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Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) responses were present in the
lungs, with Th1 cell activation as a major component of the pulmonary
Th cell response. Although a smaller component of the T cell response,
mycoplasma-specific CD8+ T cells were also a significant
component of pulmonary lymphoid responses. In vivo depletion of
CD8+ T cells resulted in dramatically more severe pulmonary
disease, while depletion of CD4+ T cells reduced its
severity, but there was no change in mycoplasma numbers in lungs after
cell depletion. Thus, mycoplasma-specific Th1 and CD8+ T
cell activation in the lung plays a critical regulatory role in
development of immunopathologic reactions in Mycoplasma
respiratory disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanisms that determine whether generated immune responses are protective or immunopathologic are unknown for any mycoplasma disease. T cell activation is most likely pivotal in determining the impact of these responses on mycoplasma infection and disease. A role for T cells in immunopathologic responses is supported by studies demonstrating T cell-deficient mice or hamsters have less severe mycoplasma respiratory disease than their immunocompetent counterparts (11, 13). We similarly found that SCID mice, lacking in functional B or T cells, develop significantly less severe M. pulmonis lung disease than immunocompetent mice (14). Interestingly, the numbers of mycoplasma in the lungs of SCID mice and immunocompetent mice did not differ, suggesting that lymphoid responses, not an uncontrolled infection, promoted inflammatory lesions due to mycoplasma infection. In fact, reconstitution of SCID mice with splenic lymphocytes resulted in disease as severe as in normal mice (14), and preliminary studies (unpublished data) suggest that components of these lesions were due to T cell activity alone. Although these studies demonstrated that lung disease due to mycoplasma infection was immunopathologic, adaptive immune responses were also shown to be important in preventing extrapulmonary dissemination of infection, leading to arthritis and the eventual death of SCID or nude mice (11, 14). Furthermore, adaptive immunity, also dependent on functional T cells, can promote resistance and recovery from M. pneumoniae and M. pulmonis disease (8, 17, 18). Thus, adaptive immune responses, mediated by T cell activity, are critical in promoting protective immunity and immunopathologic reactions associated with mycoplasma respiratory disease. However, little is know about these mechanisms, and more work is needed to understand the apparent conflicting roles of immune responses in mycoplasma disease, facilitating the eventual development of vaccines and therapies for this important group of respiratory diseases.
The role of T cell subsets is unknown for any mycoplasma disease.
However, M. pulmonis infection in mice provides an
opportunity to determine the function of these T cell subpopulations in
mycoplasma disease. Based on our previous studies of M.
pulmonis disease in two strains of rats with differing disease
susceptibilities (16, 19, 20, 21, 22, 23, 24, 25), we proposed that regulatory
T cells play a major role in determining the outcome of mycoplasma
infection, although this hypothesis has yet to be directly addressed.
The purpose of the present study was to delineate the changes in T cell
populations and their activation after mycoplasma infection and
determine their impact on the pathogenesis of M. pulmonis
respiratory disease. We focused on Th cells, as they are known to play
a critical role in protection and pathogenesis of many infectious
diseases through their ability to mediate humoral and cell-mediated
responses. Importantly, Th cells are roughly divided into two major
functional populations. Th1 cells promote cell-mediated and
inflammatory responses through their production of cytokines, such as
IFN- (26). In contrast, Th2 cells control Ab responses
through their secretion of characteristic cytokines (e.g., IL-4).
Studies have shown that Th cell subset activation is important in the
control of other infectious diseases (27, 28, 29, 30, 31, 32, 33, 34, 35), but their
impact on mycoplasma respiratory disease is unknown. In addition,
CD8+ T cells are also known to impact on the
progression of inflammatory diseases, as they can regulate these
responses (36, 37) or cause lung injury through
CD8+ T cell-mediated apoptosis
(38, 39, 40). Thus, we also examined whether changes in
CD8+ T cell responses occurred and their
contribution to mycoplasma disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C3H/HeN viral- and mycoplasma free mice were obtained from Harlan Sprague-Dawley (Indianapolis, IN). Mice were housed in sterile microisolator cages and supplied with sterile bedding, and food and water were given ad libitum. Mice used in studies were between 8 and 12 wk of age. Before experimental manipulation, mice were anesthetized with an i.m. injection of ketamine/xylazine.
