Mast Cells and Innate Immunity

Matthew J Hamilton; Richard L Stevens

Mast Cells and Innate Immunity1

Matthew J Hamilton,

Richard L Stevens

INTRODUCTION

Mast cells (MCs) are immune cells that have prominent adverse roles in allergic inflammation and systemic anaphylaxis. Cross-linking of the high-affinity IgE receptors (Fc∊RI) on the MC's plasma membrane results in the rapid release of granule mediators (e.g., histamine and protease•proteoglycan complexes) and lipid mediators (e.g., leukotriene B₄, leukotriene C₄, prostaglandin D₂, platelet-activating factor, and thromboxanes). Starting 1–4 h later, Fc∊RI-activated MCs generate substantial amounts of a diverse array of cytokines and chemokines. The accumulated effects of these exocytosed mediators result in the classical acute- and late-phase reactions seen in allergic individuals. Because of their abundance in the eye, MCs play a significant role in IgE/allergen-dependent ocular inflammation.

The release of too many MC-derived mediators at an inappropriate time can result in life-threatening anaphylaxis. While this is a serious adverse consequence of MC activation, recent studies uncovered beneficial roles for MCs in innate immunity. In this regard, it was recently shown that the “test” cells in the Styela plicata¹ and Ciona intestinalis (Stevens, unpublished data) sea squirts store substantial amounts of histamine and protease•heparin proteoglycan complexes in their cytoplasmic granules analogous to mouse, rat, and human cutaneous MCs. These primitive MC-like cells in urochordates also generate prostaglandin D₂ when exposed ex vivo to the MC-activating factor compound 48/80. Thus, cutaneous-like MCs evolved more than 500 million years ago, predating the appearance of B cells, T cells, and the development of acquired immunity. Presumably, sea squirts use their MC-like test cells to help combat the infectious organisms encountered in their ocean environments.

In support of the perceived importance of MCs for our survival, no human has been found to lack MCs. The participation of MCs in innate immunity explains why these connective tissue-residing immune cells developed prior to adaptive immunity and why they have been conserved throughout evolution. MCs are strategically positioned to confront and combat potential environmental 2pathogens. MCs recognize bacteria and their products, and these immune cells are equipped with a complex armament to carry out their vital roles in immunity. MCs display numerous receptors on their surfaces that allow these cells to respond to pathogen-induced changes in their environments. Once activated, MCs can respond in a calculated manner using a variety of amines, proteases, eicosanoids, cytokines, and chemokines. MCs therefore possess the ability to combat bacteria and other infectious organisms in a direct manner or in an indirect manner by recruiting other cell types into the infected tissue. Here, we review data that document the roles of MCs in innate immunity, with particular emphasis on the participation of the cell's tryptases in bacterial infections.

MORPHOLOGY, HISTOCHEMISTRY, AND GRANULE PROTEASE•PROTEOGLYCAN COMPLEXES OF MOUSE AND HUMAN MCs

Mature mammalian MCs are unique granulocytes that reside in connective tissues in close proximity to nerves and blood vessels, in contrast to eosinophils, neutrophils, and basophils that are preferentially found in peripheral blood. Providing the initial clue as to their immunoprotective roles, MCs are most prevalent at sites of interface with the outside environment. The lung, skin, and gastrointestinal tract have 500–4,000, 7,000–12,000, and ∼20,000 MCs/mm³, respectively. In regard to the eye, MCs are present in the choroid (the vascular layer between the retina and the sclera), but they are most abundant in the conjunctiva and eyelid.

MCs can be distinguished from other cell types based on their morphology^2–6 and histochemistry when stained with cationic dyes.^7,8 Mature MCs generally possess a monolobed nucleus and a surface architecture composed of prominent ridge-like folds of plasmalemma known as microplicae. MCs also can be distinguished from other cell types due to their numerous electron-dense, cytoplasmic secretory granules. These granules in human MCs usually contain crystalline structures that exhibit novel scrolls, gratings, and lattice patterns.⁶ Using different histochemistry approaches, Enerbäck first demonstrated the heterogeneous nature of MCs.⁸ It is now known that the histochemical differences seen in different populations of rodent MCs primarily are a consequence of serglycin proteoglycans⁹ that possess different types of glycosaminoglycan side chains [e.g., heparin¹⁰, chondroitin sulfate diB¹¹, and chondroitin sulfate E¹²].

