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,§
Departments of
*
Medicine and
Pathology, Harvard Medical School, Boston, MA 02115; and
Division of Rheumatology, Immunology, and Allergy and
§
Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, RANTES,
IL-1, or IL-6. However, the tryptase induced endothelial cells to
express large amounts of IL-8 continually over a 40-h period. Neither
enzymatically active mMCP-7 nor enzymatically inactive pro-mMCP-6 was
able to induce endothelial cells to increase their expression of IL-8.
Although the mechanism by which mMCP-6 induces neutrophil accumulation
in tissues remains to be determined, the finding that mMCP-6 induces
cultured human endothelial cells to selectively release large amounts
of IL-8 raises the possibility that this tryptase regulates the steady
state levels of neutrophil-specific chemokines in vivo during mast
cell-mediated inflammatory events. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Like the mast cell tryptases in other species (12, 13, 14, 15, 16, 17, 18), mMCP-6 is initially translated as a zymogen that possesses a hydrophobic signal peptide and a 10-residue pro-peptide whose C-terminal amino acid is Gly (1, 2). After undergoing varied post-translational modification events, mMCP-6 is stored in the acidic granules of mouse mast cells in its mature, enzymatically active form ionically bound to serglycin proteoglycans. Exocytosed mMCP-6 and mMCP-7 are metabolized quite differently in mice undergoing passive systemic anaphylaxis because of the different rates of dissociation of these two tryptases from heparin-containing serglycin proteoglycans (19). Shortly after the relevant Ag is administered to IgE-sensitized mice, mMCP-6/proteoglycan macromolecular complexes appear in the extracellular matrix adjacent to activated mast cells in the tongue, skin, spleen, and heart of normal BALB/c mice and in the spleen and liver of V3 mastocytosis mice. Because exocytosed mMCP-6 does not dissociate readily from serglycin proteoglycans, this tryptase is retained in extracellular matrixes for >1 h. The prolonged retention of mMCP-6 in tissues around activated mast cells implies that mMCP-6 preferentially exerts its effects locally.
Mast cell tryptases have been purified from various species in an attempt to deduce their preferred substrates. Unfortunately, the number of mature mast cells that can be isolated from a mouse is inadequate to obtain enough mMCP-6 for study. The nearly impossible task of removing 100% of the other mMCPs from the starting preparations of mast cell lysates also has hindered studies that address the function of the tryptase. We now describe the derivation of a pseudozymogen form of recombinant mMCP-6 from baculovirus-infected insect cells that can be easily purified, activated ex vivo by enterokinase (EK), and then studied immediately. We show that recombinant mMCP-6 and mMCP-7 are functionally distinct tryptases by multiple critera and that mMCP-6 can selectively induce large numbers of neutrophils to extravasate into the peritoneal cavity of varied mouse strains. We also show that mMCP-6, but not mMCP-7 or pro-mMCP-6, induces cultured endothelial cells to selectively increase their expression of the neutrophil chemoattractant IL-8. Finally, we show that the substrate specificity of mMCP-6 is restricted by heparin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The bioengineering approach recently developed to obtain a pseudozymogen form of mMCP-7 that could be activated with EK after its purification from the conditioned medium of insect cells (6) was used to obtain a similar pseudozymogen (pro-EK-mMCP-6) form of mMCP-6 as well as the nonmodified zymogen (pro-mMCP-6). Expressed pro-EK-mMCP-6 has an EK-susceptible peptide (Asp-Asp-Asp-Asp-Lys) between the domain that encodes the endogenous pro-peptide and the N-terminal Ile residue of the mature tryptase. It also has the eight-residue FLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (20) attached to its C-terminus to facilitate its purification. The relevant cDNA constructs, created with standard PCR approaches, were inserted in the correct orientation into the multiple cloning site of pVL1393 (PharMingen, San Diego, CA) downstream of the promoter of the polyhedrin gene. The nucleotide sequences of the resulting constructs were determined to rule out any PCR-induced artifact.
Insect cells were induced to express pro-mMCP-6 and pro-EK-mMCP-6, as
described previously for pro-mMCP-7 (21). Briefly, purified plasmid DNA
(
5 µg) was mixed with 0.5 µg of linearized BaculoGold DNA
(PharMingen) and calcium phosphate. Each resulting DNA solution was
then added to 3 x 106 adherent Spodoptera
frugiperda 9 insect cells (Invitrogen, San Diego, CA) that were in
the log phase of growth, and the infected cells were cultured for 7
days at 27°C in medium (Invitrogen) supplemented with 10%
heat-inactivated FCS (Sigma Chemcial Co., St. Louis, MO). Recombinant
virus particles (
3 x 107) from these insect cells
were added to a new culture dish containing 6 x 106
Trichoplusia ni High Five insect cells (Invitrogen) in the
log phase of growth, and the infected cells were cultured in
serum-free, Xpress medium (BioWhittaker, Walkersville, MD). Generally 4
days later, the conditioned medium was centrifuged at 1500 x
g for 15 min at room temperature before purification of the
secreted recombinant protein was attempted.
Purification of pro-mMCP-6 and pro-EK-mMCP-6 from insect cell-conditioned medium and EK activation of the pseudozymogen
Pro-mMCP-6 and pro-EK-mMCP-6 were purified by heparin-Sepharose chromatography, with a modification of that described for recombinant pro-mMCP-7 (21). Conditioned medium obtained from insect cells infected with the relevant baculovirus was dialyzed against 0.1 M NaCl and 50 mM sodium acetate, pH 5.0, and then applied to replicate heparin-Sepharose columns that had been equilibrated in the same buffer. After the column was washed, bound proteins were eluted on a linear gradient, with the NaCl concentration in the buffer increasing to 3.1 M.