Mycoplasma
The UAB CT strain of M. pulmonis was used in all experiments. Stock cultures were grown, as previously described (41), in mycoplasma broth and frozen in 1-ml aliquots at -80°C. For inoculation, thawed aliquots containing 2 x 107 CFU of M. pulmonis per milliliter were diluted to 105 CFU/20 µl. Inoculations of 20 µl diluted mycoplasma were given nasally.
Cell isolation
Mononuclear cells were isolated from lungs, as previously described (23, 42, 43). Lungs were perfused with PBS without magnesium or calcium to minimize contamination of the final lung cell population with those from blood. The lungs were separated into individual lobes and finely minced. The tissues were suspended in RPMI 1640 (HyClone Laboratories, Logan, UT) medium containing 300 U/ml Clostridium histolyticum type I collagenase (Worthington Biochemical, Freehold, NJ), 50 U/ml DNase (Sigma-Aldrich, St. Louis, MO), 10% FBS (HyClone Laboratories), HEPES, and antibiotic/antimycotic solution (Life Technologies, Grand Island, NY). The tissues were incubated at 37°C while mixing on a Nutator (Fisher Scientific, Pittsburgh, PA) for 90–120 min. During the incubation period, the tissue was vigorously pipetted every 20 min. After incubation, the digestion mixture was passed through a 250-µm nylon mesh to remove undigested tissue. Mononuclear cells were purified from cell suspensions by density gradient centrifugation using Lympholyte M (Accurate Chemicals, Westbury, NY).
Spleen cells and lower respiratory (mediastinal and hilar) lymph nodes (LRN)3 were isolated after centrifugation of cell suspensions, followed by red cell removal using ACK (ammonium chloride potassium) lysis buffer, as previously described (44).
Immunofluorescent characterization of lymphocyte populations
Two-color and three-color immunofluorescent staining was performed to identify both B and T cell populations using FITC-labeled anti-murine B220 mAb (RA-6B2; Beckman Coulter, Miami, FL), PE-labeled anti-murine CD3 mAb (KT3; Beckman Coulter), PerCP-labeled anti-CD4 mAb (L3T4, RM4-5; BD PharMingen, San Diego, CA), and FITC-labeled anti-CD8 mAb (Lyt-2, 53-6.7; BD PharMingen). Briefly, 2 x 106 cells per tube were incubated with purified 2.4G2 mAb (BD PharMingen) for 5 min at 4°C to reduce nonspecific binding of FcII/IIIRs before fluorescent Ab staining. The cells were incubated for 30 min at 4°C with 250 µl fluorescent Ab (2 µg/ml). The cells were washed in staining buffer (Mg2+-free, Ca2+-free PBS with 0.05% sodium azide, 1% FBS (HyClone Laboratories)) and fixed with 4% paraformaldehyde solution for 30 min, and the cells were then resuspended in staining buffer until analysis.
The cells were analyzed using an EPICS XL-MCL flow cytometer (Beckman Coulter). Data collection was done using System 2 software (Beckman Coulter), with further analysis done using Expo 2 analysis software (Beckman Coulter). Lymphocyte gates and detector voltages were set using unstained (control) lung and splenic cells. The proportion of each cell population was expressed as the percentage of the number of stained cells. To determine the total number of a specific lymphocyte population, their percentage was multiplied by the total number of lymphocytes isolated from a tissue.