Mature mouse and human MCs have distinct phenotypes based on the panel of proteases they store in their secretory granules bound to serglycin proteoglycans. For example, it is now known that mouse MCs store varied combinations of at least sixteen proteases in their secretory granules [designated as mouse MC protease (mMCP) 1-10, mMCP-11/Prss34, transmembrane tryptase/tryptase γ/Prss31, carboxypeptidase A3 (CPA3), cathepsin G, 3granzyme B, and neuropsin/Prss19]. The genes that encode cathepsin G, granzyme B, and mMCPs 1-5 and mMCPs 8-10 reside on chromosome 14C3. This is the second largest group of protease genes in the mouse's genome. Because these serine proteases are more similar to chymotrypsin than trypsin, the chromosome 14C3 locus historically has been named the “chymase” locus. However, it must be pointed out that some chromosome 14C3 family members (e.g., granzyme B) lack chymotryptic activity. mMCP-6,^13,14 mMCP-7,¹⁵ Prss31,¹⁶ and Prss34¹⁷ are MC-restricted tryptases, and their genes reside on chromosome 17A3.3, which contains the third largest cluster of protease genes in the mouse's genome. Fourteen genes are present on chromosome 17A3.3 that encode thirteen functional tryptic-like serine proteases, each of which exhibit distinct tissue and cell expression patterns.¹⁷

Human MCs also store numerous proteases in their secretory granules. These include cathepsin G, granzyme B, CPA3, chymase-1, Prss31, and multiple tetramer-forming tryptases. In regard to the latter family of serine proteases, five cDNAs have been isolated that encode the homologous human tryptases known as α, β1, β2, β3, and δ.^18–21 It tentatively has been concluded that these cDNAs originate from three genes on chromosome 16p13.3.²² The uncertainty is due to the fact that this region of the human genome is undergoing a high rate of mutation and recombination.²³ While it is likely that there is evolutionary pressure to expand the number of beneficial tryptases and to eliminate potentially harmful ones, the high rate of recombination at chromosome 16p13.3 probably is due to a chromosomal break and inversion event that took place at the locus after mice and humans separated in evolution.¹⁷

hTryptase-β1^20,24,25 is the functional ortholog of mMCP-6.^13,14,26 Both MC-restricted proteases are initially expressed as glycoprotein zymogens that in each instance have a signal peptide that is removed in the endoplasmic reticulum and a 10-mer propeptide that is removed in the secretory granule. The resulting mature domain of each tryptase consists of 245 amino acids. When activated, mMCP-6 and hTryptase-β1 spontaneously form tetramers with the active site of each monomer facing inside the doughnut-shaped tetramer unit. Formation of the novel tetramer unit is dependent on conserved Tyr-, Pro-, and Trp-rich domains within each monomer,^27–29 whereas interaction of the tetramer unit with the glycosaminoglycan side chain of a serglycin proteoglycan is dependent on spatially conserved His, Arg, and Lys residues.^27,28,30 The functional significance of the binding of the MC's granule proteases to serglycin proteoglycans was conclusively demonstrated when it was discovered that the MCs in the skin and peritoneal cavity of serglycin-null³¹ and heparin-null^32,33 mice are protease deficient. Because their proteoglycan-binding domains are Lys/Arg-rich rather than His-rich as in mMCP-7, exocytosed mMCP-6 and hTryptase-β1 remain tightly bound to their serglycin proteoglycans.³⁴ This feature prevents diffusion of exocytosed mMCP-46/hTryptase-β1•proteoglycan macromolecular complexes, thereby allowing these tryptases to carry out their functions locally for hours. Serglycin proteoglycans also play significant roles in regulating the substrate specificity of their bound proteases.^26,35

mMCP-6 and mMCP-7 are packaged in MCs as homotypic and heterotypic tetramers ionically bound to serglycin proteoglycans.²⁹ The fact that these tryptases can be released as heterotypic complexes suggests they have complementary activity in vivo. The role of mMCP-7 is explained by its potent anticoagulant activity. In this regard, the α chain of fibrinogen is a physiologic substrate of mMCP-7, and no protease inhibitor in blood exists to rapidly inactivate this tryptase.³⁶ mMCP-7 therefore works in concert with mMCP-6 to assist in the recruitment of other immune cells into the inflammatory area by preventing the deposition of fibrin-platelet clots, which have the ability to block cellular extravasation physically.

