of expression prior to the onset of clinical disease, and increased IL-10 seemed to be produced in the recovery stage. Rizzo and colleagues 23 reported that treatment of EAU mice with IL-10 ameliorated ocular inflammation and that concomitant treatment with IL-4 further reduced disease. Therefore, EAU has been considered to be Th1-predominant, and the suppression of Th1 response proved successful in reducing the inflammation.
Th-cell dominance has been demonstrated in patients with uveitis. Ohta and colleagues 24,25 reported T-cell subsets in aqueous humor and peripheral blood from patients with active uveitis associated with sarcoidosis, Vogt-Koyanagi-Harada (VKH) disease, HLA-B27+ uveitis, Behçet's disease, and diabetes mellitus. These researchers showed a significantly higher percentage of activated memory Th cells in inflamed eyes as compared to peripheral blood. Deschênes and associates 26 demonstrated that most patients with uveitis had significantly more activated T cells in intraocular fluid, peripheral blood, or both, and that activated Th cells were significantly increased in most forms of systemic and idiopathic uveitis.
Just as multiple sclerosis, rheumatoid arthritis, and insulin-dependent diabetes mellitus now are considered to be Th1-mediated diseases, Th1 dominance in patients with uveitis has also been reported. Lacomba and coworkers 27 measured the concentrations of IFN-γ, IL-2, IL-4, and IL-10 in aqueous humor and serum samples from patients with uveitis, including those with Behçet's disease, Fuchs' uveitic syndrome, chronic juvenile iridocyclitis, birdshot retinochoroidopathy, ankylosing spondylitis, and idiopathic uveitis. These investigators showed higher levels of IFN-γ and IL-2 in both aqueous and serum samples from uveitic patients as compared to control patients undergoing uncomplicated cataract removal. They also analyzed the correlation between cytokine concentrations and clinical characteristics and suggested that elevated serum levels of IFN-γ seemed to predispose patients with uveitis to more serious loss of vision. Moreover, it was reported that aqueous and vitreous levels of IL-12 were significantly higher in patients with active uveitis as compared to patients with moderate or quiet uveitis or uncomplicated cataract patients. 28 Among patients with active uveitis, linear regression analysis indicated a significant and linear relationship between IL-12 levels and the severity of inflammation. In this study, uveitis patients included those with HLA-B27+ uveitis, sarcoidosis, multifocal choroiditis, juvenile rheumatoid arthritis, intermediate uveitis, idiopathic anterior uveitis, panuveitis, and retinal vasculitis. These results suggested a predominantly Th1 response in patients with uveitis. However, because the number of patients with each disease was not large enough to allow for statistical comparisons, it remains unknown whether most uveitic diseases are Th1-predominant. Two exceptions are Behçet's disease and VKH disease.
It has been reported that patients with Behçet's disease have increased serum levels of IFN-γ and enhanced IFN-γ production in peripheral blood lymphocytes (PBLs). 29,30 Serum levels of soluble IL-2 receptor and TNF-α were also increased, 31 and peripheral blood monocytes isolated from the patients had the ability to produce a high level of TNF-α. 32 TNF-α is one of the proinflammatory cytokines produced from activated mononuclear phagocytes or T cells, and IFN-γ enhances the production of this cytokine. TNF-α has several functions relevant to ocular inflammation, including breakdown of the blood-ocular barrier. Nakamura 33 demonstrated that PBLs from patients with Behçet's disease were slightly activated in vivo and that they were defective at Fas and Fas ligand rather than induced apoptosis. It was suggested, therefore, that slightly activated PBLs may promote Behçet's disease and that insufficient apoptosis of these cells may cause the disorder to be more chronic. Moreover, Sugi-Ikai and associates 34 demonstrated that Th1 and Tc1 cytokines were predominant in PBLs from patients with Behçet's disease. Together, these findings suggest that activated Th1 and Tc1 cells, assumed to be resistant to apoptosis, continuously produce type 1 cytokines, thereby causing the chronic inflammation of Behçet's disease.
We investigated Th-cell subsets in VKH disease and suggested that VKH disease also is associated with a Th1-dominant response. VKH disease is well-known to be a cell-mediated autoimmune disease directed against melanocytes. The majority of cerebrospinal fluid (CSF) cells from VKH patients are lymphocytes, especially Th cells, 35 and the CD4+/CD8+ ratio in the CSF is significantly higher than that in peripheral blood. 16,35 Furthermore, cells in the CSF appear to be primarily activated memory Th cells. 36 Th cells are therefore assumed to play an important role in the pathogenesis of VKH disease. Regarding T-cell cytokines in VKH disease, it has been reported that the serum level of IFN-γ was higher in patients with VKH disease, as well as in those with Behçet's disease. 37
We demonstrated that PBLs of patients with VKH disease, especially when they are stimulated have the ability to produce primarily Th1 cytokines. 38 Cytokine production of PBLs was investigated to clarify the Th cytokine production at the gene level, at the level of intracellular expression, and at the level of secretion, by reverse transcriptase–polymerase chain reaction, flow cytometry, and enzyme-linked immunosorbent assay, respectively. We found that Th cells from patients with VKH disease tended to deviate toward a Th1 type and that activated Th cells in the inflammatory foci produced Th1 cytokines, thereby promoting inflammation. Yamaki and colleagues 39,40 reported that the target of the autoimmune process in VKH disease is a tyrosinase-related protein (TRP1, TRP2), and they developed a rat model of the disease by immunization with TRP1 and TRP2. Their findings strongly support a need for further investigation to clarify the mechanism of this disease.