RNA isolation from lungs
Total RNA was isolated from the entire lungs of mice using the Ultraspec-II RNA Isolation System (Biotecx Laboratories, Houston, TX), which is based on a previously described method (45). Briefly, the lungs were homogenized in the Ultraspec-II RNA reagent using a PRO 200 homogenizer (PRO Scientific, Monroe, CT). Chloroform was added to the homogenate and centrifuged at 12,000 x g (4°C) for 30 min. The RNA was precipitated by adding isopropanol to the aqueous phase and centrifuging the samples at 12,000 x g (4°C) for 10 min. The RNA pellet for each sample was washed twice with 75% ethanol by vortexing and subsequent centrifugation for 5 min at 7,500 x g and then resuspended in diethylpyrocarbonate-treated water. The concentration and quality of RNA in each of the samples were determined spectrophotometrically (GeneQuant II; Amersham Pharmacia Biotech, Piscataway, NJ) and by gel electrophoresis. The RNA samples were stored at -80°C until used.
Cytokine mRNA detection by RT-PCR
RT-PCR was performed using 100 ng RNA for each sample, as
previously described (46). The sequences of the primers
and the size of the resulting PCR fragments (in parentheses) for IL-2,
IL-4, IFN-, and the housekeeping gene,
2-microglobulin (2m)
are given as follows (47): IL-4 (216 bp),
5'-TCGGCATTTTGAACGAGGTC and 5'-GAAAAGCCCGAAAGAGTCTC; IFN- (227
bp), 5'-GCTCGAGACAATGAACGCT and 5'-AAAGAGATAATCTGGCTCTGC; and
2m (222 bp), 5'-TGACCGGCTTGTATGCTATC and
5'-CAGTGTGAGCCAGGATATAG.
The increase in expression of cytokine mRNA after immunization was
determined by the number of cycles of amplification that resulted in
little or no PCR product for each cytokine in total lung RNA from
sham-inoculated, control mice, as previously described (46, 48). For IFN-, IL-5, and 2m, the
samples were amplified for 30 cycles, and for IL-2 and IL-4, the
samples were run for 35 cycles. The PCR products were separated on
1.8% agarose gels and stained with ethidium bromide. Gels were
visualized using Alpha Image 2000 Documentation and Analysis System
(Alpha Innotech, San Leandro, CA). The intensity of each band was
determined using densitometry, and the relative cytokine mRNA reactions
were compared after normalization to the housekeeping gene,
2m.
Preparation of M. pulmonis Ag
A crude preparation of M. pulmonis membranes was used for in vitro stimulation and prepared as previously described (22). Briefly, M. pulmonis was cultured at 37°C in mycoplasma broth medium and harvested at pH 7. Cells were then centrifuged at 10,000 rpm for 20 min, and pellets were resuspended in 5 ml sterile 0.25 M NaCl. Following a second centrifugation at 9000 rpm for 20 min, pellets were resuspended in a total of 4 ml 2 M glycerol at 37°C. Cells were then sonicated at the highest setting for 15 s using Vibra cell sonicator (Sonics & Materials/Vibrio Cell, Newtown, CT), followed by incubation at 37°C for 10 min. For cell lysis, 0.5 ml of the cell preps was then forced through a 27-gauge needle into 25-ml aliquots of distilled water. To remove unlysed organisms, cells were centrifuged at 10,000 rpm for 20 min. Supernatants were again centrifuged at 20,000 rpm for 1 h. Membranes were resuspended in 5 ml sterile PBS (HyClone Laboratories) and stored at -80°C. All centrifugations were done at 4°C. Protein concentration was determined by standardization using Bradford protein assay (Bio-Rad, Hercules, CA). A final concentration of 5 µg/ml M. pulmonis membrane Ag was used for in vitro studies, as indicated in Results.
In vitro depletion and isolation of T lymphocyte populations
A 100-µl aliquot of mouse anti-CD4 and/or anti-CD8 mAb-labeled magnetic beads (Dynabeads mouse CD4 (L3T4), mouse CD8 (Lyt-2); Dynal Biotech, Lake Success, NY) was added directly to lung and splenic cell suspensions at a final concentration of 1 x 107 cells/4 x 107 beads. Lymphoid cells were incubated at 4°C for 20 min on a rotator. After incubation, tubes containing lymphocytes were placed on a magnet (MPC magnet; Dynal Biotech) for 2 min, and both positive and negative fractions were collected for further studies. Confirmation of cell purity was determined using flow cytometry. Cell fractions with a depletion of >95% were used for subsequent experimentation.