In most instances, the purification of pro-EK-mMCP-6 was conducted with an affinity column containing the anti-FLAG M2 mAb (International Biotechnology, New Haven, CT) (22, 23). This latter immunoaffinity column (2 ml) was washed with 0.1 M glycine, pH 3.5, and then with 50 mM Tris-HCl and 150 mM NaCl, pH 7.4. After application of the insect cell-conditioned medium, the column was washed briefly with the above pH 7.4 buffer, and then bound pro-EK-mMCP-6 was eluted with 0.1 M glycine, pH 3.5. The eluate was collected into tubes that contained 0.1 M Tris-HCl, pH 7.0, to minimize acid-mediated denaturation of pro-EK-mMCP-6. The protein concentration of the eluate was estimated by measuring the absorbance at 280 nm.
Purified pro-EK-mMCP-6 (100 µg in 100 µl) was separately mixed
with 100 µl of a pH 6.0 buffer containing 10 mM Tris-HCl, 5 mM
calcium chloride, 0.1% Triton X-100, and 50 µg of heparin. The
commercial preparation of heparin (Sigma) used in the study was first
suspended in 4 M GnHCl, 0.1 M sodium sulfate, and 0.1 M Tris-HCl, pH
7.0, and then subjected to a 48-h CsCl density gradient centrifugation
step (24) to denature and remove any trace protein contaminants that
might be present in Sigma’s preparation of this glycosaminoglycan. In
most cases, 1 µl of a solution containing 320 U of calf intestine EK
(Biozyme, San Diego, CA) was added, and the mixture was incubated at
room temperature for 17 h to allow EK to activate the mMCP-6
pseudozymogen. To determine the stability of the generated mMCP-6, in
one experiment the mixture was incubated at 37°C for up to 30 h.
At various times during the activation process, samples of the digest
were evaluated for the ability of the activated mMCP-6 to cleave
tosyl-Gly-Pro-Lys-p-nitroanilide in the 3-min assay
described below. In some cases, mMCP-6 was activated with EK in the
absence of heparin.
Insect cell-conditioned medium and column fractions (20 µl)
containing pro-mMCP-6, pro-EK-mMCP-6, or EK-activated mMCP-6 (1
µl) were diluted in SDS-PAGE buffer (2% SDS, 1% 2-ME, 0.1%
bromophenol blue, and 100 mM Tris-HCl, pH 6.8), boiled for 5 min, and
loaded onto 12% polyacrylamide gels. After SDS-PAGE, the resolved
proteins were stained with Coomassie blue or were transferred in 20 mM
Tris-HCl and 150 mM glycine containing 20% methanol, pH 8.3, for 2 to
4 h at 200 mA to polyvinylidine difluoride membranes (Millipore
Corp., Bedford, MA) with a Bio-Rad (Richmond, CA) immunoblotting
apparatus. For immunoanalysis of the resulting protein blots, each
membrane was sequentially incubated for 1 h in 5% nonfat milk for
1 h with a 1/500 dilution of affinity-purified rabbit
anti-mMCP-6 Ig (19) in Tris-buffered saline containing 0.01%
Tween-20 (TBST buffer), TBST buffer alone, and then for 1 h with a
1/1000 dilution of anti-rabbit IgG alkaline phosphatase conjugate
(1 ng/ml, final concentration) in TBST buffer. Immunoreactive
proteins were visualized with nitroblue tetrazolium (0.2 mg/ml) and
5-bromo-4-chloro-3-indolyl phosphate (0.1 mg/ml) as substrates. For
N-terminal amino acid analysis, SDS-PAGE-resolved proteins were
electroblotted onto polyvinylidine difluoride membranes and briefly
stained with 0.5% Ponceau S red, and the relevant protein/peptide
bands were subjected to automated Edman degradation by the Harvard
Microchemistry Facility (Harvard Biologic Laboratories, Cambridge,
MA).
Analysis of the substrate specificity of mMCP-6
The spectrophotometric method of Svendsen and co-workers (25)
was used to determine whether mMCP-6 is enzymatically active in the
absence or the presence of heparin glycosaminoglycan or normal mouse
serum. Generally, 1-µl samples of each reaction mixture were placed
in 1 ml of a pH 7.4 buffer containing 25 mM sodium phosphate, 1 mM
EDTA, and 50 µg tosyl-Gly-Pro-Lys-p-nitroanilide. The
change in optical density at 405 nm was determined after a 3-min
incubation at room temperature. In this assay, 1 U of enzymatic
activity is defined as a change in OD at 405 nm of 0.001/min. One
microgram of bovine pancreatic trypsin (Sigma) is equivalent to 350
U when tosyl-Gly-Pro-Lys-p-nitroanilide is the test
substrate. With the more widely used substrate
N-benzoyl-L-Arg ethyl ester, the
reference preparation of trypsin employed in our study for comparative
purposes has 11 U/µg. The ability of mMCP-6 to cleave the
trypsin-susceptible substrates
tosyl-Gly-Pro-Arg-p-nitroanilide,
benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide,
benzoyl-Pro-Phe-Arg-p-nitroanilide, and
acetyl-Ile-Glu-Ala-Arg-p-nitroanilide was also
evaluated.
A substrate phage display library that encodes an altered pIII
containing at its N-terminus the FLAG peptide followed by an
eight-residue hypervariable peptide was screened with recombinant
mMCP-6 in the presence or the absence of heparin glycosaminoglycan to
deduce which amino acid sequences are preferred for cleavage and
whether heparin influences the substrate specificity of this tryptase.
