American Journal of Respiratory and Critical Care Medicine

Tryptase, a serine protease released exclusively from activated mast cells, has been implicated as a potential causative agent in asthma. Enzymatically active tryptase is comprised of four subunits, and heparin stabilizes the associated tetramer. Lactoferrin, a cationic protein released from activated neutrophils, binds tightly to heparin, therefore we investigated lactoferrin as an inhibitor of tryptase and found that it is both a potent (Ki′ is 24 nM) and selective inhibitor. Size exclusion chromatography studies revealed that lactoferrin disrupted the quaternary structure of active tryptase. Lactoferrin was tested in an allergic sheep model of asthma; aerosolized lactoferrin (10 mg in 3 ml phosphate-buffered saline, 0.5 h before as well as 4 and 24 h after inhalation challenge by Ascaris suum) abolished both late-phase bronchoconstriction (no significant increase in specific lung resistance 4 to 8 h following provocation, p < 0.05 versus vehicle treatment) and airway hyperresponsiveness (no detectable increase in airway sensitivity to carbachol challenge 24 h after antigen challenge, p < 0.05 versus vehicle). These data suggest tryptase involvement in both late-phase bronchoconstriction and airway hyperreactivity and furthermore suggest that a physiological function of neutrophil lactoferrin is the inhibition of tryptase released from mast cells.

Tryptase, a neutral serine protease that is localized in mast cell granules, constitutes up to 20% of total cellular protein (1). The active enzyme is released with other mediators (heparin, histamine, and chymase) upon mast cell degranulation. Tryptase is unusual for two reasons: the enzyme is a tetramer composed of four active subunits, and there are no known potent endogenous inhibitors of tryptase enzyme activity (2).

The quaternary structure of the tryptase tetramer (Mr of 140,000) is stabilized by several glycosaminoglycans, particularly heparin (2). Dissociation of the tetramer abolishes enzyme activity irreversibly, suggesting that bound heparin stabilizes the catalytically active quaternary structure (2). Moreover, tryptase is partially inhibited by the heparin-binding protein antithrombin III (AT III), though inhibition is incomplete at AT III concentrations greater than that normally present in circulation (3). Other studies have confirmed that heparin antagonists are capable of inhibiting mast cell tryptase (4). Tryptase activity is unaffected by secretory leukocyte protease inhibitor (5) or α1 protease inhibitor (the major protease inhibitors found in the airway), α2 macroglobulin, or by inhibitors from lima bean, soybean, chicken ovomucoid (6), and human urine (7). Spontaneous dissociation of the tryptase tetramer has been proposed to be the mechanism by which it is regulated in vivo (2, 8).

The normal, physiological function of tryptase is unknown. However, tryptase cleaves biologically important proteins and peptides in vitro, and, thus, several pro-inflammatory properties have been proposed for the enzyme: the activation of collagenase (9) and gelatinase (10); the degradation of fibronectin (10), fibrinogen (11), and vasoactive intestinal peptide (12); and the release of bradykinin from kininogens (13, 14). Furthermore, tryptase promotes the proliferation of fibroblasts (15) and epithelial cells (16) and, therefore, may be involved in tissue remodeling.

The presence of tryptase in biological matrices is an indicator of mast cell degranulation in anaphylaxis and allergic reaction (17, 18). Mast cells are present in large numbers in lung tissue and are located in the bronchial mucosa, submucosa (19) and alveolar walls (20). Several studies have shown that both tryptase levels (21-23) and mast cell numbers (24, 25) are elevated in the bronchoalveolar lavage fluid (BAL) from atopic asthmatics, and BAL tryptase levels in these individuals are increased within minutes after antigen challenge (25, 26).

It has recently been demonstrated that inhaled tryptase causes bronchoconstriction and airway hyperresponsiveness in sheep (27), and that aerosol administration of a selective tryptase inhibitor, APC 366, abolishes late-phase bronchoconstriction and the associated airway hyperresponsiveness that ensues following airway antigen provocation (28). In addition, 1 ng of tryptase injected into the skin of allergic sheep causes an immediate cutaneous response; prior intradermal administration of APC 366 substantially reduces the severity of wheal formation (29). These data strongly support the proposal that tryptase is pro-inflammatory and represents a potential therapeutic target for the treatment of asthma, as well as other mast cell-mediated diseases (30).

