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Pulmonary and Critical Care Medicine Division, Departments of Internal Medicine and
Pathology, Ann Arbor Veterans Affairs Medical Center and University of Michigan Medical Center, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12. uPA-/- mice had
decreased levels of IFN- and IL-12, and increased IL-5, a type 2
cytokine. In the absence of uPA, the cytokine profile of regional lymph
nodes shifted from a T1 pattern characterized by IFN- and IL-2 to a
weak, nonpolarized response. We conclude that in the absence of uPA,
lymphocyte proliferative responses are diminished, and mice fail to
generate protective T1 cytokines, resulting in impaired antimicrobial
activity. This study provides novel evidence that uPA is a critical
modulator of immune responses and of immune cell effector functions in
vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both mononuclear phagocytes (macrophages (M
)) and neutrophils
express uPA constitutively, and substantially up-regulate uPA
expression in response to activating stimuli. By contrast, only
activated lymphocytes express uPA (7, 10, 11). Thus, in
lymphocytes uPA is a strict activation Ag, which suggests that uPA is
intrinsically involved in the process(es) by which lymphocytes become
activated.
In vivo investigation of the role uPA plays in pulmonary host defenses has previously been limited by the inability to completely and irreversibly eliminate uPA. This limitation has been overcome by the development of transgenic mice lacking the uPA gene (12). To determine whether uPA is required for effective pulmonary immune responses, we have established a murine model of pulmonary Cryptococcus neoformans infection (13). This model is well suited for the study of immune competency for several reasons. First, C. neoformans rarely causes disease in the immunocompetent, but is an important lethal fungal pathogen in immunocompromised patients and therefore has clinical relevance; second, C. neoformans strain 52D is not lethal in immunocompetent resistant mice but disseminates and causes death by meningitis when mice are immunocompromised (14, 15), paralleling the pattern of host susceptibility seen clinically; and third, host defense against C. neoformans is dependent on intact cell-mediated immunity (16).
We have previously demonstrated that uPA is required for protective pulmonary defenses against C. neoformans. In the absence of uPA, recruitment of inflammatory and immune cells in response to pulmonary C. neoformans infection is markedly diminished and the infection is not adequately combated; fungal CFUs in the lungs increase unchecked, and the infection disseminates to spleen and brain. The uPA-/- animals do not survive, and they die of fungal meningitis by day 85 post-intratracheal (IT) inoculation. In contrast, the wild type (WT) survive and clear the infection completely (17).
The requirement for intact cell-mediated immunity against C.
neoformans is demonstrated clinically in HIV-infected patients
whose incidence of C. neoformans infection increases
dramatically as they become CD4 lymphocyte deficient (16).
Similarly, athymic nude mice and mice that are CD4 lymphocyte depleted
have markedly increased susceptibility to C. neoformans
infections (14, 15). More recent work has shown that
several specific cytokines are of particular importance in protective
host defenses against C. neoformans. IFN- activates
alveolar macrophages, increasing uptake of the fungus and inhibition of
its growth in vitro, and in vivo, anti-IFN- Abs blocked lung
clearance of C. neoformans in immunocompetent mice
(18, 19). IL-2 has been shown to increase anticryptococcal
activity of PBMCs (20). Collectively, these data have led
to an appreciation of a requirement for the generation of a type 1 (T1)
profile of cytokines for protective host defense against C.
neoformans, characterized by the above-mentioned cytokines in
addition to IL-12 (21).
uPA-deficient transgenic mice are not CD4 lymphocyte depleted and actually have increased numbers of pulmonary CD4 lymphocytes at baseline compared with WT mice (17). Despite this, uPA-/- mice lack protective host defense against C. neoformans and behave like lymphocyte-depleted animals. This raises the possibility that the immune defect in the uPA-/- mice in response to pulmonary C. neoformans infection is not due only to a defect in cell number but also to a defect in cellular effector functions. In this study, we sought to determine whether the presence of uPA is required for the generation of a protective T1 profile of inflammatory cytokines in response to pulmonary C. neoformans infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice were housed in specific pathogen-free isolation rooms in the University of Michigan Department of Laboratory Animal Medicine (Ann Arbor, MI), which is fully accredited by the American Association for Accreditation of Laboratory Animal Care. This study was approved by the University of Michigan Committee on Use and Care of Animals. Mice were periodically checked for murine hepatitis virus and were found to be negative, and were fed standard animal chow (rodent lab chow 5008; Purina, St. Louis, MO) and chlorinated tap water ad libitum. Mice were used at 8–12 wk of age.