To prepare the phage column used in the screening process, 10 ml of the
phage-enriched supernatant was added to 2 ml of 20% polyethylene
glycol (8 kDa; Sigma) and 2.5 M NaCl, and the mixture was incubated at
4°C for 30 min. After a 30-min centrifugation at 10,000 x
g, the recombinant phage in the pellet were resuspended in 2
ml of 150 mM NaCl, 1 mM CaCl2, and 10 mM sodium phosphate,
pH 7.0, and applied to a 1-ml affinity column containing the
anti-FLAG M1 mAb. The column was washed three times with 10 ml of
the same pH 7.0 buffer to remove unbound phage. EK-activated mMCP-6
(50 µg in 200 µl buffer) in the absence or the presence of
heparin glycosaminoglycan (50 µg) was added, and the column was
sealed and incubated at room temperature for 90 min. After protease
treatment, the column was washed with 2 ml of the pH 7.0 buffer to
recover those phage that possessed protease-susceptible pIII fusion
proteins. Log-phase Escherichia coli were infected with the
obtained phage to produce phagemid. Bacteria were again grown in 2x YT
medium containing 2% glucose, and the phagemid in the bacteria were
converted to phage with the addition of helper phage. This screening
procedure was repeated up to four times to select those phage in the
library whose pIII fusion proteins were most susceptible to degradation
by mMCP-6. After cloning of the isolated phage, the nucleotide sequence
that encodes the 8-mer, protease-susceptible peptide domains in the
fusion protein of each isolated clone was then determined.
Mouse MCP-6-induced, neutrophil-specific peritonitis
Samples of purified mMCP-7 (2000 U), mMCP-6 (800 U), or
pro-EK-mMCP-6 (amount equivalent to that of mature mMCP-6 on a weight
basis) were injected separately into the peritoneal cavity of BALB/c,
C5-deficient (B10.D2-H2dTlac Hc0/oSn)
(26), mast cell-deficient
WCB6F1-Sl/Sld (also known as
WCBF1-MgfSl/MgfSl-d),
and mMCP-5-null (27) mice. All mice, except mMCP-5-null mice, were
obtained from The Jackson Laboratory (Bar Harbor, ME). Because mMCP-6
is bound to heparin-containing serglycin proteoglycans in vivo (1, 19)
and because analysis of a tryptase-specific, phage display library (see
below) revealed that heparin restricts the substrate specificity of
mMCP-6, activated mMCP-6 was allowed to form a 1/1 to 2/1 (w/w)
macromolecular complex with heparin before being injected into the
peritoneal cavity. Although heparin does not appear to influence the
substrate specificity of mMCP-7 at neutral pH (6), this
glycosaminoglycan also was present as a control when no mMCP,
pro-EK-mMCP-6, or mMCP-7 was injected into the peritoneal cavity of
separate mice. At specific time points (e.g., 36 h) after mMCP
treatment, the mouse was killed by CO2 asphyxiation, and 10
ml of Tyrode’s buffer containing 0.1% (w/v) gelatin was injected into
its peritoneum. The injected fluid was aspirated 30 s later. The
cells in the peritoneal lavage fluid were counted with a hemocytometer
and then were subjected to a 5-min centrifugation at 28 x
g onto glass slides with a Cytospin 2 cytocentrifuge
(Shandon, Pittsburgh, PA). The resulting slides were stained with
Diff-Quik (Baxter Healthcare Corp., McGaw Park, IL). In one experiment,
the peritoneal membrane from a BALB/c mouse exposed to mMCP-6 for
36 h was removed, spread on a glass slide, and stained with
methylene blue. The number of membrane-bound neutrophils was compared
with that of a peritoneal membrane isolated from a control BALB/c mouse
exposed to medium alone.
In vitro neutrophil chemotaxis assay, and evaluation of cytokine and chemokine expression by human endothelial cells exposed to mMCP-6
ChemoTx microplates (Neuro Probe, Cabin John, MD) containing membranes with 8-µm standard pore sizes were used to determine whether mMCP-6 is a chemotactic factor that can act directly or indirectly on mouse peripheral blood neutrophils. In the first experimental protocol, 29-µl portions of DMEM containing 2% FCS with or without FMLP (5.4 µM; Sigma), mature mMCP-7 (200 U), mature mMCP-6 (90 U), or pro-EK-mMCP-6 (amount equivalent to that of mature mMCP-6 on a weight basis) were added to separate wells in the bottom chambers of the assay plate. In the second experimental protocol, HUVEC (Clonetics, San Diego, CA) were placed in the bottom chambers of the assay plate 24 h before the addition of the test agent to confluent monolayers. Because FMLP is a strong chemoattractant for neutrophils (28), this three-residue peptide was used as a positive control in these in vitro experiments. Peripheral blood was collected from the retro-orbital plexus of BALB/c mice into pipettes containing EDTA anticoagulant, and the neutrophils were enriched to a purity of >75% by gradient centrifugation technology with the NIM-2 leukocyte isolation kit from Cardinal Associates (Santa Fe, NM). Samples of DMEM (25 µl) containing 2% FCS and a constant number of freshly isolated neutrophils (3,000–10,000 cells depending on the experiment) were then placed into each top well of the assay plate. After a 30-min incubation at 37°C, the membrane separating the two chambers was removed and placed in Diff-Quik stain, and the neutrophils that had migrated through it were counted.
ELISA kits from Endogen (Cambridge, MA) were used as described by the
manufacturer to quantitate the amounts of human TNF-, IL-1, IL-6,
and IL-8 in samples (1–10 µl) of the conditioned medium of replicate
endothelial cell cultures exposed for various periods of time to no
mMCP, mature mMCP-7 (200 U), mature mMCP-6 (90 U), or pro-EK-mMCP-6
(amount equivalent to that of mature mMCP-6 on a weight basis). Total
RNA was isolated according to the method of Chomczynski and Sacchi (29)
from endothelial cells exposed for 2 h to no mMCP, mMCP-6,
pro-EK-mMCP-6, or mMCP-7. The RNA samples were denatured in
formaldehyde/formamide, electrophoresed in 1.3% formaldehyde-agarose
gels, and transferred to nylon membranes (Micron Separations, Inc.,
Westboro, MA). The resulting blots were incubated in QuickHyb buffer
(Stratagene Cloning Systems, La Jolla, CA) containing a radiolabeled,
gene-specific probe for either IL-8 (30) or ß-actin (31). After the
RNA blots were washed at 60°C in 0.1x SSC buffer and 0.1% SDS, they
were analyzed by autoradiography. Reverse transcriptase (RT)-PCR also
was performed with assay kits from BioSource International (Camarillo,
CA) to evaluate the presence of IL-8, RANTES, and TNF- transcripts
in human endothelial cells that had been exposed to mMCP-6 for 1, 2, or
3 h. The IL-8-, RANTES-, and TNF--specific primers used in
these assays will result in the generation of 227-, 197-, and 342-bp
DNA fragments if the respective chemokine/cytokine transcripts are
present in a cell lysate.