MC DEVELOPMENT

All mouse MCs originate from hematopoietic progenitors that reside in the bone marrow and fetal liver.^37,38 The systemic and local factors that regulate the tissue recruitment of these progenitors, and subsequently their proliferation and development into mature MCs, are major areas of investigation. Much of this work has been carried out on cultured cells, and many of the factors that regulate the development of mouse and human MCs have been identified. In the mouse, interleukin (IL) 3^39,40 and Kit ligand (KitL)/stem cell factor/steel factor^41–43 are the two most important cytokines that regulate the retention of MC-committed progenitors in tissues and/or their proliferation, differentiation, and granule maturation. However, accessory cytokines such as IL-4,^44–46 IL-6,⁴⁷ IL-9,^48,49 IL-10,^50,51 IL-16,⁵² and transforming growth factor-β1,^53,54 also play significant roles in the final stages of development of human and mouse MCs. Although human MCs and their progenitors are less responsive to IL-3 than mouse MCs, IL-3 is routinely used to generate mouse MCs by culturing their unfractionated bone marrow cells in the presence of the cytokine for 3-6 weeks.^39,40,55,56 Arguably, the most important discovery in the MC field in the last 30 years was the identification of IL-3 and the use of this T cell-derived cytokine to generate large numbers of mouse bone marrow-derived MCs (mBMMCs) for varied in vitro and in vivo studies. Although IL-3 is not needed for the development of constitutive heparin⁺ MCs, this cytokine is required for the rapid expansion of chondroitin sulfate⁺ mouse MCs in the small intestine during a helminth infection.⁵⁷

KitL^41–43 binds to the tyrosine kinase-type surface receptor CD117/Kit⁵⁸, and KitL/Kit signaling is essential for the development of the heparin⁺ population of MCs in mice and humans.⁵⁹ In this regard, WBB6F₁Kit^W/Kit^W-v (W/W^v) mice are MC-deficient due to an inactivating point mutation in the intracellular domain of 5Kit.⁶⁰ WCB6F₁-Kitl^Sl/Kitl^Sl-d mice are MC-deficient because their mesenchymal cells can not produce the membrane isoform of the KitL, which is needed to retain MC-committed progenitors in tissues.^61,62 On the other hand, tg/tg mice are MC-deficient due to a defect in expression of microphthalmia-associated transcription factor (MITF)^63,64, which acts downstream of Kit. MITF is essential for transcription of the mMCP-6 gene⁶⁵ and numerous other MC-restricted genes. The discovery of an activating mutation at residue 816 in Kit that leads to systemic mastocytosis in humans^66,67 supports the mouse data, which led to the conclusion that KitL/Kit/MITF-signaling pathways control the development of the constitutive MCs found in most tissues.

MCs exhibit substantial plasticity in their development, and it is now known that the phenotype of a MC at any stage of its life-cycle is highly dependent on the combination of factors the mature cell and its progenitor encounter in different tissue microenvironments. Mature MCs differ from other immune cells in that they can reversibly alter what mediators they express. This property was elegantly shown in the small intestine of Trichinella spiralis-infected mice.^5,68,69 A transient mastocytosis occurs in the jejunum of helminth-infected mice^5,70 and rats.^71,72 The expanded MCs display specific combinations of proteases depending on their location within the jejunum and the time course of the infection. Most mature MCs in the jejunum reside in the muscle and submucosa at baseline, and these cells express mMCP-5, mMCP-6, and mMCP-7, but not mMCP-1 or mMCP-2. During the inductive phase of the infection, jejunal MCs cease expressing mMCP-5, mMCP-6, and mMCP-7, and begin expressing mMCP-1 and mMCP-2 as they transverse from the submucosa to the tips of the villus. This differentiation process is reversed during the expulsion phase of the infection as the senescent MCs slowly make their way from the mucosa to draining lymph nodes and ultimately to the spleen. Based on these and other data, it is now apparent that MCs can reversibly alter their protease phenotype in a time- and strata-dependent manner. The observed changes in protease expression have functional significance, as the expulsion of T. spiralis is delayed in mMCP-1-null mice.⁷³ Similar to their protease mediators, MCs also can reversibly alter their expression of prostaglandins and leukotrienes in a cytokine-dependent manner.^74,75

