Our studies demonstrate that IL-4 has impressively different inflammatory, alveolar remodeling, and fibrotic effects in lungs from Balb/c and C57BL/6 mice. They also demonstrate that IL-4 differentially regulates and/or activates TIMP-2, -3, and -4, α1-AT, and TGF-β1 and has impressively different effects on adenosine accumulation, ADA activity, and adenosine receptor expression in these animals. Last, they demonstrate that ADA therapy diminishes the differences between the phenotypes induced by IL-4 in Balb/c and C57BL/6 animals. When viewed in combination, these findings demonstrate that genetic factors have a profound influence on the tissue effects of IL-4 and that differences in the regulation of antiproteases and TGF-β1 may contribute to these different responses. They also highlight impressive differences in adenosine metabolism in C57BL/6 and Balb/c mice and demonstrate that these differences contribute to the pathogenesis of the different responses that IL-4 induces in C57BL/6 and Balb/c animals. These observations provide insights into factors that can determine whether IL-4 plays a significant or insignificant role in the generation, severity, and/or persistence of the inflammatory and remodeling responses that occur at sites of Th2-induced pathology. In so doing, they provide at least a partial explanation for the impressive patient-to-patient and animal-to-animal variability that is seen in Th2 responses in humans and animal models of human disease.
The differences in the ability of inbred murine strains to mount Th1 and/or Th2 inflammatory responses is well described in the literature, with Balb/c mice being described as Th2-prone while C57BL/6 mice are described as Th1-prone animals (4
). To date, these differences have been attributed to the differential ability of T cells from these mice to produce Th2 and/or Th1 cytokines (4
) with increased numbers of T cells from Balb/c mice committing to Th2 cytokine elaboration (4
). To determine if there are additional layers of control of these responses, studies were undertaken to determine if inbred mice respond differently to comparable amounts of IL-4. In these experiments, care was taken to use Tg animals that produced levels of IL-4 that are in accord with the levels seen during the course of aeroallergen-induced Th2 pulmonary inflammation (36
). These studies demonstrated that Balb/c and C57BL/6 mice differ in their ability to respond to equal amounts of IL-4 and that these differences are mediated by postreceptor target cell differences between these inbred animals that do not involve STAT6 signaling. Interestingly, IL-4–induced inflammation was more pronounced in C57BL/6 than in Balb/c animals. On superficial analysis, this would appear to be contradictory to the described Th2 versus Th1 propensity of these animals. In accord with our findings, however, the eosinophilic inflammation in C57BL/6 mice is more pronounced than in Balb/c mice after antigen sensitization and challenge in some Th2 modeling systems (14
). These studies suggest that IL-4, in addition to its prominent role in the initiation of Th2 cell development, can induce greater or lesser tissue effector responses depending on the genetic context.
Although cigarette smoke is the most important cause of pulmonary emphysema in the western world, only 20% of cigarette smokers experience significant chronic obstructive pulmonary disease (COPD) (the disease spectrum that includes emphysema and chronic bronchitis). Similarly, inbred mice vary significantly in their susceptibility to cigarette smoke–induced emphysema, with AKR/J and C57BL/6 mice showing various degrees of susceptibility while NZW and Balb/c mice are resistant to this toxic insult (8
). In accord with these findings, our studies add to our understanding of the processes that regulate alveolar destruction by demonstrating that IL-4 is a powerful stimulator of alveolar remodeling and emphysematous pulmonary destruction in C57BL/6 but not in Balb/c animals. These findings provide insight into the circumstances in which IL-4 could contribute to the pathogenesis of human emphysema. This may be a very important issue because IL-4 overexpression has been documented in emphysematous human lung tissues (40
) and cigarette smoke–exposed murine lungs (42
), and increased levels of IL-4 that correlate inversely with pulmonary function and directly with clinical manifestations have been noted in the plasma of patients with COPD (43
). These observations also have implications for extrapulmonary disorders such as abdominal aortic aneurysms, where IL-4–induced Th2 responses are believed to play an essential role in disease pathogenesis (24
). In both cases, disease onset, severity, and/or progression may be regulated by genetic factors that control IL-4 effector responses.
Since the mid 1960s, the protease-antiprotease hypothesis has dominated thinking as regards COPD disease pathogenesis. According to this theory, there is a balance between proteases and antiproteases in the normal lung, and an increase in proteases or a decrease in antiproteases can lead to alveolar destruction and emphysema (44
). To gain insight into the mechanisms that might be responsible for the enhanced emphysema in C57BL/6 versus Balb/c Tg mice, we characterized the expression of COPD-relevant proteases and antiproteases in these animals. Interestingly, IL-4 had comparable effects on a variety of MMPs and cathepsins. In contrast, impressively different effects on antiproteases were noted. Specifically, IL-4 inhibited α1-AT and TIMP-2 in C57BL/6 but not in Balb/c animals. In addition, IL-4 was a less potent stimulator of TIMP-3 and -4 in C57BL/6 versus Balb/c mice. These are interesting findings because pulmonary emphysema can be caused by a genetic deficiency of α1-AT, and the proteolytic effects of MMPs are inhibited by TIMPs (44
). When viewed in combination, these findings suggest that the differences in IL-4–induced alveolar remodeling that were noted are due, at least in part, to the different antiprotease responses that are induced by IL-4 in C57BL/6 and Balb/c animals.
