Initiation and Maintenance of the Granulomatous Response

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Initiation and Maintenance of the Granulomatous Response* Steven L. Kunkel, Ph.D .; &bert M. Strieter; M.D., F.C.C .P.; Niclwlas Lukac&, Ph.D .; and Stephen W Chenwe, M.D., Ph.D.

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he initiation and maintenance of a chronic granulomatous response is due to a dynamic interaction between the inciting agent, inflammatory mediators, leukocytes, and resident structural cells of a given tissue. Clinically, granulomatous diseases are difficult to treat and often require therapies which can immunocompromise the host. The limited therapeutic modalities which are available for the patient with granulomatous inflammation reflects the level of understanding of these diseases. Treatment of infectious granulomatous diseases has targeted the known agent, while treatment of idiopathic granulomatous diseases has usually targeted the inflammatory/immune cells. Independent of the etiology, granulomatous inflammation usually possesses a characteristic pathologic response. The histologic presentation of a granuloma at the time of clinical diagnosis is a mixed cellular response, typified by the presence of mononuclear cells, including both monocyte/macrophages and lymphocytes. 1 However, fibroblasts, epitheloid cells, giant cells, and other leukocyte populations are also likely to be found in the granulomatous lesion.' While various granulomatous responses can be characterized by a common "histologic picture;' these responses can vary dramatically with regard to the intensity of the infiltrating and proliferating cells. The intensity of the infiltrates and reactivity of these participating cells have aided in defining granulomas as either a foreign body-type or a hypersensitivity/immune-type response . The definition of either a foreign body or hypersensitivity granuloma is based on the degree of antigen-specific lymphocyte sensitization associated with the evolving lesion.' The true immune or hypersensitivity granuloma is a delayed-type, antigen-derived reaction with an active recruitment response, leading to a high turnover of the cellular components of the granuloma. On the contrary, a foreign body granulomatous response lacks antigen-specific sensitization and does not have an aggressive recruitment/proliferative response, and the participating cells possess a relatively low turnover rate . Although granuloma lesions have been well characterized at the histologic level, most of the soluble mediators and cellular mechanisms which lead to the initiation, maintenance, and resolution of the response remain an enigma. A number of key, basic questions regarding pulmonary granulomatous inflammation have not been fully addressed. For example, what is the etiology of certain granulomatous diseases such as sarcoidosis and Wegener's granulomatosis? What is the combination of adherence polypeptides and chemotactic factors which first initiates leukocyte infiltration ·From ~~ Department of Pathology and Division of Pulmonary and Cntical Care, Department of Internal Medicine The University of Michigan Medical School, Ann Arbor. ' This study was supported in part by NIH grants IP50HlA6487 HL02402, HL31693, HL35276, DK38149, and the Council fo; TobaccoResearch. Dr. Strieter is a RJR Nabisco Research Scholar. Reprint requesta: Dr. Kunkel, Department of Internal Medicine ' University of Michigan Medical School, Ann Arbor 48109

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and then maintains leukocyte infiltration? What are the multifunctional roles of these mediators during lymphocyte sensitization and proliferation? Why does the leukocyte recruitment process last for months or often years? What is the role of nonimmune, resident cells in the maintenance of the granulomatous response? What are the clonal alterations which occur in these cells, leading to an "inflammatory phenotype?" What are the critical mediatorslcytokines which lead to the maintenance of the granulomatous response? Why are physiologic alterations so intimately associated with active granulomatous disease? Why do many granulomas resolve while others progress to debilitating end-stage disease? The above questions reflect lack of the knowledge which is necessary to both clearly understand the mechanisms of granuloma development, and more importantly, to strategically design more efficacious therapies. In the following sections, we will attempt to address some of the fundamental mechanism(s) that are needed for the evolving granulomatous response. ADHERENCE PROTEINS AND THE GRANULOMATOUS RESPONSE