Application of Cytokine Systems to Immunosuppressive Therapy
Most therapies for uveitis are nonspecific. These include corticosteroids, antimetabolites (e.g., methotrexate, azathioprine), inhibitors of transcription factors (e.g., cyclosporine, tacrolimus), and DNA cross-linking agents (e.g., cyclophosphamide, chlorambucil). 41 New agents directed against cytokines and their receptors, many of which act by inhibiting important Th1 cytokine rather than signaling pathways, are, however, beginning to be used for treatment of patients with uveitis. These include TNF inhibitors such as Infliximab (Centocor, Malvern, PA) and Etanercept (Immunex, Seattle, WA) and specific inhibitors of IL-2 signaling, including Daclizumab (Zenapax, Roche Laboratories, Nutley, NJ) and Basiliximab (Simulect, Novartis Pharmaceutical Co., East Hanover, NJ). Ongoing and future efforts to elucidate the mechanisms of Th1 and Th2 signaling responses hold great promise for improved therapeutics for patients with uveitis.
We greatly appreciate Dr. E. Cunningham for reviewing the manuscript and for his helpful suggestions.
1. Mosmann TR, Cherwinski H, Bond MW, et al. Two types of murine helper T cell clone: I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986; 136: 2348–2357
2. Fiorentino DF, Bond MW, Mosmann TR. Two types of mouse T helper cell: IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J Exp Med 1989; 170: 2081–2095
3. Sornase T, Larenas PV, Davis KA, et al. Differentiation and stability of T helper 1 and 2 cells derived from naive human neonatal CD4+ T cells, analyzed at the single-cell level. J Exp Med 1996; 184: 473–483
4. Mosmann TR, Moore KW. The role of IL-10 in cross regulation of TH1 and TH2 responses. Immunol Today 1991; 12: 49–53
5. Chen Y, Kuchroo VK, Inobe J, et al. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 1994; 265: 1237–1240
6. Hans HS, Jun HS, Utsugi T, et al. A new type of CD4+ suppressor T cell completely prevents spontaneous autoimmune diabetes and recurrent diabetes in syngeneic islet-transplanted NOD mice. J Autoimmun 1996; 9: 331–339
7. Fukaura H, Kent SC, Pietrusewicz MJ, et al. Induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor-β1-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients. J Clin Invest 1996; 98: 70–77
8. Powrie F, Carlino J, Leach MW, et al. A critical role for transforming growth factor-β but not interleukin 4 in the suppression of T helper type 1–mediated colitis by CD45RB low
CD4+ T cells. J Exp Med 1996; 183: 2669–2674
9. Seder RA, Paul WE. Acquisition of lymphokine-producing phenotype by CD4+ T cells. Annu Rev Immunol 1994; 12: 635–673
10. Yoshimoto T, Paul WE. CD4pos, NK1. 1pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3. J Exp Med 1994; 179: 1285–1295
11. Yanagida T, Kato T, Igarashi O, et al. Second signal activity of IL-12 on the proliferation and IL-2R expression of T helper cell-1 clone. J Immunol 1994; 152: 4919–4928
12. Taira S, Kato T, Yamamoto K, et al. Differential requirement for humoral factors for IL-2R expression of murine T cell subsets, Th1, Th2 and CD8Th clones. Cell Immunol 1993; 147: 41–50
13. Wagner DH, Stout RD, Suttles J. Role of the CD40-CD40 ligand interaction in CD4+ T cell contact–dependent activation of monocyte interleukin-1 synthesis. Eur J Immunol 1994; 24: 3148–3154
14. Shu U, Kiniwa M, Wu CY, et al. Activated T cells induce interleukin-12 production by monocytes via CD40-CD40 ligand interaction. Eur J Immunol 1995; 25: 1125–1128
15. Kato T, Hakamada R, Yamane H, Nariuchi H. Induction of IL-12 p40 mRNA and IL-12 production of monocytes via CD40-CD40 ligand interaction. J Immunol 1996; 156: 3932–3938
16. Norose K, Yano A, Aosai F, et al. Immunologic analysis of cerebrospinal fluid lymphocytes in Vogt-Koyanagi-Harada disease. Invest Ophthalmol Vis Sci 1990; 31: 1210–1216
17. Wang XC, Norose K, Yano A, et al. Two-color flow cytometric analysis of activated T lymphocytes in aqueous humour of patients with endogenous vs exogenous uveitis. Curr Eye Res 1995; 14: 425–433