The experimental model of MC adaptation in the response to a helminth infection has not yet been studied in a bacterial infection and in different inflammatory disorders. Nevertheless, the ability of a MC to alter its expression of the proinflammatory tryptases mMCP-6 and mMCP-7 in a reversible manner is likely to impact the animal's ability to recruit peripheral blood neutrophils and eosinophils into a tissue to combat an infectious organism. It also remains to be determined if the MCs in the conjunctiva can reversibly alter their phenotype in response to different pathogens as occurs in the jejunum. However, at the mRNA level, the two most abundant MC-restricted proteases in the conjunctiva 6are the tryptases mMCP-6 and mMCP-7 (Miyazaki, Stevens, and Ono, unpublished data), thereby suggesting a prominent role for these tryptases in ocular inflammation.

RECEPTORS THAT MCs USE TO RECOGNIZE BACTERIA AND OTHER INFECTIOUS ORGANISMS

Mature MCs use numerous receptors to recognize and respond to factors produced by bacteria and other infectious organisms (Fig. 1-1). The MCs response to these foreign factors can be opsonin-dependent or opsonin-independent.

Fig. 1-1: MC participation in innate and acquired immunity. The MCs in the eye and other tissue sites express a diverse array of receptors (e.g., TLR2, TLR4, TLR6, CD48, PAR-1, and PAR-2) that recognize factors that originate from bacteria, yeast, and other infectious organisms. MCs also possess receptors (e.g., Fc∊RI, FcγRII, and FcγRIII) that recognize immunoglobulins generated in the host against pathogens. Finally, the coating of bacteria and other infectious organisms with complement proteins results in the generation of anaphylatoxins which bind to the C3a and C5a/CD88 receptors on the MC's surface. MCs release varied combinations of biologically active factors when they become activated via these signaling pathways. Cross-linking of a surface receptor such as Fc∊RI results in the rapid release of the preformed mediators stored in the cell's granules [e.g., histamine, serotonin, and protease•serglycin proteoglycan (PG) complexes]. Mouse MCs store 16 proteases in their granules, and mMCP-6 and mMCP-7 are the cell's tetramer-forming tryptases. Homotypic mMCP-6 tetramers remain bound to serglycin proteoglycans when exocytosed from activated MCs, whereas homotypic mMCP-7 tetramers dissociate from their macromolecular complexes. A few minutes after the cell's high-affinity IgE receptors are cross-linked, activated MCs release varied combinations of prostaglandins, leukotrienes, and other lipid mediators. Hours later, Fc∊RI-activated MCs markedly increase their expression of varied combinations of at least 31 different cytokines and chemokines. Thus, activated MCs produce more biologically active factors than any other cell type in the body

As an example of their participation in acquired immunity, MCs can bind to helminths in an opsonin-dependent manner. In this scenario, helminths evoke a humoral response that results in the generation of helminth-specific IgE. The binding of this immunoglobulin to surface Fc∊RI receptors allows MCs the ability to recognize and bind to the helminth.⁷⁶ After a bacterial infection, mice and humans normally produce pathogen-specific IgG. Nevertheless, in some instances, MCs also can respond to bacteria in an IgE/Fc∊RI-dependent manner. For example, the generation of IgE that recognizes Staphylococcus aureus-derived exotoxin can induce the release of mediators from Fc∊RI-bearing MCs and basophils, which, in turn, can lead to atopic dermatitis.⁷⁷ Mouse⁷⁸ and human⁷⁹ MCs also express the low-affinity IgG receptors FcγRII and FcγRIII on their surfaces. Some populations of MCs become activated via these Fcγ receptors when they encounter immune complexes,⁸⁰ and the inflammatory Arthus reaction to antibody-antigen complexes is a consequence of the activation of cutaneous MCs via their FcγRIII receptors.⁸¹ Bullous pemphigoid is an autoimmune disorder in which autoreactive IgG recognizes the hemidesmosomal proteins BP230 and BP180. The binding of anti-BP230 and/or anti-BP180 IgG to the MC's FcγR receptors results in MC degranulation, neutrophil infiltration, and skin blistering.⁸²