The initiation of an inflammatory event which results in a chronic response possesses many similarities of an acute response in that specific leukocyte populations must recognize and actively interact with "inflamed" or activated endothelial cells. However, the specific proteins which are involved in mediating either an acute or chronic inflammatory response are quite different. The COII/COI8 family (beta-2 integrins) and L-selectin are adherence molecules found on the surface of polymorphonuclear cells, which enable this cell to actively interact with the endothelium," The beta-2 integrins expressed on the surface of the neutrophil interact with their counter-receptor lCAM-l and/or ICAM-2 on the endothelium. Although ICAM-l is constitutively present on the surface of the endothelium, early response cytokines, such as interleukin-l (ILl) and tumor necrosis factor (TNF), are known to be important signals for the quantitative induction of endothelial cell-derived ICAM1. The counter-receptor for L-selectin on the endothelial cells is still not clearly defined. L-selectin is known to mediate the phenomenon known as "neutrophil rolling" on the endothelium. This initial L-selectin-dependent interaction between the neutrophil and the endothelium is likely important for the subsequent COII/COI8 complex to function in vivo, since the CO II/CO18 adherence molecules do not operate well in a high f10wlhigh shear vascular system. Thus, L-selectin aids in promoting the initial neutrophil-endothelial cell interactions, while the CDII/CDI8 complex tenaciously binds the neutrophil to the endothelium . While active adherence of neutrophils to endothelium is present only during inflammation, the lymphocyte-endothelial cell adherence system is interactive both under normal homeostasis and inflammatory conditions. This difference allows the lymphocyte to play an important role in immune surveillance by constantly trafficking through normal tissue and lymph nodes, as well as in response to an inflammatory signal leading to its participation in an immune process. CHEST I 103 I 3 I FEBRUARY, 1993 I Supplement

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Interestingly, certain adherence molecules appear to be important in both neutrophil- and lymphocyte-mediated migration, such as L-selectin on the lymphocyte and neutrophil and ICAM-l and ICAM-2 on the endothelial cell. However, the lymphocyte also can express VLA-4 and CD44, binding to endothelial cell VCAM-l and glycoaminoglycanlhyaluronic acid , respectively. These adherence systems are likely to playa role in adherence under both normal and inOammatory conditions.' During the evolution of a hypersensitivity granulomatous response, lymphocyte adherence appears to follow a number of principals which are important in regulating their migration pattern. For example, note the following: (a) naive lymphocytes usually migrate via lymph nodes, while memory lymphocytes migrate via nonlymphoid tissue; (b) memory lymphocytes can migrate to tissues where they were previously sensitized; and (c) inOamedlactivated endothelium increases the migration of lymphocytes to the site of inOammation preferentially over normal homeostasis trafficking patterns. These general aspects of lymphocyte recruitment are important to the success of a granulomatous reaction, since nonsensitized 'virgin' lymphocytes are needed to interact with antigen-presenting cells in the context of antigen in lymph nodes which drain an inOammed area. This process insures that antigen presentation, lymphocyte sensitization, and clonal expansion of lymphocyte pools will occur in an orderly fashion and is an important step in the development of a hypersensitivity granuloma. While naive peripheral blood lymphocytes can be rapidly elicited to regional lymph nodes, previously sensitized lymphocytes are needed as sentinel cells to migrate in tissue and respond to a potential challenge. This surveillance mechanism of sensitized lymphocytes is an important feature of an accelerated immune response and is likely a key mechanism in the clinical phenomenon of the 'flare' response associated with a number of chronic inOammatory diseases. Interestingly, once an area of inOammation has been established, the activated endothelium now takes precedence for migration of lymphocytes and targets these cells to a site of inOammation. Both naive and sensitized lymphocytes which would either be involved with physiologic trafficking or tissue-specific movement are susceptible to interacting with activated endothelial cells which are associated with an area of in8ammation. The peripheral blood monocyte is an additional mononuclear cell which is crucial to the success of an evolving granuloma. The binding and recruitment of blood monocytes share characteristics that are common to both neutrophils and lymphocytes. Like the neutrophil, the monocyte can utilize the CDll1CD18 family of adhesion polypeptides. However, both the lymphocyte and the monocyte utilize VLA-4 during the evolution of a migratory response. Interestingly, monocytes appear to greatly rely on this shared adherence system (VLA-4which binds to its ligand, VCAM1, on the endothelial cells). In addition, both mononuclear cell types may normally traffic via tissue during noninOammatory conditions. Since the initiation and maintenance of the immune granuloma is highly dependent upon lymphocyte-monocyte/macrophage interactions, the ability of both mononuclear cell types to utilize similar adherence systems insures the participation of both cells at the site of inOam-

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mation. The ability of lymphocytes and monocytes to use common adherence mechanism(s), which are different from the molecules used by neutrophils, may explain the characteristic pattern of lymphocyte/monocyte recruitment, independent of neutrophil recruitment associated with different disease states. CHEMOTACTIC CYI'OKINES AND TIlE GRANULOMATOUS RESPONSE

