1. Background to the Th1/Th2 paradigm
In 1986 two types of murine helper T cell clones were described that have had enormous effect on the way exploration of immune responses to pathogen invasion is understood (3). Helpful review material is referenced at the end of this article (1, 2, 4). Initially the paradigm was adopted enthusiastically and it seemed as though immune responses to various classes of infecting organisms could be neatly classified as either eliciting a Th1 or a Th2 response and that immunity to an organism required one of these specific responses for clearance of infection. Then it became apparent that in many cases a balance of these two types of T-cell responses was actually required to eliminate infection. Now it is beginning to emerge that in fact, the paradigm may not be nearly as clear-cut or 'neat' as was initially thought. This brief review will endeavour to introduce the paradigm as initially described in mice, illustrate it in the prototypical murine experimental Leishmaniasis model and then trace how our grasp of the T-helper cell subset responses has evolved toward understanding the need for balance. Finally we will examine some evidence that illustrates that the paradigm may well be more complex than initially thought.
The Th1/Th2 dogma can be simply described in the following way: CD4+ T-cells have been categorised into two subsets (Th1 and Th2) according to the profile of cytokines they produce. Although the initial work was done in mice, published work upholds the principle in man, dogs and cats as well. Generally, intracellular infections rely on a Th1 type response to resolve infection and extracellular parasites (mainly helminths) elicit a Th2 type response. Prototype Th1 cytokines include IL-12, IFNγ and TNF. These cytokines are pro-inflammatory and evoke cell-mediated immune responses and these cytokines are typically needed to resolve bacterial, viral and protozoal infections. Th2 cytokines include IL-4, IL-5 and IL-10. These cytokines are anti-inflammatory and typically evoke humoral immune responses and Th2 responses are classically described in immune responses to helminths.
An important question raised is: What factors are responsible for determining into which class of T helper cell a naive (Th0) CD4+ cell will differentiate? Factors that play a role include the nature of the antigen, the dose of the antigen and the organ of initial antigen exposure. Perhaps the most important determinant however is the cytokine milieu in which antigen presentation occurs. This is determined by the cytokines released by the antigen presenting cell (APC) into the immunologic synapse. Of the three APC's known, the dendritic cell (DC) is emerging as key and hence the biology of this phenotype is being closely examined. The early production of IL-12 in the immunological synapse between APC and Th0 T-cell has been shown to prime Th1 responses in response to intracellular microbial infections and the secretion of IL-10 to bias Th2 T-cell responses.
The infection in which this paradigm of immunity is best studied and most easily understood is Leishmania and so we will examine this infection as prototypical and use it to illustrate the general principles.
It is well known that resolution of a murine Leishmania infection depends on an early Th1 response and that susceptibility to disease is associated with early Th2 domination. This has been shown in various ways, namely: in mice that are genetically susceptible; using blocking antibodies and using cytokine gene knockout mice. Crucial to outcome is the nature and behavior of the APC during infection and it is has become clear that the dendritic cell population is the key player in this regard. In the skin, epidermal Langerhans cells internalise L. major amastigotes and transport them to the regional lymph node. DC's exposed to Leishmania promastigotes or promastigote culture supernatant stimulate proliferative responses in lymph node T cells from naïve mice. In the spleen a role for DC's in disease resistance has been shown in a mouse model of Leishmania donovani infection. A decrease in spleen red pulp parasite load was associated with a movement of DC's into the red pulp and dramatic increases in IL-12 and IFNγ production. The secretion of Th1 type cytokines in to the immunological synapse at the time of antigen presentation to Th0 T-cells determines the development of a Th1 phenotype. The most important cytokine in this regard seems to be IL-12 although a role for IL-18 and IFNγ have also been implied.