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Results |
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Insect cells infected with the relevant construct secreted forms
of pro-mMCP-6 and pro-EK-mMCP-6 (Fig. 1
)
into the conditioned medium that could be purified by heparin-Sepharose
chromatography (Fig. 2). Like pro-mMCP-6,
pro-EK-mMCP-6 bound to a heparin-Sepharose column that had been
equilibrated in 100 mM NaCl/10 mM sodium phosphate, pH 5.5. Because
both recombinant proteins dissociated from the heparin-Sepharose column
when the NaCl concentration of the buffer was raised to 1.4 M, it was
concluded that most of the pro-mMCP-6 and pro-EK-mMCP-6 secreted by the
insect cells into the conditioned medium were properly folded.
Pro-EK-mMCP-6 also could be purified with the anti-FLAG
immunoaffinity column (Fig. 3A).
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2 kDa when the mMCP-6 pseudozymogen was incubated for
3 h with EK (data not shown). Even if exposed to EK at pH 6.0 for
up to 24 h, the recombinant protein did not undergo substantial
autolysis. Amino acid sequence analysis revealed that the resulting
product possessed an N-terminal sequence of
Ile-Val-Gly-Gly-His-Glu-Ala-Ser-Glu, which is identical with that of
mature, native mMCP-6 (1, 2). Mouse MCP-6 exhibited good enzymatic
activity after a 2-h incubation with EK (Fig. 3
B) and
optimal enzymatic activity at pH 7.2 to 7.6 (data not shown), and
readily cleaved tosyl-Gly-Pro-Lys-p-nitroanilide and
tosyl-Gly-Pro-Arg-p-nitroanilide in the absence of heparin
glycosaminoglycan. However, in the absence or the presence of heparin,
mMCP-6 did not effectively cleave
benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide,
benzoyl-Pro-Phe-Arg-p-nitroanilide, or
acetyl-Ile-Glu-Ala-Arg-p-nitroanilide. When a
tryptase-specific, phage display library was subjected to four rounds
of treatment with enzymatically active mMCP-6 in the absence of heparin
glycosaminoglycan, no specific peptide sequence in the hypervariable
domain of the pIII fusion protein was obtained in the 30 arbitrarily
selected clones (Table I). The two most
prominent features of the peptides obtained by screening the library
with mMCP-6 alone were the over- and under-representation of positively
and negatively charged residues, respectively. One-half of the selected
clones had three or more Lys and/or Arg residues in the susceptible
peptide, and two of the clones actually had five positively charged
residues. When the same library was subjected to two to four rounds of
treatment with mMCP-6 in the presence of an equal amount of heparin
glycosaminoglycan, a much more limited number of sequences was obtained
(Table II).
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Mouse MCP-6-induced, neutrophil-specific peritonitis
Neutrophils represented only 1 ± 1% (mean ± SD;
n = 3) of the 1.0 ± 0.2 x 107
cells in the peritoneal lavage fluid of untreated BALB/c mice.
Thirty-six hours after 800 U of enzymatically active mMCP-6 (an amount
that corresponds to 2 µg or 0.6 nM trypsin) was injected i.p. into
BALB/c mice, the number of cells in the exudates was comparable to
those in untreated, mMCP-7-treated, and pro-EK-mMCP-6-treated BALB/c
mice. Nevertheless, neutrophils now represented 54 ± 9%
(mean ± SD; n = 6) of the total cells in the
exudates of the mMCP-6-treated mice. Thus, mMCP-6 induced a significant
(p < 0.001) >50-fold increase in the number
of neutrophils in the peritoneal cavity of this mouse strain. Not
buffer alone, pro-EK-mMCP-6, or mature mMCP-7 could induce substantial
numbers of neutrophils to accumulate in the peritoneal cavity of a
BALB/c mouse. Neutrophils represented only 1 ± 1, 6 ± 2,
and 4 ± 4% (mean ± SD or 1/2 range;
n = 2 or 3) of the total cells in the peritoneal cavity
exudates of BALB/c mice exposed for 36 h to medium alone, medium
containing pro-EK-mMCP-6, and medium containing mMCP-7, respectively.
The mMCP-6-induced neutrophil accumulation in BALB/c mice was prominent
after 7 h, was maximal after 1 day, and persisted for at least 2
days (Fig. 4). As shown in Figure 5, the peritoneal membrane of a BALB/c
mouse examined 36 h after mMCP-6 treatment also contained 58-fold
more neutrophils than that of the control mouse. When three other mouse
strains were examined 36 h after 800 U of mMCP-6 was injected once
into the peritoneal cavity of each animal, neutrophils represented
64 ± 1, 50 ± 2, and 50 ± 17% (mean ±
1/2 range), respectively, of the cells in the peritoneal
exudates of mMCP-5-null (n = 2), C5-deficient
(n = 2), and mast cell-deficient
WCB6F1-Sl/Sld
(n = 2) mice. At no time were large numbers of
eosinophils, basophils, or platelets detected in the peritoneal exudate
of any mMCP-6-treated mouse. Mast cells are normal resident cells of
the peritoneal cavity, and their numbers in the lavage fluids were
similar in all treated mice.