In regard to their role in innate immunity, MCs can respond to complement-derived anaphylatoxins due to their C3a and C5a/CD88 receptors. Because sea squirts express complement⁸³, it is likely that their MC-like “test” cells also are regulated by complement-derived factors like C3a and C5a. Sepsis occurs in the mouse when its cecum is ligated and punctured (CLP). The resultant accumulation of neutrophils into the bacteria-infected peritoneal cavity is highly dependent on factors released from the peritoneal MCs.⁸⁴ Prodeus and coworkers⁸⁵ noted that C3-deficient mice are more sensitive to CLP-induced sepsis than wild-type mice. In this bacterial infection model, fewer neutrophils and fewer degranulated MCs were found in CLP-treated C3-deficient mice. The ability to rescue the mouse's impaired neutrophil response to the bacterial infection by treating the C3-deficient mice with purified C3 protein confirmed the key role for complement pathways in MC-dependent sepsis.

MCs respond to fimbriated Escherichia coli in an opsonin-independent manner. FimH is the mannose-binding subunit of type 1 fimbriae, which are rod-like adhesive structures that project from the outer surface of E. coli and other enterobacteria. Malaviya and coworkers reported that MCs express CD48 and use this SLAM (signaling lymphocyte-activation molecule) family member to recognize and phagocytose FimH-expressing E. coli.^86,87 The biologic relevance of the CD48-FimH interaction remains to be determined. Although it was initially proposed that MCs use CD48 primarily to kill E. coli, neutrophils are the primary cells in the body that eliminate bacteria. Thus, a more likely scenario is that FimH-CD48-dependent signaling events induce MCs to release factors that induce the recruitment of large numbers of neutrophils into the bacteria-infected site.8

MCs also can recognize factors produced by infectious organisms via their Toll-like receptors (TLRs). In vitro-differentiated mBMMCs express TLR2, TLR4, and TLR6.^88,89 TLR2 forms a dimer with TLR6, and this receptor complex recognizes bacterial peptidoglycan and yeast zymosan, whereas TLR4 recognizes bacterial lipopolysaccharide (LPS). mBMMCs increase their expression of granulocyte-macrophage-colony stimulating factor, IL-1β, and cysteinyl leukotrienes when exposed to peptidoglycan and zymosan, but not LPS. In contrast, in vitro-differentiated human MCs that had been exposed to LPS increased their expression of tumor necrosis factor-α (TNF-α), IL-5, IL-10, and IL-13, as did peptidoglycan-treated human MCs.⁹⁰ Peptidoglycan additionally induced histamine release from this population of cells. It therefore is apparent that the mediators released from MCs by bacterial products are dependent on what TLR is used in the signal-transduction event. Because MCs are heterogeneous, it remains to be determined if the MCs in every human and every mouse respond to TLR and CD48 agonists in the same way. In this regard, mouse fetal skin-derived MCs express much more TLR3 and TLR7 than mBMMCs. Nevertheless, the cytokine responses that have been observed so far in TLR-activated mouse and human MCs are distinct from the histamine and eicosanoid responses evoked by cross-linking of the cell's high-affinity IgE receptors. The accumulated CD48 and TLR data highlight the many ways in which mouse and human MCs are able to recognize various bacteria and illicit diverse responses.

An unexplored area of investigation is the ability of MCs to recognize bacteria via NOD1/CARD4 and NOD2/CARD15. NOD2 recognizes bacterial muramyldipeptide, whereas NOD1 recognizes the peptidoglycan-specific dipeptide γ-D-glutamyl-meso-diaminopimelic acid. MCs have been implicated in inflammatory bowel disease⁹¹, and many patients with Crohn's disease have a mutated NOD2 gene.^92,93 While it remains to be seen if MCs use NOD1 and/or NOD2 to recognize microbial products, we recently discovered that in vitro-differentiated mouse and human MCs express NOD1 and NOD2 (Hamilton, Krilis, and Stevens, unpublished findings).