Despite the fact that an earlier study indicated that macrophages were the most efficient presenters of Leishmanial antigen in an in-vitro system, several subsequent studies have shown the DC's to be the principal antigen presenting cell and source of IL-12. In one study it was found that that DC's can take up organisms and, after this internalization, undergo changes in surface phenotype suggesting maturation. Using flow cytometry to analyze cytokine production at the single-cell level, it was found that infected DC's, but not monocytes, produce large amounts of IL-12p70 in a CD40 ligand (CD40L)-dependent manner. Macrophages fail to make IL-12 following infection. They actually become refractory to stimuli that normally cause IL-12 production after having internalised the organism. The IL-12 production occurs very early in the course of infection, peaking on day one and returning to base line levels by day 3-post infection. It was proposed that a DC-T cell cluster might thus provide the initial stimulus for IFNγ production by NK cells. Interestingly, the fate of Leishmania promastigotes is different in DC's compared to macrophages. Macrophages take up large numbers of promastigotes that mature to amastigotes in parasitophorous vacuoles. DC's take up small numbers of promastigotes that do not differentiate to amastigotes but rather appear to be degraded. Research has reinforced the idea that to impact the course of an infection through immunomodulation, it is the early events that must be impacted. Influencing later events, even in the same measure, is ineffectual.
DC's from susceptible mouse strains are an early source of the Th2 biasing cytokine IL-4. Leishmania amazonensis amastigotes and metacyclics are able to enter and activate DC's of both susceptible and resistant mouse strains. DC's from susceptible BALB/c mice however fail to produce CD40 induced IL-12 but rather produce IL-4.
Transfer of these BALB/c DC's into syngeneic amastigote exposed mice results in a significantly higher parasite specific Th2 response than that seen in resistant mouse strains. DC's taken from IL-4-/- mice indicate that this Th2 biasing is at least partially due to amastigote carrying DC's. Thus host susceptibility, at least in part, may be due to the Th2 biasing effect of infected DC's.
Similar findings have been demonstrated for several infectious agents including Toxoplasma, Trypanosoma cruzi and Cryptosporidium parvum.
The need for both T-cell phenotypes acting sequentially has been recognised in Plasmodium chabaudi AS infection of the resistant C57BL/6 and susceptible AJ mouse strains. It has become clear that early Th1 responses cause a rapid drop in parasitaemia and a later Th2 response illicits an antibody response that finally clears parasitaemia. Several studies have shown an important role for early IL-12 production and DC's in Plasmodium infections. In a mouse model of this disease it has been shown that the spleen is the primary source of IL-12. Resistant C57BL/6 mice show an early Th1 response in the spleen as shown by increased levels of TNFα and IFNγ mRNA expression and low levels of IL-4 expression in splenic lymphocytes. TNFα mRNA levels correlate with cytokine concentrations in blood. In addition anti-TNF antibody given early in the infection renders resistant mice susceptible. The susceptibility of AJ mice however is correlated with lower levels of early Th1 cytokine mRNA expression in the spleen and also later on in the disease around the time of death. Hence it is apparent that the timing and the site of Th1 cytokine release is crucial in outcome. In resistant mice, if the early Th1 response is not followed by a Th2 response, effective antiparasite antibodies are not generated and the infection persists.
2. Questioning the paradigm
The simple way of understanding the Th1/Th2 paradigm of immune response to protozoan infections, namely, early Th1 cytokines clear the infection and later Th2 cytokines prevent serious ongoing inflammation, may well be an over simplification of the truth. Cytokine interactions are complex and timing as well as specific location of release are just two factors that may impact strongly on the effect of a particular cytokine. The "good guys-bad guys" view of Th1 versus Th2 cytokines is less and less a view held onto as a dogma useful in the explanation of immunopathogenesis of disease.