After mast cell exposure to an allergen, neutrophils are recruited to the site of mast cell activation where they release the contents of their secretory granules. Tryptase itself has been shown to be a chemoattractant for leukocytes (31). Lactoferrin, a cationic 78 kDa member of the transferrin class of proteins, is released from serous cells and the secondary granules of neutrophils in response to inflammatory stimuli (32). Though the primary sequence of lactoferrin is 85% homologous to the sequence of transferrin, lactoferrin possesses heparin-binding domains (33) that are unique within the class. Lactoferrin that is released from activated neutrophils could inhibit mast cell tryptase activity by associating with enzyme-bound heparin.

In this study, we investigated lactoferrin as an inhibitor of tryptase in vitro and found that it is both potent (Ki′ is 24 nM) and selective. We demonstrate that lactoferrin causes disruption of the tryptase tetramer and that tryptase inhibition by lactoferrin is blocked by the presence of excess heparin. These results suggest that tryptase inhibition is mediated through lactoferrin's heparin-binding domains. Previous reports showed that a potent and selective tryptase inhibitor, APC 366, is efficacious in a sheep model of asthma (28). In our studies, aerosolized lactoferrin abolished both late-phase bronchoconstriction (no significant increase in specific lung resistance 4–8 h following provocation, p < 0.05 versus vehicle treatment) and airway hyperresponsiveness and, thus, has a pharmacological profile similar to APC 366. These data suggest that lactoferrin may be an endogenous regulator of tryptase activity and, therefore, a natural suppresser of mast cell-induced allergic disease.

Materials

Human lung tryptase and rat skin tryptase were purified from tissue samples as described (34, 35). An immortalized human mast cell line (HMC-1) was provided by Dr. Joseph H. Butterfield of the Mayo Foundation (36); tryptase was purified from this cell line by modification of a published procedure (37). Human trypsin (Athens Research and Technology, Athens, GA), thrombin (CalBiochem, San Diego, CA), and plasmin (Boehringer Mannheim, Indianapolis, IN) were purchased from the indicated commercial sources. Porcine intestinal mucosa heparin, Tosyl-Gly-Pro-Lys-pNA, human lactoferrin, and apo-transferrin are products of the Sigma Chemical Company, St. Louis, MO. A TSK-G3000SWXL size-exclusion column was purchased from Tosohaas, Montgomeryville, PA, and gel-filtration molecular weight markers (high and low) from Pharmacia, Uppsala, Sweden. Rat anti-tryptase was produced at Arris.

Enzyme Kinetics

Enzyme (0.5 nM) and inhibitor at varying concentrations were pre- incubated for 1 h (unless otherwise indicated) in either low phosphate (120 mM NaCl, 2.7 mM KCl, 0.13 mM NaH2PO4, 0.90 mM Na2HPO4, pH 7.5) or Tris buffer (100 mM NaCl, 50 mM Tris, 0.05% Tween-20, 10% DMSO, pH 8.2) at room temperature before adding substrate (0.5 mM Tosyl-Gly-Pro-Lys-p-nitroanilide). The change in the rate of absorbance (405 nm) was measured using a UV/MAX Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA) interfaced with a Macintosh Quadra 840 computer. Apparent inhibition constants (38), Ki′, were calculated from the enzyme progress curves using the software package BatchKi (provided by Dr. Petr Kuzmic, School of Pharmacy, University of Wisconsin). The software package Enzyme Kinetics v 1.11 (Trinity Software, Campton, NH) was used to transform the kinetic data into double reciprocal plots.

Size-Exclusion Chromatography

The TSK-G3000SWXL column was calibrated using gel infiltration markers. Tryptase (340 nM) was mixed with excess lactoferrin (15 μM) in low phosphate buffer (460 μl). At 1, 3, and 18 h after mixing, samples (100 to 300 μl) were injected at a flow rate of 1 ml/min into a TSK-G3000SWXL column that was previously equilibrated with buffer (0.1 M NaH2PO4/Na2HPO4, 0.1 M Na2SO4, pH 6.5). Detection of protein in the eluent was accomplished spectrophotometrically (A280) and fluorometrically (λex, 280 nm; λem, 335 nm) and fractions (1 ml) were collected. Tryptase activity in the fractions was assayed by diluting an aliquot (50 μl) into a low phosphate buffer solution (50 μl) containing substrate (0.5 mM). Enzyme activity was measured as previously described.

Western Slot Blot

Tryptase protein was detected in column fractions by immunoblot staining. A slot blot apparatus (Baxter, McGaw Park, IL) was used to transfer samples (55 μl) of the size-exclusion chromatography fractions to a nitrocellulose membrane that was treated and blocked with buffer and gelatin as described by the manufacturer (Immunoblot Assay Procedure; Bio-Rad, Hercules, CA). A rat mAb to human tryptase was added (5 μg/ml, final concentration) and incubated with the membrane for 1 h. After washing, a mouse anti-rat-alkaline phosphatase conjugate (Jackson ImmunoResearch, West Grove, PA) was added for 1 h. Following another buffer wash, the membrane was developed (BioRad AP color development kit; Bio-Rad) until the immunoblots were visible (approximately 5 min).

