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Chemokines in the Pathogenesis of Asthma
Hypothesis: The pathophysiologic pulmonary responses of asthma are due to excessive local production and release of chemokines
Specific aim I: To determine if repeated intratracheal injections of chemokines into mice will induce asthma-like chronic pulmonary inflammation.
Specific aim II: To determine if immunization with household dust containing cockroach allergens followed by repeated aerosol exposure to the dust will result in asthma-like chronic pulmonary inflammation.
Specific aim III: To determine if reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI) are responsible for enhanced chemokine gene expression following exposure to cockroach allergens
Specific aim IV: To determine if specific inhibition of chemokine biological activity will prevent asthma-like chronic pulmonary inflammation following exposure to allergens
Asthma represents an important health problem in the United States since it affects nearly 15 million Americans. It is among the most common respiratory complaints seen by physicians in the outpatient setting. Despite the magnitude of the problem, understanding of the pathogenesis of asthma at this time is limited. This lack of knowledge has contributed to the morbidity and mortality of asthma. The health care costs associated with asthma are on the increase and were estimated to be $6.2 billion in the United States in 1990. Allergen exposure is becoming increasingly recognized as an important component of asthma. Asthma most likely occurs in patients who are exposed to the allergen. Upon re-exposure to these same allergens present in aerosol, an asthmatic attack is initiated. A recent publication has indicated that exposure to cockroach antigens in inner cities can result in significant exacerbations of asthma in children (1). While these general concepts of the immunology of asthma are accepted, the precise signals within the lung which dictate the progression and development of the pathophysiologic consequences are still being determined. Understanding the local mediators which dictate the progression of disease will help to define which targets should be blocked. This represents an attractive therapeutic strategy since only those mediators felt to be important will be decreased, rather than the brute force immunosuppression which is so frequently used to treat severe asthma. Cytokines are emerging as potent mediators of multiple aspects of inflammation. They may be considered to be the hormones of the immune system, where cytokines are made by one cell, move to another cell where binding to a specific receptor induces a response. The cytokines are proteins whose gene regulation has been extensively studied at both the cellular and molecular level. Numerous studies have demonstrated the importance of cytokines in the inflammatory response. These studies include experiments where exogenous cytokines are given to animals or patients with the result of inducing acute immunologic and physiologic alterations. In patients with diseases, or in experimental animal models of disease, blockade of the cytokines results in amelioration of disease. Thus the cytokines represent excellent targets for modulation since reagents are available to specifically inhibit the biological activity each of the cytokines. In experimental animal models of allergic pulmonary inflammation it has been shown that inhibition of either interleukin 1 (IL-1) or TNF will decrease the pulmonary inflammation (2,3). The immunology of cytokines often reveals a cascade of events, where initiation of the early "alarm" cytokines such as TNF and IL-1 induces subsequent cytokines (4,5). A group of these cytokines, the chemokines, have assumed increasing importance and are represented by IL-8, or its murine homologs KC or MIP-2α (6), discussed in more detail below. These chemokines have been found at sites of inflammation (7), including finding elevated levels in the lavage fluid from patients with asthma (8). A role for the chemokines in the causing organ injury has begun to be established with the development of new reagents. Lupine IL-8 has been described, and in rabbit models of acute inflammation blockade of IL-8 has been shown to reduce injury (9). Additionally, anti-IL-8 will prevent pulmonary ischemia/reperfusion injury (10), and anti-IL-8 reduces proteinuria observed during experimental glomerulonephritis (11). Thus there is ample evidence that the chemokines may be causative agents in the pathogenesis of inflammatory diseases. The chemokines are divided into C-C and C-X-C families (12). Members of C-X-C chemokines have been cloned from rodent cDNA or genomic libraries. They included the KC gene product(6) which shows approximately 90% sequence homology with rat cytokine-induced neutrophil chemoattractant (CINC) (13,14). Additionally, the nucleic acid sequence for mouse (15) and rat (16,17) MIP-2α has been published. Both CINC and KC are approximately 70% homologous to human GROα. Similarly, rat and mouse MIP-2α are approximately 70% homologous to human GROβ. All 4 rodent chemokines are approximately 40-45% homologous to human IL-8 (18,19). Despite extensive efforts, no exact structural homolog to IL-8 has been published for rodents. The biological activity of rat CINC closely resembles that of human IL-8 and GROα. Rat CINC is a potent chemoattractant for neutrophils in vitro and induces a rapid but transient influx of neutrophils exclusively when injected intradermally into rats (18). Using a highly specific ELISA for rat CINC a rapid rise in local levels of CINC (maximal at 8 hours) has been reported with a corresponding influx of neutrophils into carraggeenin-injected subcutaneous air pouches in rats (20). Rat CINC messenger RNA (mRNA) is also rapidly up-regulated in vitro in alveolar macrophages and in vivo in total lung homogenates after LPS challenge (7). In vivo, neutrophil influx into the lung follows the increase in CINC mRNA, supporting the importance of CINC as a neutrophil chemotaxin in rats (7). Safirstein et al. (21) also reported a rapid increase in the expression of CINC mRNA in rat kidneys after an ischemic insult and reperfusion. Mouse KC resembles human IL-8 in that it is expressed in numerous different cell types including macrophages (22,23), fibroblasts (24,25), and endothelial cells (26) in response to inflammatory stimuli such as LPS and IL-1. KC is down-regulated by corticosteroids (27). Based on this evidence, we propose to test the hypothesis that KC and MIP-2α act as important regulators of inflammation and will serve as the target for these investigations. Our investigations will establish the precise role of the chemokines in the pathogenesis of chronic pulmonary inflammation. One of the significant alterations that occurs in asthma is abnormal innervation of the bronchus with resulting