Tissue fibrosis is a major cause of morbidity and mortality in pulmonary and extrapulmonary disorders. As a result of recent studies, the type II cytokine hypothesis has been formulated, which proposes that fibrosis occurs when cytokine balance shifts in a Th2 direction (26
). Although IL-4 is known to stimulate fibroblast proliferation, myofibroblast differentiation, and collagen and proteoglycan production in experimental systems (23
), and fibroblasts at sites of Th2 inflammation express IL-4R components (48
), IL-4 is not thought to be a particularly powerful fibrogenic mediator when compared with other Th2 cytokines, such as IL-13 (18
). Our studies shed light on the process that may be responsible for this by demonstrating that the fibrogenic effects of IL-4 are impressively dependent on genetic modifiers. Specifically, they demonstrate that IL-4 is a powerful stimulator of pulmonary fibrosis in C57BL/6 mice, but not in Balb/c animals. These findings are in accord with the fibrogenic capacity of other stimuli, such as bleomycin and radiation in these inbred murine strains (3
). Our studies also demonstrate that IL-4 is a more powerful stimulator and activator of the powerful fibrogenic mediator TGF-β1 in C57BL/6 than in Balb/c animals. This suggests that these different effects of IL-4 are due in part to the differential ability of IL-4 to stimulate and activate TGF-β1 in these inbred animals. It is important to point out, however, that the differences in collagen accumulation that were noted exceeded the differences in TGF-β1 production and activation that were seen. This raises the possibility that other cellular and molecular events may also contribute to the differential fibrotic effects of IL-4 in these different genetic settings.
Adenosine is a nucleoside that is generated by ATP catabolism at sites of tissue stress and injury that signals via G protein–coupled receptors that regulate a wide array of physiologic and immune responses (50
). Elevated levels of adenosine are found in BAL fluids from patients with asthma (57
), and adenosine regulates the functions of a number of cell types central to Th2 responses, including eosinophils, mast cells, and smooth muscle cells (51
). Aerosol adenosine exposure also induces bronchospasm in patients with asthma and COPD but not in normal controls (60
). The present studies demonstrate that IL-4 stimulates tissue adenosine accumulation, inhibits ADA activity, and enhances the expression of the A1
, and A3
adenosine receptors in C57BL/6 but not in Balb/c animals. These studies also demonstrate that these alterations play a key role in the pathogenesis of the differential effector functions of IL-4 in these inbred murine stains because ADA therapy decreased the levels of IL-4–induced inflammation, alveolar remodeling, and tissue fibrosis in C57BL/6 animals to levels that approximated those in Balb/c animals. This is, to our knowledge, the first demonstration that adenosine plays an important role in the pathogenesis of these key IL-4 effector activities and the first demonstration that genetic factors regulate tissue adenosine metabolism. We previously demonstrated that interventions that increased the levels of tissue adenosine generated a phenotype that is similar in many ways to the responses in asthma and COPD, including eosinophil- and macrophage-rich inflammation, alveolar enlargement, airway remodeling, mucus metaplasia, airways hyperresponsiveness, and tissue fibrosis (62
). We also demonstrated that IL-13 is a powerful stimulator of tissue adenosine accumulation and that this increase in adenosine is an essential contributor to the pathogenesis of IL-13–induced tissue alterations (51
). The present studies demonstrate that IL-4 has similar effects on adenosine metabolism and induces similar tissue responses in C57BL/6 but not in Balb/c mice. This raises the interesting possibility that, in appropriate genetic circumstances, IL-4–induced alterations in adenosine metabolism may be an essential part of Th2 inflammatory and remodeling responses.
In summary, these studies demonstrate that genetic factors can modify IL-4 effector responses, resulting in qualitatively and quantitatively different inflammatory, destructive, and fibrotic tissue responses. They also highlight the importance of differences in antiprotease induction and adenosine metabolism in the pathogenesis of these different tissue responses. Since IL-4, antiprotease, and adenosine dysregulation are well documented in COPD and asthma and other Th2 disorders, it is tempting to speculate that similar genetic modifiers are responsible for the variable presentations, natural histories, and responses to therapy that clinicians see in caring for patients with these disorders. Additional investigations of the genes that mediate and the mechanisms that are responsible for the different responses induced by IL-4 in inbred mice and their relevance in humans are warranted.