Transgenic uPA-depleted mice (uPA-/-) and background-matched control mice uPA+/+ (WT) were generous gifts from P. Carmeliet (Center for Transgene Technology and Gene Therapy, Leuven, Belgium) (12). The uPA gene was knocked out by homologous recombination with the uPA/neomycin construct in embryonic stem cells derived from strain 129 mice. The embryonic stem cells were injected into C57/B6 blastocysts, and the resulting chimeric males were bred with C57/B6 females to produce transgenic mice heterozygous for the uPA gene. Breeding of these progeny produced the homozygous mice used to establish the uPA-/- and uPA+/+ lines used to generate colonies. Mice of this background (C57B6/129) are immunocompetent and have preservation of complement-dependent acute lung injury (22). WT mice of this background (C57B6/129), inoculated with the same strain of C. neoformans used in this study, are resistant to the infection—the mice survive, the fungus does not disseminate to brain, and the organism is cleared (17); furthermore, they have been shown to generate T1 cytokine responses (23) similar to those described in WT mice in this work.
Confirmation of genotype of uPA-/- and WT mice
We confirmed the genotype of the uPA-/- and WT mice by PCR analysis, as previously described (17). Genomic DNA was extracted from tail samples by proteinase K digestion, followed by chloroform extraction and ethanol precipitation (24). Samples were amplified with primers specific for the WT uPA gene (sense, 5'-ctgtctgtcatccaaccagtcc; antisense, 5'-gacacgcatacacctccgttct) (25) or the knockout uPA/neomycin construct (sense, 5'-ctgtctgtcatccaaccagtcc; antisense, 5'-ctcctgtcatctcaccttgctc; sequence pMC1Neo from Stratagene, La Jolla, CA), designed with the aid of the program Primer 2 (Scientific and Educational Software, Stateline, PA). Reaction mixtures (0.02 mg/ml DNA; 50 mM KCl; 10 mM Tris-HCl, pH 8.3; 2.5 mM MgCl2; 0.25 mM each nucleotide (dCTP, dATP, dTTP, dGTP); 0.2 mM sense and antisense primers; 0.025 U/ml Taq polymerase (Life Technologies, Gaithersburg, MD)) were denatured (94°C for 3 min) and amplified through 35 cycles (93°C for 30 s, 62°C for 45 s, 72°C for 120 s) with a one-cycle extension of 72°C for 5 min. Products were electrophoresed in agarose, stained with ethidium bromide, Southern blotted, and hybridized with labeled gene-specific internal oligonucleotide probes (26) to check product length and sequence specificity. Amplification products of the WT and knockout genes are 1153 or 1292 bp, respectively.
C. neoformans
C. neoformans strain 52 D was obtained from the American Type Culture Collection (Manassas, VA) (ATCC 24067; serotype D) (27). Stock cultures of C. neoformans were passed monthly on Sabouraud’s dextrose agar slants (Difco, Detroit, MI) and stored at 4°C. For infection, yeast was grown from stock in Sabouraud’s dextrose broth medium (1% neopeptone, 2% dextrose; Difco) for 48 h at 35°C on a shaker, washed twice in nonpyrogenic saline (NPS; Travenol, Deerfield, IL), counted on a hemocytometer, and diluted to 3.3 x 105 organisms/ml in NPS.
IT inoculation
Mice were lightly anesthetized with pentobarbital (64 mg/kg i.p.; Butler, Columbus, OH) and restrained on a small board. Each mouse received an IT inoculum of 1 x 104 C. neoformans in 30 µl of NPS, as previously described (28). This technique results in a highly reproducible pulmonary infection (13). Aliquots of the inoculum were serially diluted and plated out to confirm the number of CFU of C. neoformans being delivered. Control mice that received an IT inoculation with normal saline were examined histologically at various times after inoculation and showed no evidence of pulmonary inflammation.