18. Ohta K, Norose K, Wang XC, et al. Abnormal naive and memory T lymphocyte subsets in the peripheral blood of the patients with uveitis. Curr Eye Res 1997; 16: 650–655
19. Calder VL, Shaer B, Muhaya M, et al. Increased CD4+ expression and decreased IL-10 in the anterior chamber in idiopathic uveitis. Invest Ophthalmol Vis Sci 1999; 40: 2019–2024
20. Muhaya M, Calder V, Towler HMA, et al. Characterization of T cells and cytokines in the aqueous humor (AH) in patients with Fuchs' heterochromic cyclitis (FHC) and idiopathic anterior uveitis (IAU). Clin Exp Immunol 1998; 111: 123–128
21. Mochizuki M, Kuwabara T, McAllister C, et al. Adoptive transfer of experimental autoimmune uveoretinitis in rats. Invest Ophthalmol Vis Sci 1985; 26: 1–9
22. Barton K, McLauchlan MT, Calder VL, et al. The kinetics of cytokine mRNA expression in the retina during experimental autoimmune uveoretinitis. Cell Immunol 1995; 164: 133–140
23. Rizzo LV, Xu H, Chan CC, et al. IL-10 has a protective role in experimental autoimmune uveoretinitis. Int Immunol 1998; 10: 807–814
24. Ohta K. T lymphocyte subsets in aqueous humor from patients with uveitis [in Japanese]. J Jpn Ophthalmol Soc 1996; 100: 899–904
25. Ohta K, Norose K, Wang XC, et al. Apoptosis-related Fas antigen on memory T cells in aqueous humor of uveitis patients. Curr Eye Res 1996; 15: 299–306
26. Deschênes J, Char DH, Kaleta S. Activated T lymphocytes in uveitis. Br J Ophthalmol 1988; 72: 83–87
27. Lacomba MS, Martin CM, Chamond RR, et al. Aqueous and serum interferon gamma, interleukin (IL) 2, IL-4, and IL-10 in patients with uveitis. Arch Ophthalmol 2000; 118: 768–772
28. El-Shabrawi Y, Livir-Rallatos C, Christen W, et al. High levels of interleukin-12 in the aqueous humor and vitreous of patients with uveitis. Ophthalmology 1998; 105: 1659–1663
29. Ohno S, Kato F, Matsuda H, et al. Detection of gamma interferon in the sera of patients with Behçet's disease. Infect Immun 1982; 36: 202–208
30. Ohno S, Kato F, Matsuda H, et al. Studies on spontaneous production of gamma-interferon in Behçet's disease. Ophthalmologica 1982; 185: 187–192
31. Sayinalp N, Ozcebo O, Ozdemir O, et al. Cytokines in Behçet's disease. J Rheumatol 1995; 22: 904–907
32. Nakamura S, Sugita M, Tanaka S, Ohno S. Enhanced production of in vitro tumor necrosis factor-alpha in Behçet's disease. In: Dernouchamps JP, et al., eds. Recent advances in uveitis. Amsterdam: Kugler Publications, 1993: 71–74
33. Nakamura S. Cell biology in endogenous uveitis [in Japanese]. J Jpn Ophthalmol Soc 1997; 101: 975–986
34. Sugi-Ikai N, Nakazawa M, Nakamura S, et al. Increased frequencies of interleukin-2-and interferon-γ-producing T cells in patients with active Behçet's disease. Invest Ophthalmol Vis Sci 1998; 39: 996–1004
35. Ariga H, Ohno S, Higuchi M, et al. Immunological studies on lymphocytes in the cerebrospinal fluid of patients with Vogt-Koyanagi-Harada disease and sympathetic ophthalmia [in Japanese]. J Jpn Ophthalmol Soc 1988; 92: 225–228
36. Ohta K, Yoshimura N. Expression of Fas antigen on helper T lymphocytes in Vogt-Koyanagi-Harada disease. Graefes Arch Clin Exp Ophthalmol 1998; 236: 434–439
37. Ohno S. Immunological aspects of Behçet's and Vogt-Koyanagi-Harada's disease. Trans Ophthalmol Soc UK 1981; 101: 335–341
38. Imai Y, Sugita M, Nakamura S, et al. Cytokine production and helper T cell subsets in Vogt-Koyanagi-Harada's disease. Curr Eye Res 2001 (in press)
39. Yamaki K, Kondo I, Nakamura H, et al. Ocular and extraocular inflammation induced by immunization of tyrosinase related protein 1 and 2 in Lewis rats. Exp Eye Res 2000; 71: 361–369
40. Yamaki K, Gocho K, Hayakawa K, et al. Tyrosinase family proteins are antigens specific to Vogt-Koyanagi-Harada disease. J Immunol 2000; 165: 7323–7329
41. Solomon SD, Cunningham ET. Use of corticosteroids and noncorticosteroid immunosuppressive agents in patients with uveitis. Comp Ophthalmol Update 2000; 1: 273–286