Several studies have shown a role for classical Th2 cytokines in an early appropriate disease protecting immune response against intracellular protozoal infections. IL-4, when present during initial activation of DC's, can instruct them to produce IL-12 and promote a Th1 response thus protecting normally susceptible BALB/c mice against Leishmania major infection. If IL-4 is present later during the period of T cell priming, a Th2 response develops and mice succumb to progressive disease. This example shows that the timing of exposure to a particular cytokine may be more important than to which side of the traditional Th1/Th2 spectrum it has been classified. Stat4 is a second messenger important in signaling Th1 cytokine production. The Th2 cytokines, IL-4 and IL-10, suppress Stat4 induction in DC's and macrophages if present during maturation and activation, respectively, thus diminishing IFN-gamma production. In contrast, IL-4 has no effect on Stat4 levels in mature DC's and actually augments IFNγ production by DC's during Ag presentation, indicating that IL-4 acts differently in a spatiotemporal manner.
A phosphorylcholine-containing glycoprotein secreted by the filarial nematode, Acanthocheilonema viteae, which generates a Th2 antibody response in vivo, has (as would be expected) been found to induce the maturation of dendritic cells with the capacity to induce Th2 responses (increased IL-4 and decreased IFN-gamma). The switch to either Th1 or Th2 responses is not affected by differential regulation through CD80 or CD86 and the Th2 response is achieved in the presence of IL-12. This is counter-intuitive, as the traditional model would imply that the presence of IL-12 would dominate and bias a Th1 response.
Surprisingly, and somewhat contrary to what was hypothesised (based on the simplistic view of Th1/Th2), IL-2 knock out mice develop severe Th1 mediated autoimmune mediated disease (haemolytic anaemia, Crohn's disease). TNF is a prototype proinflammatory Th1 cytokine and yet under some circumstances TNF can cause immunosuppression. Anti-TNF therapy has been associated with dramatic improvements in many rheumatoid arthritis and Crohn's disease patients. The same treatment however has been associated with worsening multiple sclerosis with enhanced demyelination in some human patients. Under some circumstances and in some animal models, TNF treatment can reduce the severity of some prototypic Th1 type diseases, including diabetes, experimental autoimmune encephalomyelitis and arthritis. Typically TNF enhances antigen presentation by APC's. It has however been shown that in some circumstances TNF can inhibit the function of mature DC's and might induce their apoptosis and inhibit antigen presentation. It is hypothesized that the timing and duration of TNF production are important in deciding the protective versus harmful effects of TNF.
Both type I and type II interferons are used to treat infectious disease but in up to 19% of patients treated with these cytokines, autoimmune conditions develop (typical Th1 type diseases). Paradoxically type I interferons are also used to treat autoimmune diseases like multiple sclerosis (where it is the corner stone of therapy in some countries). These are cytokines that induce APC maturation and enhance antigen presentation. The mechanisms of interferon induced immune down-regulation are not completely clear but type I interferons are known to be able to inhibit IL-12 production and enhance IL-10 production. Both IFNα and IL-10 can promote the differentiation of CD25+CD4+ regulatory T cells. IFNγ is regarded as the canonical type I cytokine and consistent with this it activates APC and promotes Th1 differentiation. Despite large amounts of evidence incriminating it as a promoter of disease, there is also evidence for its protective roles in autoimmunities. The immunosuppressive action of this and other cytokines relates to their ability to induce members of the suppressor of cytokine signaling (SOCS) family of intracellular messengers that serve as classic feedback inhibitors of cytokine signal transduction. Socs1 gene knock out mice show extensive IFNγ-dependent pathology and show excessive IFNγ responses. It thus seems possible that low-level exposure to IFNγ might attenuate subsequent responsiveness to the harmful effects of these cytokines, the net effect thus being inhibitory.
References
1. Allen, J. E., and R. M. Maizels. 1997. Th1-Th2: reliable paradigm or dangerous dogma? Immunol Today 18:387-92.
2. Jankovic, D., A. Sher, and G. Yap. 2001. Th1/Th2 effector choice in parasitic infection: decision making by committee. Curr Opin Immunol 13:403-9.
3. Mosmann, T. R., H. Cherwinski, M. W. Bond, M. A. Giedlin, and R. L. Coffman. 1986. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 136:2348-57.
4. Mosmann, T. R., and S. Sad. 1996. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today 17:138-46.