Thin section histology and transmission electron microscopy
The lungs were perfused, the trachea cannulated, and the lungs inflated in situ with 2.5% gluteraldehyde in PBS. Next, the entire thoracic contents were dissected and fixed by immersion in 2.5% gluteraldehyde in PBS. Parasagittal sections through the fixed lungs were cut, dehydrated, embedded in Polybed 812 resin (Polysciences, Warrington, PA), and sectioned at 1-µm thickness. The slides, each representative of both lungs from a single mouse, were stained with toluidine blue. For transmission electron microscopy, samples were prepared as above, stained en bloc with 20% uranyl acetate in water, sectioned at 90 nm, poststained with saturated uranyl acetate and lead citrate, and examined in a LEO EM 900 (Leo, Thornwood, NY).
C. neoformans-stimulated proliferation
In separate experiments, 10 days after IT inoculation with C. neoformans, mice were euthanized, and the hilar and mediastinal lymph nodes were collected, disaggregated to a single cell suspension, counted, and cultured at 2 x 105 cells/well/200 µl of media in 96-well culture plates (Costar, Cambridge, MA) in the presence and absence of heat-killed C. neoformans. Controls included naive lymph node cells that were harvested from unprimed WT and uPA-/- mice and cultured, as described above, in the presence and absence of heat-killed C. neoformans. The cells were cultured for 3 days. Eighteen hours before harvesting (PHD Cell Harvester; Cambridge Technology, Watertown, MA), the cells were pulsed with [3H]thymidine (1 µCi/well; Amersham, Arlington Heights, IL). Proliferation, as indicated by thymidine incorporation, was measured in counts per minute on a Beckman LS 6500 scintillation counter (Beckman Coulter, Fullerton, CA).
Cytokine determination by ELISA
At various times after inoculation, mice were killed with an overdose of pentobarbital. The lungs were subjected to bronchoalveolar lavage (BAL) by repeated instillation and removal of aliquots of PBS to a total volume of 1.5 ml. Regional lymph nodes (hilar and mediastinal) were removed, disaggregated to a single cell suspension, counted on a hemocytometer, and cultured in media (RPMI 1640, supplemented with FCS (10%), penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamicin (100 µg/ml)) at 5 x 106 cells/ml, with heat-killed C. neoformans for 24 h. The lungs were then harvested, minced, processed to a single cell suspension as previously described (17), counted on a hemocytometer, and cultured (5 x 106/ml) in media for 24 h. Cytokine concentrations in the BAL fluid and in lung cell- and lymph node cell-conditioned media were measured by ELISA (picograms per milliliter), using specific Ab pairs (BD PharMingen, San Francisco, CA).
Adoptive transfer
Three weeks after IT inoculation with C. neoformans, donor WT mice were sacrificed. The lungs and regional lymph nodes were harvested and processed to a single cell suspension, as described above, and the nonadherent cells were placed in a prewarmed nylon wool column (Robbins Scientific, Mountain View, CA) and incubated at 37° for 1 h. The nylon-wool nonadherent cells were slowly eluted with warm media, washed, pelleted, and resuspended in sterile PBS. A total of 2 x 107 cells in 0.2 ml of PBS was infused via the tail vein into each recipient uPA-/- mouse.
Assessment of DTH
Cryptococcal filtrate Ag was prepared as previously described (28). Mice were challenged with footpad injections of 30 µl of cryptococcal filtrate Ag in the right footpad and diluent in the left footpad. Forty-eight hours after injection, footpad thickness was measured by a micrometer (Lux Scientific Instrument, New York, NY) and expressed in micrometers. The thickness of the right footpad minus the thickness of the left footpad represents footpad swelling.