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Freshly isolated mouse peripheral blood neutrophils could not be induced to migrate within 30 min through a membrane with 8-µm pores when they were exposed briefly to buffer, mMCP-6, mMCP-7, endothelial cells, or the combination of endothelial cells and mMCP-7. However, migration through the membrane did occur when neutrophils were exposed to FMLP or were concomitantly exposed to mMCP-6 and human endothelial cells. In two experiments, 75- and 100-fold more neutrophils were induced to migrate through the membrane when the endothelial cells in the bottom well were exposed to enzymatically active mMCP-6 than when they were exposed to the inactive zymogen.
As assessed by ELISA, the levels of TNF-, IL-1, and IL-6 protein
produced by the mMCP-6-treated human endothelial cells all were below
the limit of detection. However, mMCP-6-treated cells dramatically
increased their expression of immunoreactive IL-8 in a dose-dependent
manner (Fig. 6A). To
up-regulate their expression of this chemokine, the endothelial cells
only had to be exposed to mMCP-6 for 20 to 50 min (Fig. 6B). Moreover, the endothelial cells continued to
produce large amounts of immunoreactive IL-8 40 h after the
initial 20- to 50-min exposure to mMCP-6. Neither pro-EK-mMCP-6 nor
mMCP-7 was able to induce these endothelial cells to increase their
expression of the neutrophil chemotactic factor (Fig. 6B). No RANTES- or TNF--specific DNA fragments
were generated from mMCP-6-treated endothelial cells even when the RNA
samples were reverse transcribed and subjected to 35 cycles of PCR. In
contrast, the IL-8-specific, 227-bp DNA fragment was readily generated
when the RT-PCR assay was performed on endothelial cells that had been
exposed to mMCP-6 for 1 (n = 1), 2
(n = 2), or 3 (n = 1) h
(data not shown). Furthermore, as assessed by RNA blot analysis (Fig. 6C), the level of IL-8 mRNA in mMCP-6-treated cells
was considerably higher than that in replicate cells exposed to medium
alone, pro-EK-mMCP-6, or mature mMCP-7.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Insect cells infected with the relevant construct secreted
pro-EK-mMCP-6 and pro-mMCP-6 (Fig. 1) into the conditioned medium. As
assessed by heparin-Sepharose chromatography (Fig. 2), most of the
mMCP-6 pseudozymogen secreted into the conditioned medium by the insect
cell is properly folded. Although recombinant (6) and native (19)
mMCP-7 exhibit good catalytic activity in the absence of heparin, it
has been reported that a human mast cell tryptase purified from the
lung does not exhibit substantial enzymatic activity unless this
glycosaminoglycan is present in the assay (32, 33). The ability to
purify pro-EK-mMCP-6 from the conditioned medium by means of the
immunoaffinity column (Fig. 3) allowed us to determine whether the
mouse tryptase exhibits enzymatic activity in the absence of heparin.
The finding that the EK-activated tryptase readily cleaved
tosyl-Gly-Pro-Lys-p-nitroanilide and
tosyl-Gly-Pro-Arg-p-nitroanilide in the absence of heparin
indicates that the broad catalytic activity of this tryptase is not
dependent on heparin-containing serglycin proteoglycans.
The observation that recombinant mMCP-6 cleaves
acetyl-Ile-Glu-Ala-Arg-p-nitroanilide less efficiently than
recombinant mMCP-7 in vitro suggested that the two mouse tryptases
might have different substrate specificities even though their overall
amino acid sequences are 71% identical. Thus, the substrate phage
display library that helped us identify a physiologic substrate of
mMCP-7 (6) was used to identify mMCP-6-preferred peptide substrates.
When the library was subjected to four rounds of treatment with
enzymatically active mMCP-6 in the absence of heparin
glycosaminoglycan, no specific peptide sequence in the hypervariable
domain of the pIII fusion protein was obtained in the 30 arbitrarily
selected clones (Table I). Nevertheless, the observation that only one
of these mMCP-6-susceptible clones had the preferred mMCP-7-susceptible
sequence in its pIII fusion protein was further evidence that the two
homologous tryptases degrade very different substrates. The two most
prominent features of the peptides obtained by screening the library
with mMCP-6 alone were the over- and under-representation of positively
and negatively charged residues, respectively. These findings are
consistent with the electrostatic properties of mMCP-6, which predicted
that the substrate-binding pocket of mMCP-6 is more negatively charged
than that in mMCP-7 (19). The difference in the electrostatic potential
of the pocket is due primarily to loop 3 which has a -3 net charge in
mMCP-6 and a 0 net charge in mMCP-7.
When the substrate phage display library was screened with a
mMCP-6/heparin binary complex, a more limited number of sequences was
obtained (Table II). Surprisingly, the two clones that were obtained
repeatedly had dissimilar sequences of Arg-Asn-Arg-Gln-Lys-Thr-Asn-Asn
and Thr-Pro-Leu-Leu-Lys-Ser-Trp-Leu. The latter favored peptide and the
other less favored peptides obtained in the selection process with the
mMCP-6/heparin complex were similar in that each had a Pro residue, at
least one Thr or Ser residue, and a net charge of only +1 or +2. The
discovery that the favored peptide in this series had a Pro residue at
its P4 site is of interest because Cromlish and co-workers (34) noted
that a human mast cell tryptase purified from the pituitary will cleave
three prohormones ex vivo that have Pro residues at their P4 sites and
Lys/Arg residues at their P1 sites.