Sheep Studies

Allergic sheep (n = 4), previously characterized as dual responders (displaying early and late phases of bronchoconstriction), were administered (by aerosol) lactoferrin (10 mg in 3 ml of phosphate-buffered saline or vehicle control, 0.5 h before and 4 h and 24 h after inhalation challenge with Ascaris suum antigen. Specific lung resistance (SRL) was monitored via an esophageal balloon catheter as described (39) and measured at the times indicated in Figure 1. Twenty-four hours after antigen challenge, airway responsiveness, defined as the cumulative carbachol dose required to increase SRL by 400% (39), was measured subsequent to the final dose of lactoferrin or vehicle (Figure 1). Airway resistance of each treated animal was compared with control (in which the same animal was dosed with vehicle only). Baseline SRL was measured just prior to antigen challenge and baseline PC400 was measured 1 to 2 d before challenge. Statistical significance was determined by paired t-test analysis, and error bars in Figure 1 indicate standard deviation from the mean.

To determine the effect of lactoferrin on tryptase, enzyme (0.5 nM) was incubated with varying concentrations of lactoferrin for 1 h and residual enzyme activity was determined. As shown in Figure 2, lactoferrin is a potent inhibitor of tryptase activity at a heparin concentration of 25 ng/ml (a 10-fold molar excess of heparin to tryptase assuming an average molecular weight of 5,000). The calculated Ki′ of lactoferrin is 24 nM. The results are similar for human lung tryptase and tryptase from HMC-1 cells. There is no detectable tryptase inhibition by apo-transferrin (which has 85% sequence homology with lactoferrin but no heparin-binding domain) at a concentration of 1 mM (data not shown) and tryptase incubated without lactoferrin for 1 h is stable (< 10% loss of enzyme activity).

To determine the importance of a heparin-lactoferrin interaction to the mechanism of enzyme inhibition, tryptase was incubated for 1 h with lactoferrin in the presence of a 200,000-fold molar excess of heparin (500 μg/ml). The results (Figure 2) indicate that excess heparin protects tryptase activity from the effects of lactoferrin.

The kinetic mechanism of tryptase inhibition by lactoferrin was determined. A double reciprocal plot (Figure 3) of the inhibition data indicates that lactoferrin appears to be a non-competitive inhibitor (Km of substrate is unchanged in the presence of inhibitor) when pre-incubated with tryptase for 1 h prior to the addition of substrate.

The capacity of lactoferrin to inhibit other trypsin-like serine proteases (in addition to tryptase) was also investigated (Table 1). Lactoferrin is a poor inhibitor of trypsin, thrombin, plasmin, and rat tryptase (35)—enzymes that do not require heparin for activity.

Table 1. Ki′  OF LACTOFERRIN FOR TRYPSIN-LIKE PROTEASES

Enzyme* Ki′ (nM)
Human Tryptase24
Rat Tryptase546,000
Trypsin> 1,000,000
Thrombin> 1,000,000
Plasmin> 1,000,000

*Enzyme (human tryptase, 0.5 nM; rat tryptase, 2.5 nM; trypsin, 15.7 nM; thrombin, 12.9 nM; plasmin, 7.6 nM) was incubated with varying concentrations of lactoferrin for 1 h in Tris assay buffer and the reaction was started by the addition of substrate (Tosyl-Gly-Pro-Lys-pNA, 0.5 mM final concentration).

Ki′ values were determined as described in Methods section.

Because tryptase inhibition by lactoferrin appeared to be mediated via a heparin-dependent mechanism, the effect of lactoferrin on the stability of the tryptase tetramer was assessed by use of size-exclusion chromatography. Size-exclusion is useful for separating and measuring relative amounts of tryptase tetramer (retention time, 7.5 min) and monomer (retention time, 9.5 min). A mixture of tryptase (340 nM) and porcine heparin (24 μg/ml) was incubated in the presence (15 μM) and absence of lactoferrin for 18 h. Samples were removed from both solutions at 1, 3, and 18 h after mixing for the determination of residual tryptase activity (Table 2) and tryptase tetramer decomposition (Figure 4).