Statistical analysis
Comparisons between group means were performed by an unpaired
Student’s t test. Statistical calculations were done using
StatView 4.5 software (Abacus Concepts, Berkeley, CA). Data are
expressed ± SEM. Statistical difference was accepted at
p 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously demonstrated that uPA-/-
mice are unable to clear pulmonary C. neoformans infection
and that the number of pulmonary inflammatory and immune cells are
strikingly reduced in uPA-/- compared with WT
mice during the first 3 wk of infection. However, histologically, at
late time points, the uPA-/- mice had large
numbers of alveolar M (17). Despite this,
uPA-/- mice did not clear the pathogen.
Therefore, we compared the histology of WT and
uPA-/- mice at day 35 post-IT inoculation. This
time was chosen because it is a point in which we would expect to see
the result of effective M activation (microbial killing) or a lack
of effective M activation (microbial growth) clearly. We performed
histologic studies using plastic imbedding and thin sectioning, and
transmission electron microscopy. Demonstrated in Fig. 1
A, by day 35 the WT lungs
show relatively few alveolar M. Most M do not contain
intracellular C. neoformans organisms, and scattered
C. neoformans ghosts, appearing as empty, nonviable cell
capsules, were present within the alveolar spaces. By contrast, the
lungs of uPA-/- mice (Fig. 1B) have
alveolar spaces nearly filled with M, many of which have the
appearance of multinucleated giant cells. These M have obviously
ingested C. neoformans, many containing more than three
organisms. Strikingly, however, as demonstrated by transmission
electron microscopy in Fig. 1C, although the
uPA-/- M are able to ingest C.
neoformans, they have impaired intracellular killing. Not only are
there viable C. neoformans present intracellularly, but the
C. neoformans organisms are actively proliferating, as
demonstrated by copious budding off the parent fungi within the M.
Thus, during pulmonary C. neoformans infection,
uPA-deficient mice have impaired antimicrobial activity even when
pulmonary M number appears to be sufficient for host defenses. These
histologic data extend our previously published quantitative data,
which demonstrated inexorable increases in pulmonary C.
neoformans CFU in uPA-/- mice in contrast
to the progressive pulmonary clearing of the pathogen in WT mice
(17), by suggesting a failure of macrophage activation
sufficient for C. neoformans killing.
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Because M require stimulation by lymphokines to acquire
fungicidal activity, we next turned our attention to the lymphocyte
response to C. neoformans infection. Protective
cell-mediated immune defenses against C. neoformans are
characterized by robust T lymphocyte proliferation within regional
lymph nodes. Grossly, the draining hilar and mediastinal lymph nodes
from uPA-/- mice appeared small compared with
the lymph nodes from WT mice. We compared the number of cells collected
from the regional nodes of uPA-/- mice with WT
mice at 10 days post-IT inoculation C. neoformans. As shown
in Fig. 2A, the regional lymph
nodes from uPA-/- mice consistently had fewer
cells in response to C. neoformans infection compared with
WT mice (p < 0.001).
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B, there was a comparable low level of proliferation
evident in both the C. neoformans-infected (primed) WT and
uPA-/- nodal cells without rechallenge in
vitro. However, when the primed WT nodal cells were rechallenged with
C. neoformans in vitro, a robust proliferation was induced.
By contrast, when rechallenged with C. neoformans in vitro,
the primed uPA-/- nodal cells failed to
increase proliferation. Thus, T lymphocyte proliferation is impaired in
the absence of uPA in response to Ag. Trivial proliferation was
observed when unprimed cells from either WT or
uPA-/- mice were stimulated with C.
neoformans in vitro. Determination of cytokine levels in BAL fluid from C. neoformans-infected WT mice compared with uPA-/- mice
Protective host defense against C. neoformans depends
on the generation of a T1 profile of cytokines (29).
Because the T1 cytokine, IFN-, is known to be critical for
protective host defense against C. neoformans, we compared
the level of IFN- in the BAL from WT mice with that from
uPA-/- mice. As shown in Fig. 3, while the levels of IFN- were the
same in uninfected WT and uPA-/- mice (day 0),
by day 7 WT mice had nearly 10 times more IFN- in BAL than did
uPA-/- mice (p <
0.005), and while the IFN- level in BAL was low in both WT and
uPA-/- mice on day 14 post-IT, the WT mice had
higher IFN- levels in BAL compared with
uPA-/- thereafter to day 42.