Lastly, MCs express proteinase-activated receptor (PAR) 1 and PAR-2, and the cellular activation via these receptors results in degranulation and increased expression of cytokines.^94–98 While the emphasis in this area of investigation has been on the ability of thrombin, trypsin, and other endogenous proteases to activate MCs via their PARs, it is now apparent that numerous proteases produced by infectious organisms and biting insects activate mammalian cells via this family of receptors. For example, the house dust mite proteases Der p3 and Der p9 are potent activators of PAR-2.⁹⁹ House dust mites live in beds, sofas, and carpets, and feed on human skin. Their protease-rich fecal droppings elicit extreme allergic reactions in many humans who live in moist climates where the insect infestation is greatest. The accumulated data suggest that MCs also use the receptor PAR-2 in immune surveillance.9

ROLE OF MCs AND THEIR TRYPTASE MEDIATORS IN BACTERIAL INFECTIONS

Earlier in vitro studies revealed that MCs release their mediators when exposed to bacteria. However, the importance of MCs in bacterial infections did not become apparent until in vivo studies were carried out on MC-deficient W/W^v mice before and after their reconstitution with wild-type +/+ mBMMCs. Using the CLP model of sepsis, Echtenacher and coworkers⁸⁴ noted that W/W^v mice are more susceptible to E. coli than their MC-sufficient +/+ littermates. Malaviya and coworkers¹⁰⁰ obtained similar data when Klebsiella pneumoniae was injected into the peritoneal cavity or lungs of a W/W^v mouse. In the latter infection model, W/W^v mice were ∼20-fold less efficient than +/+ mice in clearing the enterobacteria. The MC-deficiency of W/W^v mice can be rescued using an adoptive transfer approach in which the mice are given unfractionated bone marrow cells or in vitro-differentiated mBMMCs from their histocompatible +/+ littermates.^101,102 The ability of reconstituted W/W^v mice to combat K. pneumoniae confirmed a key role for MCs in the anti-bacteria response.¹⁰⁰ When evaluating for a potential mechanism to explain the increased lethality seen in the W/W^v mice, Malaviya and coworkers¹⁰⁰ discovered a significant difference in the number of neutrophils in the infected peritoneal cavities of W/W^v mice compared with that in +/+ mice. Because neutrophils are the professional killers of bacteria in the body, it became evident from these and other experiments that MCs primarily were indirectly involved in combating bacterial infections by inducing the recruitment of neutrophils.

It is likely that MCs produce a diverse array of factors that work in concert to orchestrate an effective anti-bacteria response. mBMMCs can produce modest amounts of leukotriene B₄,¹⁰³ which is a potent chemotactic factor for neutrophils.¹⁰⁴ However, the fact that the MCs in the mouse and rat peritoneal cavity preferentially metabolize arachidonic acid to prostaglandin D₂¹⁰⁵ suggested that leukotriene B₄ was not the primary antimicrobial factor produced by peritoneal MCs. While MCs also release histamine when activated, this vasopermeability factor turned out not to be the MC-restricted factor needed for eliminating bacteria because histamine-null mice actually combated bacteria better than histamine-sufficient mice.¹⁰⁶ The MCs in the peritoneal cavity contain substantial amounts of mMCP-4 and mMCP-5.^13,107,108 These two chromosome 14C3 family members (as well as mMCP-1, mMCP-2, mMCP-3/4L, mMCP-8, mMCP-9, mMCP-10, cathepsin G, and granzyme B) are expressed as zymogens that contain 2-mer propeptides. Because dipeptidyl peptidase I (DPPI)/cathepsin C is used to activate the chromosome 14C3 family of serine proteases¹⁰⁹, the MCs in DPPI-null mice have very little, if any, enzymatically active mMCP-4 and mMCP-5. Like histamine-null mice, DDPI-null mice fared better than their wild-type counterparts with respect to a bacterial challenge.¹¹⁰ The reasons for these unexpected findings remain to be determined. However, it is presumed 10that histamine, mMCP-4, and/or mMCP-5 play adverse roles in bacterial infections by inducing vasopermeability, which, in turn, allows more harmful bacteria to enter the circulation. Whatever the mechanism, none of the MC's chromosome 14C3 family members appear to have a beneficial role in bacterial clearance.