Table 2. LACTOFERRIN ACCELERATES THE DECOMPOSITION OF TRYPTASE TETRAMER

Incubation Time (h)− Lactoferrin* + Lactoferrin*
% Enzyme Activity % Tryptase Tetramer % Enzyme Activity % Tryptase Tetramer
 1100756749
 37760313
186641100

*Solutions of heparin (24 μg/ml) and tryptase (340 nM) with and without lactoferrin (15 μM) were incubated for the indicated time in low phosphate buffer. Samples (25 μl to 100 μl) were removed for the determination of enzyme activity and size-exclusion chromatography.

Enzyme activity is expressed relative to the tryptase activity determined following a 1 h pre-incubation in the absence of lactoferrin.

The percentage of tryptase tetramer is estimated from area under the curve values calculated by HP Chemstation software (Hewlett-Packard, Menlo Park, CA). Calculations assume that the excitation and emission spectra for the tetramer and monomer are equivalent.

The chromatograph (Figure 4, panel A) of the control mixture (no lactoferrin) following a 1 h incubation, shows that tryptase is a mixture of tetramer (large peak) and monomer (small peak). It is not known if the tryptase monomer present in the preparation (25% of the tryptase protein, Table 2) reflects enzyme dissociation in solution or on the column or the presence of significant levels of contaminating inactive monomer in the tryptase preparation.

The chromatograph (Figure 4, panel B) of a sample from the mixture of tryptase and lactoferrin incubated for 1 h indicates that a substantial conversion of tryptase tetramer to monomer accompanies tryptase inhibition by lactoferrin (49% remaining tetramer, 33% inhibition, Table 2). In a sample from the same reaction mixture, following 18 h of incubation (Figure 4, panel C), the peak corresponding to tryptase tetramer is absent and the disappearance of tetramer is concomitant with a 90% loss of enzyme activity (Table 2).

Immunoblot assay (with a rat anti-human tryptase monoclonal antibody) of samples from fractions collected throughout the chromatographic separations (Figure 4, panel D) confirmed that both the 7.5 min peak and the 9.5 min peak are derived from tryptase; no other fractions contained tryptase protein. The fraction that corresponded to the tetramer had substantial proteolytic activity; whereas, very low but measurable enzyme activity was found in the fraction in which monomer eluted.

Lactoferrin was tested in the allergic sheep model of asthma (39). These sheep develop early- and late-phase bronchoconstriction and an associated increase in bronchial responsiveness following antigen (Ascaris suum) challenge. Sheep treated with lactoferrin (10 mg in 3 ml of phosphate buffered saline by aerosol), 0.5 h before and 4 h and 24 h following antigen challenge had substantial reductions in both late-phase bronchoconstriction (4 h to 8 h following antigen challenge, Figure 1) and airway hyperresponsiveness to carbachol (24 h following antigen challenge, Figure 1). The magnitude of the early phase (0 to 4 h) was not significantly reduced by lactoferrin treatment; however, the duration of the early phase bronchoconstriction appeared to be shortened as a result of treatment.

Our results show that lactoferrin is a potent, time-dependent inhibitor of human tryptase; the Ki′ value of lactoferrin for human tryptase is 24 nM as determined from the residual enzyme activity following a 1 h incubation of enzyme and inhibitor (Figure 2). The presence of a large excess of heparin effectively blocks inhibition (Figure 2).

We considered the possibility that lactoferrin, because it has substantial sequence homology (RILK) with the reactive site loop of a tryptase inhibitor derived from leech (KILK) (40) inhibits tryptase by a serpin-like mechanism. To test this hypothesis, tryptase that was inactivated with lactoferrin (Table 2) was subjected to gel-filtration chromatography (Figure 4) and SDS-PAGE. However, no tryptase-lactoferrin complex was observed by either method, inconsistent with a serpin-like mechanism of enzyme inhibition (41, 42). Furthermore, if tryptase inhibition by lactoferrin was due to a serpin-like mechanism it is likely that trypsin would be similarly inhibited, and it is not (Table 1). Thus, a serpin-like mechanism for the inhibition of tryptase by lactoferrin was excluded.

Lactoferrin appears to be a noncompetitive inhibitor of human tryptase (Figure 3) consistent with a mechanism of irreversible inhibition that results in a reduction in enzyme Vmax. The Km (1.0 mM) of the small molecule substrate, Tosyl-Gly-Pro-Lys-pNA, is unaffected by lactoferrin (Figure 3). Also, the values of Ki and Kis, 65 nM and 67 nM, respectively (Figure 3), are within experimental error, suggesting that the affinity of lactoferrin for tryptase is unaffected by substrate binding. Furthermore, we show that lactoferrin is a poor inhibitor of rat skin tryptase (Table 1), trypsin, thrombin, and plasmin— enzymes that do not require heparin for stabilization. We believe these data are consistent with lactoferrin, through its heparin-binding domain, interfering with the active, quaternary structure of tryptase by interacting with enzyme-bound heparin at a site removed from the enzyme active site.