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Determination of the cytokine levels generated by lung inflammatory and immune cells from C. neoformans-infected WT and uPA-/- mice in vitro
The lungs of uPA-/- mice contain only about half the number of immune and inflammatory cells compared with the lungs of WT mice during the early response to pulmonary C. neoformans infection in vivo. Therefore, we questioned whether the lack of T1 cytokines at the site of the infection (BAL) in uPA-/- mice could be due to the diminished cell numbers in the lung rather than failure of polarization to a T1 profile of cytokines.
To determine whether differences in cell number were the cause of
the diminished level of T1 cytokines expressed by
uPA-/- mice compared with WT mice, we processed
the lungs to purified single cell suspensions and then compared, in
vitro, the cytokine production by equal numbers of WT and
uPA-/- lymphocytes and M through day 28
post-IT inoculation. There was no difference in the level of cytokines
expressed by uninfected (day 0) WT compared with uninfected
uPA-/- mice for any of the
cytokines evaluated. Lung cells from WT mice generate a T1 profile of
cytokines in response to C. neoformans challenge (Fig. 4). Even at a constant cell number,
pulmonary mononuclear cells isolated from WT mice produce far more
IFN- in vitro than do cells isolated from
uPA-/- mice for all the time points tested
(post-IT day 7, p = 0.042; day 14, p =
0.047; day 21, p = 0.049; day 28, p =
0.010). Similarly, lung cells from WT mice produce far more IL-12 than
do uPA-/- mice for all the time points tested
(post-IT day 7, p = 0.001; day 14, p =
0.026; day 21, p = 0.002; day 28, p =
0.004). uPA-/- mice produce higher levels of
IL-5 than do WT mice over the experimental period, although this
increase was statistically significant only at day 21 post-IT
inoculation. Thus, the diminution of IFN- and IL-12 levels seen in
the uPA-/- mice compared with WT mice (Figs. 3 and 4) is not due to diminished lung mononuclear cell number, nor to a
global inability to elaborate cytokines, but rather to a failure of
polarization to a T1 phenotype in the absence of uPA.
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To further document that the low levels of T1 cytokines observed in the lungs of uPA-/- animals infected with C. neoformans were due to an inability to develop a T1 response, and not due to an inability to recruit T1 effector lymphocytes to the lung, we evaluated cytokine production by cells obtained from regional lymph nodes. Hilar and mediastinal lymph nodes were harvested from C. neoformans-infected WT and uPA-/- mice at various time points after IT inoculation. These primed lymph node cells were rechallenged with C. neoformans in vitro, and cytokine production was determined after 24 h.
Regional lymph node cells from WT mice generate high levels
of IFN- in response to Ag challenge (Fig. 5). The T1 polarization in WT mice begins
very early in the immune response, so that by day 7 the WT lymph node
cells produce more than four times more IFN- in response to C.
neoformans challenge in vitro than uPA-/-
nodal cells (p = 0.006). IFN- levels remain
significantly diminished in the absence of uPA at day 28
(p = 0.0202). Interestingly, whereas IL-2 was
undetectable in BAL and lung cell-conditioned media, nodal
cell-conditioned media contained IL-2. Similar to IFN-, WT
lymphocytes produced more IL-2 than did uPA-/-
lymphocytes (day 14 post-IT, p < 0.013). By contrast,
lymph node cells from uPA-/- mice generate more
IL-5. Increased levels of IL-5 (a type 2 (T2) cytokine) were seen by
day 7 in the lymph node-conditioned media of
uPA-/- mice when compared with WT mice
(p = 0.0274). Thus, in the absence of uPA, the
cytokine profile of regional lymph nodes shifts away from a protective
T1 pattern of cytokines. This change in lymphocyte cytokine profile
occurs early in the course of C. neoformans infection and
persists throughout the development of the immune response. These data
demonstrate that uPA modulates the development of central immune
responses.
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To confirm that the immune defects in uPA-/- mice described above are attributable to a T lymphocyte deficiency, adoptive transfer experiments were done. Because pulmonary host defense against C. neoformans is dependent on the development of specific T cell-mediated immunity and the generation of a T1 cytokine response (15, 18, 19, 21, 28), we sought to determine whether the provision of WT immune T lymphocytes would reverse these immune defects in uPA-/- mice. Freshly purified WT immune T lymphocytes were injected via tail veins into recipient uPA-/- mice the day after IT inoculation.