Once the appropriate peripheral blood leukocytes respond to adherence signals on activated endothelial cells and become targeted to a developing granuloma, they next engage in a process which will commit them to active participants in the inOammatory response." The blood born cells migrate between the endothelial cells, pass through the basement membrane matrix, and are lured to exact sites of in8ammation by following chemical signals. Historically, research into the formation of chemotactic gradients had centered around factors which demonstrated little specificity for moving individual leukocyte populations. These chemotactic signals included cSa, fMLp, and LTB. Recently, a collection of small polypeptides «10,000 daltons) has been identified which displays chemotactic specificity for individual populations of leukocytes. These chemotactic cytokines belong to a supergene family of related in8ammatory proteins and are likely to be the major mechanism responsible for the elicitation of particular leukocyte population(s) to a site of chronic inOammation.7-10 There is little doubt that the successful arrival of leukocytes is dictated by a dynamic sequence of events which is initiated at the surface of the endothelium and progresses to the expression of a specific chemotactic cytokine. These coordinated events form the foundation for the in8ux of leukocytes which is needed to initiate and maintain the granulomatous lesion. While it may be easy to envision the mechanism(s) responsible for the early recruitment of the necessary leukocytes to the developing granulomatous lesion, it becomes more difficult to understand the mechanism(s) responsible for the continued, chronic elicitation of the appropriate leukocyte populations. In certain chronic granulomatous diseases, it is not uncommon to 6nd monocytes and lymphocytes being continually summoned to an area of inOammation for months or years. Thus, a fundamental change has occurred which will allow peripheral blood leukocytes to chronically be susceptible to endothelial cellderived adherence molecules and chemotactic cytokines which will ensure their constant participation in the granulomatous response. However, the continued recruitment of leukocytes is ultimately associated with long-term tissue injury. This injury is often nonspecific and leukocytedependent, via the release of proteases and arachidonic acid and reactive oxygen metabolites. II In addition, the injury may be fibroblast-dependent due to the deposition of collagen and the formation of end-stage 6brosis. In either case, the normal sequel of events is often loss of normal organ function. During the maintenance phase of chronic granulomatous inOammation, which is likely to be the clinically presented phase, the disease is usually difficult to therapeutically treat. The options for any successful treatment are often limited to immunocompromising agents. This clinical phenomenon suggests that fundamental changes 35lh Amual Aspen Lung Conference

have occurred at the site of granulomatous inflammation which have likely rendered cells in the tissue phenotypically altered. NONINFLAMMATORY CELLS AND GRANULOMATOUS RESPONSE

One of the interesting aspects with regard to the elicitation of leukocytes to an area of granulomatous inflammation is the participation of nonimmune structural cells and endothelial cells...... Recent evidence now supports the idea that cells which traditionally were thought to be either targets or bystanders to active inflammation, such as endothelial, epithelial, and stromal cells, now must be considered as active effector cells to the evolving inflammation. For example, the fibroblast is an essential cell in end-stage granulomatous disease but is also an important cell during the early inflammatory response as a source of inflammatory cytokines. The role that stromal cells play during chronic inflammation is likely to be significant given the large number of cytokines which these cells can synthesize . An important regulatory aspect with regard to the generation of stromal cell-derived cytokines is that these cells depend upon an initial host immune response prior to their involvement in the inflammatory response. Thus, most stromal cells can respond to early response cytokines, interleukin-l (ILl) or TNF, but do not respond to infectious agents themselves or chemicals released by infectious agents, such as lipopolysaccharide. However, once proximal cytokines are generated, these cells can be directed to generate many important inflammatory mediators, including defined chemotactic cytokines. In addition, there is a high likelihood that the normal stromal cells may become chronically altered, perhaps by the constant exposure to an inflammatory environment, resulting in the constitutive synthesis of products which perpetuate the chronic lesion. Phenotypic changes in stromal or epithelial cells which surround a chronic inflammatory region could lead to the constant recruitment of monocytes and lymphocytes needed ' to maintain the ongoing lesion. This phenotypic alteration in cellular function could subsequently result in resistance to therapeutic agents often observed in chronic granulomatous diseases. CONCLUSIONS

Granulomatous inflammation possesses a common set of pathologic events which appear to be due, at least in part, to the continued recruitment and activation of a variety of leukocyte populations. Although the initiating insult may vary, as in the etiology of tuberculosis, sarcoidosis, or berylliosis, the subsequent immune reaction, characterized by infiltrating leukocytes, is often the same. While the exact mechanism(s) by which leukocytes are elicited to the granuloma is not entirely understood, it is likely that the local production of specific chemotactic agents are paramount to the success of the developing lesion. The recent identification of a novel supergene family of chemotactic cytokines,

which possess activity for eliciting specific leukocyte populations, has provided a potential mechanism for the constant leukocyte recruitment response associated with chronic inflammation. In addition, the expression of these chemotactic polypeptides has been identified in a number of cellular sources, including mononuclear leukocytes, fibroblasts, epithelial cells, smooth muscle cells, and endothelial cells. Studies directed at understanding the ongoing recruitment process which leads to the continued cellularity of a granulomatous lesion are likely to provide needed insight to develop more efficacious therapies to treat these chronic inflammatory diseases. REFERENCES

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