TNF-α is a neutrophil chemoattractant transiently produced by activated MCs.^111–113 Using anti-TNF-α antibody blocking approaches, Malaviya¹⁰⁰ and Echtenacher⁸⁴ and their coworkers tentatively concluded that TNF-α was the critical MC-derived factor required for efficient bacterial clearance. However, macrophages greatly outnumber MCs in the peritoneal cavity and lung. When activated with LPS, macrophages produce more TNF-α than activated MCs. Furthermore, TNF-α-null mice successfully combated a bacterial infection when their MCs were activated.¹¹⁴ It therefore became apparent that another factor more restricted to MCs had to be involved in the neutrophil recruitment seen in bacterial infections.

Administration of recombinant mMCP-6^26,115 or hTryptase-β1²⁴ into varied mouse tissues resulted in the preferential accumulation of neutrophils. Moreover, hTryptase-β1-treated W/W^v mice could combat a K. pneumoniae infection better than pro-Tryptase β1-treated W/W^v mice.²⁴ In support of a key role for the MC's tryptases in granulocyte accumulation, Oh and coworkers¹¹⁶ were able to block airway inflammation in mice given the tryptase inhibitor MOL6131. Transcription of the mMCP-6 gene is greatly diminished in tg/tg mice.⁶⁵ The fact that tg/tg mice also cannot combat bacterial infections efficiently supported the conclusion that mMCP-6 was likely the critical MC-restricted anti-microbial factor.¹¹⁷ Definitive proof that this tryptase has a critical immunoprotective role in bacterial infections occurred when it was shown that a newly created mMCP-6-null mouse could not survive a K. pneumoniae infection of its peritoneal cavity.¹¹⁸

The mechanisms by which mMCP-6 and its human ortholog hTryptase-β1 regulate granulocyte accumulation in tissues at the molecular level remain to be determined, but in vitro studies revealed that mMCP-6 can induce human endothelial cells to increase their expression of IL-8, which is a potent chemoattractant for neutrophils.²⁶ The accumulated data suggest that mMCP-6 in mice and hTryptase-β1 in humans control bacterial infections in an indirect manner by inducing bystander cells to secrete factors that recruit bactericidal neutrophils.

CONCLUSION

While recent studies on the MC's tryptase tetramers have provided exciting new insight into the beneficial roles of MCs in immunity, many questions remain unanswered. This review has highlighted some of the pertinent studies that have brought to the forefront the importance of MC-restricted tryptases in 11bacterial infections. Nevertheless, little is known about how these proteases are activated inside MCs from their zymogen precursors, how the enzymatically active mature proteases are metabolized after their exocytosis from the MC to dampen their pro-inflammatory effects, and how they interact with other mediators and cells in the body. MCs and their progenitors are highly susceptible to M-tropic strains of HIV-1^119–121, and the number of hTryptase-β1⁺ MCs are greatly reduced in the gastrointestinal tract of AIDS patients.¹²² Because AIDS patients are highly susceptible to bacteria and other infectious organisms, it is possible that recombinant hTryptase-β1 could provide beneficial therapy to these immunocompromised patients. In contrast, pharmacologic inhibition of the MC's tryptases might have benefit in various inflammatory disorders characterized by prominent neutrophil accumulation after MC activation as occurs in rheumatoid arthritis and ocular inflammation.

The ability of MC-restricted tryptases to help mammals fight off bacteria explains why their genes have been conserved. Nevertheless, mouse MCs store varied combinations of 14 other serine proteases in their granules. Some of these proteases (e.g., CPA3, Prss31, and mMCP-5) have human orthologs. The mMCP-6/hTryptase-β1 studies noted in this review raise the possibility that CPA3, Prss31, and mMCP-5/chymase-1 have been conserved for millions of years because they also possess yet unexplored beneficial roles in innate and/or acquired immunity. Mature MCs display a wide range of phenotypes in tissues and can adapt to changes in the local environment. The functional significance of MC heterogeneity has not been adequately explored. It therefore is anticipated that future studies will illuminate the importance of MCs and their diverse array of mediators in various infections, and perhaps even in situations where MCs respond inappropriately. After all, MCs today encounter a far different world than that which existed when these cells first appeared in evolution.

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Chapter Notes

Mast Cells and Innate Immunity1