Two weeks later, the delayed-type hypersensitivity (DTH) response to
C. neoformans of WT, uPA-/-, and
adoptive transfer (AT)-uPA-/- mice was
compared. By way of control, footpad swelling in naive WT and naive
uPA-/- mice in response to C.
neoformans was the same (60 µm). WT mice developed obvious DTH
reactions to C. neoformans (210 ± 10 µm), while the
uPA-/- mice had nearly no response (97 ±
20 µm; WT compared with uPA-/-,
p < 0.003, n = 3). By contrast,
AT-uPA-/- mice developed a robust DTH response
of 319 ± 45 µm (uPA-/- compared with
AT-uPA-/-, p 0.016,
n = 3). Furthermore, the DTH response of the
AT-uPA-/- mice was no different from the DTH
response in WT mice (WT compared with
AT-uPA-/-, p = NS,
n = 4). Thus, the uPA-/- mice
lacked the ability to respond to Ag with a DTH response, indicating
impaired T cell-mediated immune function; this defect could be
corrected with adoptive transfer of WT immune T lymphocytes.
Production of the T1 cytokines IFN- and IL-12 was compared among WT,
uPA-/-, and AT-uPA-/-
mice 3 wk after IT inoculation with C. neoformans. Equal
numbers of purified lung immune and inflammatory cells were
rechallenged with C. neoformans in vitro, and the levels of
IFN- and IL-12 in the conditioned media were determined 24 h
later. The uPA-/- cells produced far less
IFN- than did the WT cells (372.7 ± 61 vs 2088.4 ± 459
pg/ml, p 0.03, n = 3). In contrast,
the AT-uPA-/- mice produced 1296.3 ±
167.8 pg/ml IFN-, far more than the uPA-/-
mice (uPA-/- compared with
AT-uPA-/-, p 0.009,
n = 3). Furthermore, IFN- production by
AT-uPA-/- mice was no different from IFN-
production in WT mice (WT compared with
AT-uPA-/-, p = NS,
n = 5). Similarly, the uPA-/-
cells produced far less IL-12 than did the WT cells (497.3 ± 7.9
vs 932 ± 58.7, p 0.002, n =
3). In contrast, the AT-uPA-/- mice produced
812.6 ± 30.3 pg/ml IL-12, far more than the
uPA-/- mice (uPA-/-
compared with AT-uPA-/-, p
0.0002, n = 3). Furthermore, IL-12 production by
AT-uPA-/- mice was no different from IL-12
production in WT mice (WT compared with
AT-uPA-/-, p = NS,
n = 5).
The provision of immune WT T lymphocytes to
uPA-/- mice during the immune response to
C. neoformans substantially reverses the immune deficit seen
in uPA-/- mice. The development of a DTH
response and the generation of protective T1 cytokines in the
AT-uPA-/- mice were far greater than those in
the uPA-/- mice. Adoptive transfer of immune WT
T lymphocytes to uPA-/- mice resulted in
IFN- and IL-12 production that was the same as that seen in WT mice.
Thus, T lymphocyte defects contribute significantly to the immune
deficit noted in uPA-/- mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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50% decrease in the number of pulmonary CD4+
cells in infected uPA-/- mice compared with WT.
Because clinically overwhelming C. neoformans infection is
generally not seen until patients are critically CD4 depleted (CD4
counts of <200) (30), it is difficult to attribute the
degree of compromise seen in the uPA-/- mice to
cell number alone, making qualitative T lymphocyte defects a more
likely explanation. Clonal expansion of lymphocytes in response to Ag is perhaps the most fundamental aspect of central immune processes. Lymphocyte proliferation in response to C. neoformans in vivo was strikingly diminished in the absence of uPA, as reflected by regional lymph node cell number during infection. Furthermore, lymphocyte proliferation in response to C. neoformans in vitro was profoundly reduced. T lymphocyte proliferative responses are deficient in uPA-/- mice. Lymphocyte proliferation in response to mitogen is diminished in uPA-/- mice compared with WT (31). The reduction seen in Ag-specific proliferation in vitro is even more striking and is paralleled in vivo by the persistently diminished number of lymph node cells seen in the uPA-/- mice throughout the 42 days of C. neoformans infection (data not shown). Thus, lymphocyte proliferative responses are profoundly blunted in the absence of uPA in an Ag-specific in vivo model.