Ag-specific in vitro stimulation of mononuclear cells
Lymphoid cells were cultured in 96-well round-bottom microtiter plates in RPMI 1640 (HyClone Laboratories) supplemented with 5% FBS (HyClone Laboratories), HEPES, 10 U/ml rIL-2 (BD PharMingen; shown in during earlier studies (49) to amplify, but not shift Th cell responses), antibiotic/antimycotic solution (Life Technologies), and 50 µM 2-ME (Life Technologies). Lymphoid cells were stimulated at 37°C and 5% CO2. Cells were stimulated with or without 5 µg/ml prepared Mycoplasma Ag in a final volume of 200 µl/well culture media at a cell concentration of 2 x 106 cells/ml. Supernatants were collected 4 days later and stored at -80°C until assayed for cytokine levels.
Cytokine assays
The levels of cytokine were measured by capture ELISA. Murine
IL-4 levels were measured using OptEIA IL-4 ELISA set (BD PharMingen),
while murine IFN- levels were determined using mouse IFN- MiniKit
(Endogen, Woburn, MA). Easy-wash 96-well flat-bottom microtiter plates
(BD Biosciences, Bedford, MA) were coated overnight at 4°C with 100
µl mAb specific for either murine IL-4 or IFN- diluted in 0.1 M
Na2HPO4, pH 9. Plates were
washed and blocked with 200 µl PBS/Tween 20 supplemented with 10%
FBS solution for 30 min. Following PBS/Tween 20 wash, 100 µl sample
supernatants were placed into the appropriate wells and incubated
overnight at 4°C. Plates were washed three times with PBS/0.05%
Tween, and 100 µl biotinylated rat anti-mouse cytokine (IL-4 or
IFN-) mAb was added to each well and incubated overnight at 4°C.
To reveal the reaction, avidin-HRP and 3,3'5,5'-tetramethylbenzidine
substrate (Moss, Pasadena, MD) were used. Plates were read using MX80
plate reader (Dynatech, Chantilly, VA) at an absorbance of 630 nm. A
total of 100 µl 0.25 M HCl was also added to reaction as needed to
increase sensitivity of reaction and read at an absorbance of 450 nm.
Cytokine levels were determined by comparison with standard curves
generated from murine recombinant cytokines (IL-4 and IFN-; BD
PharMingen) after log/log quadratic linear regression analysis using
Revelation 2.0 software (Dynatech).
In vivo depletion of CD4 and CD8 cells using mAb treatment
Hybridoma cell lines, GK1.5 (L3T4) and 53-672 (LyT3) (American
Type Culture Collection (ATCC), Manassas, VA), were grown in MiniPerm
Bioreactors (Sartorius, Edgewood, NY) in IMDM culture medium (HyClone
Laboratories) containing 15% FBS at 37°C, 10%
CO2. Ab concentrations in culture supernatants
were determined using a rat IgG-specific ELISA. The ability to deplete
T cell populations in vivo (
98% depletion) was confirmed for each
batch of Ab by immunofluorescent staining of spleen cells from
treated mice.
Three days before infection, mice were given a 1 ml (1 mg) i.p.
injection of anti-CD4 (ATCC) and/or anti-CD8 mAb. Ab injections
were repeated every 5 days (50, 51). In vivo depletion of
lymphocyte populations was confirmed as 98% depletion by examining
splenic cell populations using immunofluorescent staining and flow
cytometry.
Mycoplasma numbers
The numbers of CFU in lungs and nasal passages were determined as previously described (52). Briefly, lungs were minced and placed in mycoplasma broth medium. The samples were sonicated (Vibra cell sonicator; Sonics & Materials/Vibro Cell) for 2 min at 50 amplitudes without pulsing. After sonication, 1/10 serial dilutions were prepared, and 20 µl of each dilution was plated onto Mycoplasma plate media. After 7 days of incubation at 37°C, colonies were counted, and the number of CFU recovered from each tissue was calculated.