Despite these findings, the other favored peptide from the substrate phage display library that possesses a +3 charge might be more physiologically relevant because its overall charge is more similar to that obtained when the library was screened with mMCP-6 alone. Why only one peptide with a +3 charge was obtained and why this peptide was not present in the original 30 clones isolated when the library was screened with just mMCP-6 remains to be determined experimentally. However, the electrostatic potential of the three-dimensional model of mMCP-6 suggests that the putative heparin binding domain on the surface of this tryptase resides closer to its active site than in all other mMCPs. Thus, it is likely that heparin glycosaminoglycan sterically restricts the substrate binding cleft of mMCP-6 by directly influencing one or more of the seven loops that form the pocket. The discovery that the substrate specificity of a rat mast cell chymase is also altered by heparin glycosaminoglycan (35) emphasizes the importance of the glycosaminoglycan side chains of serglycin proteoglycans in fine-tuning the substrate specificities of certain members of the chromosome 14 and chromosome 17 families of serine proteases. Although it remains to be determined which proteins are the physiologic substrates of mMCP-6, the phage display library data document that mMCP-6 and mMCP-7 have different substrate specificities. Because analysis of the substrate phage display library revealed that heparin can influence the substrate specificity of mMCP-6, the biologic activity of mMCP-6 in most experiments was evaluated in the presence of heparin. Like mMCP-7, mMCP-6 exhibited optimal enzymatic activity at approximately pH 7.4.
We previously noted that mMCP-7 is resistant to inactivation by the
diverse array of protease inhibitors in the blood of the mouse (19).
The finding that the enzymatic activity of mMCP-6 was inhibited
completely after a 30-min incubation at 37°C or a 1.5-h incubation at
room temperature in the presence of 50% mouse serum confirms other
data indicating that mMCP-6 and mMCP-7 are functionally different
enzymes. This finding also suggests that even if some exocytosed mMCP-6
is able to leave an inflamed tissue site, it probably would not stay
enzymatically active for long in the circulation. Although it remains
to be determined what are the inhibitors in mouse serum that
selectively inactivate mMCP-6, the rat homologue of mMCP-6 readily
binds the inter--trypsin inhibitor (36, 37) and 1m
(38) families of protease inhibitors.
The mast cells that reside in the peritoneal cavity of the BALB/c mouse
express mMCP-6, but not mMCP-7 (9). Because this observation suggested
that mMCP-6 cleaves specific proteins that reside in the peritoneal
cavity or its membrane, enzymatically active mMCP-6 was injected into
the peritoneal cavity of the BALB/c mouse to assess whether the
recombinant tryptase could induce a detectable biologic response in
vivo. Seven hours after mMCP-6 administration, a pronounced
accumulation of neutrophils occurred in the peritoneal cavity of this
mouse strain, as typically seen in acute inflammatory responses (39).
However, unlike inflammatory responses induced by LPS, the
mMCP-6-induced accumulation of neutrophils persisted for >2 days (Fig. 4). Thus, mMCP-6 induced a selective and persistent accumulation of
neutrophils. The fact that pro-EK-mMCP-6 could not induce the
peritonitis indicates that the inflammatory reaction is dependent on
enzymatically active mMCP-6. The finding that very few neutrophils
extravasated into the peritoneal cavity of BALB/c mice exposed to
2.5-fold more mMCP-7 than mMCP-6 documents the specificity of the
tryptase effect. The reason that large numbers of lymphocytes,
monocytes, and/or eosinophils do not extravasate into the peritoneal
cavity of mMCP-6-treated mice could be the proteolytic inactivation of
the chemokines that recruit these three populations of cells.
Alternatively, mMCP-6 could induce the local expression of a factor
that selectively attracts neutrophils or a factor (e.g., a binding
protein) that selectively inactivates all chemoattractants except those
that recruit neutrophils.
Neutrophils generally undergo apoptosis within 24 h after they extravasate into an inflammatory site (39). The presence of large numbers of neutrophils in the peritoneal cavity 2 days after a single injection of mMCP-6 probably is due to the persistent release of neutrophil-specific chemotatic factors from endothelial cells in the blood vessels lining the peritoneal membrane, from the mesothelial cells in the peritoneal cavity, or from the resident macrophages in the peritoneal cavity. However, it is also possible that the extravasated neutrophils persist in the mMCP-6-treated mice in part because their apoptosis is delayed. Coxon and co-workers (40) recently demonstrated that the neutrophils that extravasate into the peritoneal cavity of thioglycolate-treated CD11b-null mice do not undergo rapid apoptosis. Using Ab specific for ß2 integrins, these investigators demonstrated that phagocytosis and generation of an oxidative burst through the CD11b/CD18 integrin is needed for the efficient induction of apoptosis of this leukocyte. In our model, the neutrophils that extravasated into the peritoneal cavity of mMCP-6-treated mice might be longer lived because of their failure to come in contact with opsonized particles that bind to the CD11b/CD18 integrin on the surface of the neutrophil.
Mouse MCP-6 induced similar numbers of neutrophils to extravasate into the peritoneal cavity of BALB/c, mMCP-5-null, C5-deficient, and mast cell-deficient WCB6F1-Sl/Sld mice. Certain populations of mouse mast cells activated through their high affinity Fc receptors for IgE express leukotriene B4 (41), platelet-activating factor (42), and a large number of cytokines and chemokines (43, 44, 45, 46, 47). That mMCP-6, by itself, can induce neutrophil extravasation into the peritoneum of a mast cell-deficient mouse strain indicates that the tryptase does not have to induce resident mast cells to release neutrophil-specific chemoattractants. C5a anaphylatoxin is a potent neutrophil chemotactic factor (48) that is normally generated by a tryptic cleavage at Arg751 of its 190-kDa precursor protein by C5 convertase. The finding that neutrophils also accumulate in the peritoneal cavity of the mMCP-6-treated, C5-deficient mouse indicates that mMCP-6 is not functioning primarily as a C5 convertase.