We show that aerosol administration of lactoferrin to allergen-challenged sheep caused a significant reduction of both late-phase bronchoconstriction and airway hyperresponsiveness in these animals. In a previous study, a selective small molecular weight tryptase inhibitor (APC 366) was also effective in preventing the late- and hyperreactive phases of bronchoconstriction in allergic sheep (28). The in vivo efficacy of two selective tryptase inhibitors, that have substantially different physical properties and mechanisms of enzyme inhibition, further implicates tryptase as the therapeutic target. These data also validate the proposal that tryptase has a causative role in airway hypersensitivity reactions.

The elucidation of the mechanism of inhibition of tryptase by lactoferrin confirms the proposal that agents that associate tightly with heparin may promote decomposition of the tryptase tetramer (2). Therefore, other agents known to bind heparin (such as IL-8 [43], protamine [44], etc.) may also inhibit tryptase through a similar mechanism. Like lactoferrin, other proteins released from the granules of eosinophils and neutrophils are cationic (e.g., bactericidal permeability increasing protein, major basic protein, eosinophil cationic protein) and bind heparin. Therefore, a physiological function of these proteins could be the inhibition of mast cell tryptase activity.

Ironically, heparin has been shown to reduce early (45) and late phase (46) antigen-induced bronchoconstriction in sheep and has been proposed as a potential anti-inflammatory therapeutic (47). However, heparin has several biological activities including immunosuppression (48) and antagonism of the inositol 1,4,5-triphosphate (IP3) receptor (49, 50); blocking IP3 binding prevents mast cell degranulation. Thus, though both lactoferrin and heparin are efficacious in the sheep model of asthma, they exert their effects through distinctly different mechanisms.

Lactoferrin concentrations are greater in the BAL of smokers and chronic bronchitis patients than in normals (51). The lactoferrin concentrations measured in the BAL of these groups are equal to, or greater than the Ki′ (24 nM) for tryptase inhibition. Therefore, lactoferrin concentrations in BAL are sufficient to inhibit tryptase in vivo.

It has been proposed that the emigration of neutrophils into the asthmatic airway following antigen challenge represents a normal healing mechanism, possibly related to the generation of factors from neutrophils that counteract the processes that lead to hyperresponsive airways (52). For example, in asthmatic patients, allergen-induced late-phase reactions are associated with elevated serum levels of neutrophil chemotactic activity (53) and increased numbers of neutrophils in BAL fluid (54-56). Furthermore, elevated levels of circulating lactoferrin and myeloperoxidase suggest that continuous neutrophil activation occurs in mild asthmatics (57).

From the evidence presented, we propose that one possible function of lactoferrin following release from the neutrophil is to neutralize the enzymatic activity of tryptase. This proposed function, if true, would mean the existence of a feedback inhibition mechanism for the suppression of tryptase activity. Furthermore, if lactoferrin functions as a suppresser of tryptase-induced cellular recruitment and inflammatory processes, it raises the possibility that a cause of airway hypersensitivity in some asthmatics is a reduction in the effectiveness of this negative-feedback mechanism.

Our observations show that lactoferrin is a potent selective inhibitor of mast cell tryptase in vitro. We demonstrate that the mechanism of lactoferrin's inhibition is disruption of the active tryptase tetramer into inactive monomers. The specificity of lactoferrin inhibition (no activity towards proteases that do not require heparin) and the ability of heparin to block inhibition suggest that the mechanism of tryptase inhibition is displacement of stabilizing heparin. The observed effects of aerosolized lactoferrin in the allergic sheep model mimic other reported tryptase inhibitors (28) and suggests a potential role for lactoferrin in vivo.

The authors thank Drs. Adam Dubin and James Travis, University of Georgia, for the purification of human lung tryptase and Dr. William Simmons, Loyola University of Chicago, for providing purified rat skin tryptase. The authors particularly wish to acknowledge Ms. Daun Putnam for skillfully obtaining the Ki′ data displayed in Table 1.

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Correspondence and requests for reprints should be addressed to William R. Moore, Ph.D., Arris Pharmaceutical Corporation, 385 Oyster Point Blvd., Suite 3, South San Francisco, CA 94080. E-mail: William–

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