hypersensitivity of the airways. We hypothesize that the chemokines elaborated by injured bronchial epithelial cells, invading macrophages and activated interstitial cells will cause disassociation of the cholinergic motor end-plates in the bronchus. Following a brief period of deafferentation (28), re-establishment of the motor end-plate occurs with concomitant sprouting of the invading neuronal growth cone and, in the normal individual, transient hyperinnervation of the motor end-plate (29-31). It is hypothesized that continuous stimulation of the bronchial immune system by inhalation/instillation of allergens provides a milieu in which the motor end-plate is not permitted to re-establish the normal phenotype. The bronchial smooth muscle will, therefore, gain and retain hypersensitivity and responsiveness to further stimulation. It is anticipated that a consequence of altered cholinergic innervation of bronchial smooth muscle will be manifest in greater susceptibility to asthma. Morphological and histochemical confirmation of this principle is sought along with mechanistic investigation of the role of cytokines/chemokines and neural cell-adhesion molecules in the etiology and progression of asthma. Neural cell adhesion molecules (N-CAMs) are a group of 20 calcium-independent adhesion molecules broadly grouped into three molecular weight classifications (120, 140 and 180kD). Alterations in the expression of all three forms of N-CAM have been observed in a variety of experimental models of neurotoxicity. These include ethanol (32), lead (33), methylmercury and other toxic chemicals (34). Thus, altered expression of N-CAMs is a sensitive and consistent marker for a variety of neural changes incurred in chemically-mediated damage. Such damage could result from excessive local elaboration of the chemokines. The fidelity with which motor end-plates are re-established is coordinated by the recapitulation of anatomic and molecular cues reminiscent of embryonal developmental processes (35). Following either mechanical or toxicological insult, terminals of the motor neurons degenerate "Wallerian-type" for a period dependent on the degree of damage incurred. Commitment of the neuron to reinnervation of the motor end-plate requires alterations in the microenvironment of the motor end-plate, elaboration of regenerative factors from the target tissue and de novo expression of a variety of modified cell-adhesion molecules (36). Three major phases appear to predominate the injury and repair of neuromuscular junctions: 1) Retraction of the terminal nerve processes results in transient reduction in cholinesterase activity at synapses in the CNS and PNS (28), 2) Initial "Wallerian-type" degeneration of the proximal neuronal process with concomitant alteration in the expression of N-CAM to the polysialylated embryonal form. The latter isoform of N-CAM permits facile gliding of adjacent fasicles with greater efficiency of reinnervation. 3) Transient hyperinnervation of the newly-reformed neuromuscular junction followed by appropriate synaptic pruning and establishment of adult anatomical arrangements and expression of the poorly sialylated adult N-CAMs. These changes will be carefully followed in a model of asthma-like chronic pulmonary inflammation. Stimulation of cells with ROI or RNI will cause the up-regulation of chemokines (37-40). Similarly, if cells are stimulated in the presence of inhibitors/scavengers of ROI or RNI there is a selective decrease in the production of the chemokines (37-40). This raises the intriguing possibility that a link between the production of the chemokines and the development of an allergic response may mediated by the intracellular formation of ROI or RNI. It has been demonstrated that ozone-induced inflammatory responses are great in asthmatic subjects (41) and that diesel exhaust particles enhance local cytokine production in allergen challenged mice (42). This also provides an important link to the second project on asthma and exposure. Different animal models of asthma which have been used to investigate the pathogenesis of the disease are reviewed in (43,44). One of the more common models involves sensitizing animals to a specific allergen followed by aerosol challenge with the same allergen. In this setting the immunization results in a specific response to the allergen which includes acute inflammation of the airways and hypersensitivity of the airways to further challenges (45,46). Figure 1 is a schematic of the proposed steps involved in the development of the inflammation. Our work will focus on the chemokines as the critical mediators which drive the inflammation of the airways. A significant aspect of this portion of the grant is the development of a murine model of asthma based on the probable allergens found in the households of children who develop significant asthma, particularly cockroach allergens (1).
There are several important cytokines which have been investigated as potential mediators of the pathogenesis of asthma reviewed in (47). Included in this group are IL-5 (45,48), IL-12 (49), and gamma-interferon (50). While all of these may be involved we have selected the chemokines to focus our work. These are attractive mediators to investigate since they may represent the final common pathway of tissue injury. In contrast to other cytokines, the chemokines are extremely long-lived and may persist in local sites of inflammation for days or even weeks (51,52). Also, the chemokines have been demonstrated to recruit T cells and eosinophils to sites of inflammation (53,54). Another important component is the potential for the development of new therapeutic agents specifically directed against the chemokines. It has recently been identified that chemokine receptors are critical for the progression of HIV (55,56). This has raised the exciting possibility that blockade of chemokine receptors will halt the spread of HIV and has prompted a race to develop such reagents. As they become available they will be tested in our model. We intend to explore these potential relationships using a mouse model of asthma-like chronic pulmonary inflammation which are outlined in Figure 1. Mice will be immunized with house dust containing a high level of cockroach antigens. Repeated aerosol exposures to the same mixture will be performed, and the pulmonary response precisely detailed. We will then specifically block selected chemokines to prevent the local inflammation, airway damage and hyperinnervation. Successful completion of our specific aims will permit better delineation of the critical mediators of pulmonary inflammation due to repeated exposure to allergens.
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Objective:
We will determine if the mechanism of chronic pulmonary inflammation due
to repeated exposure to allergens is mediated by the excessive local
production of chemokines.