In vitro studies revealed that in the absence of endothelial cells, mMCP-6 was unable to induce freshly isolated mouse neutrophils to migrate within 30 min through a membrane with 8-µm pores. This observation coupled with the findings that mMCP-6 is inactivated by factors in the blood and that very little free or complexed mMCP-6 can be found in the blood of mice undergoing systemic anaphylaxis (19) suggest that the mMCP-6-induced extravasation of neutrophils into the peritoneal cavity of the BALB/c mouse is not primarily a consequence of the tryptase acting directly on the circulating neutrophil. By themselves, the endothelial cells used in the in vitro assay did not induce neutrophil chemotaxis. However, chemotaxis did occur when neutrophils were concomitantly exposed in vitro to mMCP-6 and endothelial cells.
Generally, neutrophil extravasation into tissues is dependent on the
CXC family of chemokines (30, 49, 50, 51, 52). While IL-8/NAP-1 is the most
potent neutrophil chemoattractant in humans, other CXC chemokines
(e.g., neutrophil-activating peptide-2, epithelial cell-derived
neutrophil-activating peptide-78, and growth-related oncogene , ß,
and 
) also are potent chemoattractants for human neutrophils. One
human chemokine receptor is relatively IL-8 specific, whereas the other
recognizes numerous members of the IL-8 chemokine family (53). The
precise number of IL-8-like chemokines in the mouse has not been
determined, but there appears to be only one IL-8-like receptor in this
species, and the acute migration of neutrophils into the
thioglycolate-treated peritoneum is severely compromised in transgenic
mice lacking this chemokine receptor (54).
Because mouse IL-8 has not been cloned and because ELISAs to this and
related mouse chemokines are not currently available, we evaluated the
ability of mMCP-6 to induce human endothelial cells to increase their
expression of an IL-8-like chemokine that would interact with the only
receptor on the surface of the mouse neutrophil that reacts with this
family of chemokines. As assessed by ELISA and/or RT-PCR,
mMCP-6-treated endothelial cells did not produce TNF-, RANTES,
IL-1, or IL-6. Nevertheless, these treated cells dramatically
increased their expression of IL-8 protein and mRNA (Fig. 6). Cairns
and Walls (55) noted that a mast cell tryptase preparation isolated
from human lung tissue also can induce a cultured human epithelial cell
line to increase its expression of IL-8. Four cDNAs have been isolated
by two groups (13, 14, 15) that encode highly homologous human mast cell
tryptases (designated tryptases I, II/ß, III, and ). While it
remains to be determined whether these human tryptases are derived from
distinct genes or are allelic variants of a smaller number of genes,
two distinct mast cell tryptase genes exist in varied inbred mouse
strains (2, 3, 7, 8, 9). Even though the amino acid sequences of mMCP-6
and mMCP-7 are 71% identical, mMCP-7 was unable to induce neutrophil
extravasation and was unable to induce human endothelial cells to
produce IL-8. Because mMCP-6 and mMCP-7 often are coexpressed in the
same mast cell in many tissues (2, 3, 9, 10, 19), caution now must be
exercised in drawing conclusions about the function of a mast cell
tryptase that has not been evaluated in its recombinant form.
Mouse MCP-6 could cleave receptors on the plasma membrane of the
endothelial cell (e.g., the thrombin receptor) (56) or
receptor-associated proteins in the matrix (e.g., fibronectin) (57),
thereby inducing a signal transduction cascade that ultimately results
in the increased transcription of the IL-8 gene or the increased
stabilization of its transcript. Because human IL-8 often is activated
after a tryptic-like cleavage of its precursor at Arg28,
mMCP-6 also could act directly on the precursor of this chemokine.
Whatever the mechanism(s), the finding that levels of IL-8 are
increased in the lungs of patients with asthma and other lung disorders
(58, 59, 60, 61) now raises the possibility that the level of this chemokine in
certain allergic situations is regulated in part by the human homologue
of mMCP-6. Because mMCP-6 induces neutrophil extravasation and because
mMCP-7 inhibits the formation of fibrin/platelet clots, these two
homologous tryptases appear to have evolved in the mouse to work in
concert with mast cell-derived TNF- (62) and leukotriene
B4 (41) to facilitate the influx of neutrophils into
inflammatory sites.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard L. Stevens, Brigham and Women’s Hospital, Department of Medicine, Smith Building, Room 616, 75 Francis St., Boston, MA 02115. E-mail address:
3 Abbreviations used in this paper: mMCP, mouse mast cell protease; EK, enterokinase; FLAG, the peptide whose amino acid sequence is Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys; TBST buffer, Tris-buffered saline containing 0.01% Tween-20; RT, reverse transcriptase.
Received for publication June 27, 1997. Accepted for publication October 29, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ali, W.-T. Qiu Matsumoto, R. L. Stevens. 1997. The tryptase, mouse mast cell protease 7, exhibits anticoagulant activity in vivo and in vitro due to its ability to degrade fibrinogen in the presence of the diverse array of protease inhibitors in plasma. J. Biol. Chem. 272:31885.ali, M. F. Gurish. 1996. Fate of two mast cell tryptases in V3 mastocytosis and normal BALB/c mice undergoing passive systemic anaphylaxis: prolonged retention of exocytosed mMCP-6 in connective tissues and rapid accumulation of enzymatically active mMCP-7 in the blood. J. Exp. Med. 184:1061.-trypsin inhibitor light chain. J. Biol. Chem. 269:3818.1-macroglobulin in vivo: mast cell tryptase is a major component of the 1-macroglobulin-proteinase complex endocytosed into rat liver lysosomes. Biochem. J. 298:79.