In addition to proliferating, T lymphocytes express cytokines critical
to the generation of specific immune responses and to macrophage
activation. The importance of the T1 cytokine IFN- is well
established. IFN- mediates M activation and C.
neoformans killing in vitro, and animals depleted of IFN- by Ab
treatment are unable to defend against C. neoformans in vivo
(18, 19, 32, 33, 34). It is likely that the poor intracellular
killing evident in M from uPA-/- mice in
vivo is largely due to diminished IFN--stimulation. IL-2 also plays
an important role. IL-2 has been shown to increase fungistatic activity
of human PBMCs (20), and the development of amplified
anticryptococcal DTH response in recipients of adoptively transfered
spleen cells occurs when the donor cells express high levels of both
IL-2 and IFN- (35). We show that uPA has profound
regulatory effects on cytokine expression in vivo. The WT mice express
a T1 profile of cytokine with high levels of IFN- and IL-12 in BAL
fluid. Similarly, a T1 profile is clearly dominant when cell number is
rigorously controlled using isolated lung cells or lymph nodes cells
stimulated with C. neoformans in vitro. By contrast, the
uPA-/- mice did not polarize the immune
response to a T1 profile of cytokines. The
uPA-/- mice had far lower levels of IFN- and
IL-12 in BAL fluid and in supernatants from lung cells in culture;
additionally, higher levels of IL-5, a T2 cytokine, were present.
Similarly, the nodal cells from uPA-/- mice
produced less IFN- and IL-2 than did WT mice, but higher levels of
IL-5. Providing WT immune T lymphocytes by adoptive transfer to
C. neoformans-infected uPA-/- mice
substantially reverses the immune deficits in
uPA-/- mice, as demonstrated by the restoration
of the DTH reaction to C. neoformans, and the generation of
a T1 cytokine profile in AT-uPA-/- mice that
was the same as that seen in WT mice.
These findings demonstrate that, in the absence of uPA, polarization to a protective T1 response fails to occur. This alone is sufficient to explain the death of C. neoformans-infected uPA-/- animals. Interestingly, however, the uPA-/- mice also fail to develop a well-polarized T2 response. Notably, they did not produce any detectable IL-4, nor did they have increased numbers of pulmonary eosinophils (17). WT mice of this background, inoculated with the same strain of C. neoformans, have recently been shown to generate T1 responses similar to those described in this work; however, mice of this background, when rendered genetically CCR2 deficient, generate robust T2 responses to the same pathogen (23). Thus, the uPA-/- mice are aberrant not only in their lack of a T1 response but, perhaps more importantly, in their striking unresponsiveness to Ag stimulation, and the lack of polarization to either a T1 or a T2 response.
The biologic action of uPA is traditionally conceptualized in terms of its enzymatic capacities that permit pericellular proteolysis. In vitro support for the role of uPA in cell migration has been demonstrated in hemopoietic and tumor cells, in which migration can be impeded by inhibitors to uPA or plasmin (4, 36, 37, 38). In vitro monocyte chemotaxis in the absence of matrix proteins is not diminished by inhibiting uPA enzyme activity, but is diminished by antisense-uPA oligonucleotides, suggesting that uPA binding to its receptor is involved in cell movement, perhaps by providing activation signaling (39). The binding of uPA to its receptor has been shown to activate calcium flux, prime for superoxide production, and induce tyrosine phosphorylation events (40, 41, 42). Collectively, these data argue that the biologic effects of uPA are not limited to its known ability to mediate pericellular proteolysis, but extend to cellular signaling and cell activation events.