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, GROß, and GRO activate human neutrophil and basophil leukocytes. J. Biol. Chem. 268:15419.This article has been cited by other articles:
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R. I. Zuberi, J. R. Apgar, S.-S. Chen, and F.-T. Liu Role for IgE in Airway Secretions: IgE Immune Complexes Are More Potent Inducers Than Antigen Alone of Airway Inflammation in a Murine Model J. Immunol., March 1, 2000; 164(5): 2667 - 2673. [Abstract] [Full Text] [PDF]
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C. Huang, G. Morales, A. Vagi, K. Chanasyk, M. Ferrazzi, C. Burklow, W.-T. Qiu, E. Feyfant, A. Sali, and R. L. Stevens Formation of Enzymatically Active, Homotypic, and Heterotypic Tetramers of Mouse Mast Cell Tryptases. DEPENDENCE ON A CONSERVED Trp-RICH DOMAIN ON THE SURFACE J. Biol. Chem., January 7, 2000; 275(1): 351 - 358. [Abstract] [Full Text] [PDF]
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E. Razin, Z. C. Zhang, H. Nechushtan, S. Frenkel, Y.-N. Lee, R. Arudchandran, and J. Rivera Suppression of Microphthalmia Transcriptional Activity by Its Association with Protein Kinase C-interacting Protein 1 in Mast Cells J. Biol. Chem., November 26, 1999; 274(48): 34272 - 34276. [Abstract] [Full Text] [PDF]
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A. D. Befus, C. Mowat, M. Gilchrist, J. Hu, S. Solomon, and A. Bateman Neutrophil Defensins Induce Histamine Secretion from Mast Cells: Mechanisms of Action J. Immunol., July 15, 1999; 163(2): 947 - 953. [Abstract] [Full Text] [PDF]
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C. Huang, L. Li, S. A. Krilis, K. Chanasyk, Y. Tang, Z. Li, J. E. Hunt, and R. L. Stevens Human Tryptases alpha and beta /II Are Functionally Distinct Due, in Part, to a Single Amino Acid Difference in One of the Surface Loops That Forms the Substrate-binding Cleft J. Biol. Chem., July 9, 1999; 274(28): 19670 - 19676. [Abstract] [Full Text] [PDF]
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 K. P. Valchanov and G. B. Proctor Enzyme Histochemistry of Tryptase in Stomach Mucosal Mast Cells of the Mouse J. Histochem. Cytochem., May 1, 1999; 47(5): 617 - 622. [Abstract] [Full Text]
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L. Li, Y. Li, S. W. Reddel, M. Cherrian, D. S. Friend, R. L. Stevens, and S. A. Krilis Identification of Basophilic Cells that Express Mast Cell Granule Proteases in the Peripheral Blood of Asthma, Allergy, and Drug-Reactive Patients J. Immunol., November 1, 1998; 161(9): 5079 - 5086. [Abstract] [Full Text] [PDF]
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D. S. Friend, N. Ghildyal, M. F. Gurish, J. Hunt, X. Hu, K. F. Austen, and R. L. Stevens Reversible Expression of Tryptases and Chymases in the Jejunal Mast Cells of Mice Infected with Trichinella spiralis J. Immunol., June 1, 1998; 160(11): 5537 - 5545. [Abstract] [Full Text] [PDF]
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E. Tchougounova, E. Forsberg, G. Angelborg, L. Kjellen, and G. Pejler Altered Processing of Fibronectin in Mice Lacking Heparin. A ROLE FOR HEPARIN-DEPENDENT MAST CELL CHYMASE IN FIBRONECTIN DEGRADATION J. Biol. Chem., February 2, 2001; 276(6): 3772 - 3777. [Abstract] [Full Text] [PDF]
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G. W. Wong, L. Li, M. S. Madhusudhan, S. A. Krilis, M. F. Gurish, M. E. Rothenberg, A. Sali, and R. L. Stevens Tryptase 4, a New Member of the Chromosome 17 Family of Mouse Serine Proteases J. Biol. Chem., June 1, 2001; 276(23): 20648 - 20658. [Abstract] [Full Text] [PDF]
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C. Huang, G. T. De Sanctis, P. J. O'Brien, J. P. Mizgerd, D. S. Friend, J. M. Drazen, L. F. Brass, and R. L. Stevens Evaluation of the Substrate Specificity of Human Mast Cell Tryptase beta I and Demonstration of Its Importance in Bacterial Infections of the Lung J. Biol. Chem., July 6, 2001; 276(28): 26276 - 26284. [Abstract] [Full Text] [PDF]
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G. W. Wong, S. Yasuda, M. S. Madhusudhan, L. Li, Y. Yang, S. A. Krilis, A. Sali, and R. L. Stevens Human Tryptase epsilon (PRSS22), a New Member of the Chromosome 16p13.3 Family of Human Serine Proteases Expressed in Airway Epithelial Cells J. Biol. Chem., December 28, 2001; 276(52): 49169 - 49182. [Abstract] [Full Text] [PDF]
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C. Levy, H. Nechushtan, and E. Razin A New Role for the STAT3 Inhibitor, PIAS3. A REPRESSOR OF MICROPHTHALMIA TRANSCRIPTION FACTOR J. Biol. Chem., January 11, 2002; 277(3): 1962 - 1966. [Abstract] [Full Text] [PDF]
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) or pro-EK-mMCP-6 (•) were applied to
replicate 18-ml heparin-Sepharose columns. The columns were washed, and
the bound proteins were eluted with a salt gradient. Depicted is the
OD280 of the eluate 5-ml fractions of each column. The
inset (top right) depicts the
SDS-PAGE-immunoblot analysis obtained with anti-mMCP-6 Ig on
samples (20 µl) of fractions 15 to 20 of the gradient eluate of the
pro-EK-mMCP-6-treated column. Similar findings were obtained in two
other experiments with pro-mMCP-6.
), lymphocytes (
), macrophages (
), mast cells
(•), and eosinophils (*). In each mouse, >300 cells in the
peritoneal lavage fluid were examined. The depicted data are the mean
of the results (±range) obtained from the two animals at each time
point, and from three untreated mice (time zero). As a control, two
additional BALB/c mice were given medium alone. When these control mice
were examined 36 h later, neutrophils, lymphocytes, macrophages,
mast cells, and eosinophils represented 1 ± 1, 59 ± 6,
36 ± 6, 3 ± 1, and 2 ± 1%, respectively, of the
cells in their peritoneal lavage fluid.