There is substantial evidence that the uPA/uPAR system and the immune
system interact. Both uPA and uPAR expression are modulated by
inflammatory mediators. M uPA expression is increased by TNF-
,
IFN-, IL-1, and IL-2 (43, 44). Activated T lymphocytes
express uPA and uPAR (7, 10, 11). Stimulation with phorbol
esters and TCR-mediated stimulation result in substantial up-regulation
of uPA and uPAR in T cells (7, 10, 11). uPAR is
coexpressed with CD25 (IL-2R), establishing its expression in
TCR-mediated T cell activation (7). Furthermore, uPAR is
up-regulated by exposure to IL-2 and IL-4, but not to several other
cytokines (7). Interestingly, exposure to IL-2 or IL-4
up-regulates uPAR on only a subset of lymphocytes, perhaps
corresponding to T1 or T2 phenotypes, respectively. Conversely, there
is substantial, but fragmented, evidence suggesting that uPA is a
modulator of immune and inflammatory responses. For example, early
reports showed that plasminogen activator could act as a lymphocyte
mitogen (45), that uPA was a neutrophil chemotaxin
(46), and that IL-2 synthesis by Jurkat T cells requires a
membrane-associated serine protease (47). Many of these
early studies were limited by an inability to characterize the involved
proteases completely. uPA amplifies TNF- production on the mRNA
level by U937 M (48), establishing that uPA can act as
a regulator of cytokine expression in vitro. Thus, bidirectional
communication links the uPA/uPAR system and inflammatory cytokine
networks.
The profound effects that uPA deficiency has on T lymphocyte proliferation in response to TCR-mediated signaling, and the effects of uPA deficiency on T1/T2 differentiation during C. neoformans infection place uPA in essential T lymphocyte signaling pathways. The defect in the immune response in the absence of uPA has several characteristics. First, the defect is evident in central lymphocyte differentiation (in the regional lymph nodes); second, it is present early (by day 7) and is persistent; third, it is characterized by a profound defect in proliferation; fourth, there is diminished IL-2 production; and finally, fifth, there is a failure to polarize to a T1 phenotype. Collectively, these characteristics are remarkably similar to the responses seen when the anergic state is induced in pluripotential precursor clones (Th0) in vitro. Anergic Th0 clones exhibit reduced proliferative responses and diminished expression of T1 cytokines with retention of T2 cytokines (49). In our in vivo model, specific T lymphocyte proliferation is nearly absent, and T1 cytokines are profoundly reduced, while IL-5 levels are minimally increased in the absence of uPA, fully consistent with anergic Th0 clones. However, whereas in vitro the separation of anergic Th0 clones and T2 clones is sometimes controversial, in vivo the uPA-/- mice clearly fail to develop a classic T2 phenotype; they remain immunologically unresponsive, despite continued Ag stimulation from proliferating microbes. This suggests that uPA deficiency may induce a functional anergy-like state in a clinically relevant, in vivo, infectious model.
In summary, uPA-replete mice develop classic T1 responses in lung and regional lymph nodes in response to C. neoformans and are protected. In the absence of uPA, T1 responses fail to develop either in the lung or in regional lymph nodes, Ag-specific lymphocyte proliferation is markedly blunted, and the uPA-/- mice fail to mount adequate immune defense against C. neoformans. These studies define uPA as a novel and required immunomodulatory molecule for the development of protective T1 immune responses to a clinically relevant fungal pathogen.
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2 Address correspondence and reprint requests to Dr. Margaret R. Gyetko, Departments of Internal Medicine and Pathology, Pulmonary and Critical Care Medicine Division, Ann Arbor Veterans Affairs Medical Center and University of Michigan Medical Center, 2215 Fuller Road (111G), Ann Arbor, MI 48105. E-mail address: mgyetko{at}umich.edu
3 Abbreviations used in this paper: uPA, urokinase-type plasminogen activator; AT, adoptive transfer; BAL, bronchoalveolar lavage; DTH, delayed-type hypersensitivity; IT, intratracheal; M, macrophage; NPS, nonpyrogenic saline; T1, type 1; T2, type 2; uPAR, uPA receptor; WT, wild type.
Received for publication March 16, 2001. Accepted for publication November 2, 2001.
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