Assessment of gross lesions
Lungs were removed, and each lobe was examined by two observers for the presence of gross lesions. The percentage of each lobe with gross lesions was recorded. The gross lesion scores were weighted by the percentage that each lobe contributes to the total lung weight in arriving at the gross lesion index for lungs (53).
Statistical analysis
Data were evaluated by ANOVA, followed by Fisher protected least
square differences multigroup comparison. These analyses were performed
using the StatView (SAS Institute, Cary, NC) computer programs. A
p value 
0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the changes in lymphocyte populations in mice infected with M. pulmonis, mononuclear cells were isolated from the lungs, LRN, and spleens at various time intervals (days 0, 7, and 14) after infection. The numbers of B and T cells, including CD3+CD4+ Th cells and CD3+CD8+ T cells, were determined.
T cells were the major lymphocyte population that changed after
mycoplasma infection. By 7 days after infection, there was a 3- to
4-fold increase in T cell numbers within the LRN, but no changes were
seen in lungs or spleens at this time (Fig. 1
). The numbers of B cells, however, did
not significantly change in any tissue at this time. Increases in
pulmonary T cell numbers were apparent by 14 days after infection. At
this time point, there was about a 10-fold increase in T cells in the
lungs of infected mice. There was also a 3-fold increase in B cells
from lungs of infected mice. In contrast, there was no significant
increase in the number of B or T cells isolated from spleens throughout
the course of infection. Thus, it is likely that T cells are a major
population of lymphocytes contributing to the inflammatory
infiltrate.
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). At this time, there was a small, but not
statistically significant, increase in
CD3+CD8+ T cells. At 14
days after infection, there was a significant increase in both T cell
populations in the lungs of mice infected. There was, however, no
significant increase in
CD3+CD4+ or
CD3+CD8+ T cells in the LRN
and spleen between 7 and 14 days after infection. Importantly, there
was a higher percentage of
CD3+CD4+ Th cells than
CD3+CD8+ T cells in all
tissues at every time interval after mycoplasma infection (Table I). Furthermore, the percentage of
CD3+CD4+ Th cells
significantly increased at 7 days after infection in the LRN and at 14
days in the lung. Thus, Th cells were the major T cell population
present in lungs and LRN throughout the course of disease, and there
was a preferential increase in Th cells in both tissues at selected
stages of disease. However, the number of CD8+ T
cells concomitantly increased in the lungs. These changes in T cell
populations coincided with the massive cellular infiltrate within the
lungs after M. pulmonis infection.
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cytokine mRNA expression in the lungs of mice
infected with M. pulmonis
IL-4 is the characteristic cytokine produced by Th2 cells, while
Th1 and CD8+ T cells produce IFN-
(26). We examined IL-4 and IFN- expression within LRN
and lungs during different stages of disease. LRN and lungs from
uninfected and M. pulmonis-infected mice were collected at
different time points (days 0, 7, and 14) after infection. Total RNA
was isolated from these tissues, and the expression of IFN- and IL-4
mRNA was determined using RT-PCR. There was a significant increase in
IL-4 mRNA levels in LRN, but not in lungs, at 7 days after infection
(Fig. 2a). In contrast, there
was no significant increase in IFN- mRNA levels within LRN or lungs
at that time point. At 14 days after infection, IL-4 and IFN- mRNA
levels were significantly higher within LRN and lungs.
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can be produced by CD4+ and
CD8+ T cells and NK cells,
CD4+ and CD8+ T lymphocytes
were isolated from uninfected and mice infected with mycoplasma 14 days
earlier, and IFN- cytokine mRNA expression was determined by RT-PCR.
There was an increase in IFN- mRNA levels in both
CD4+and CD8+ T cell
populations (Fig. 2b). In addition, there was an increase in
IL-4 mRNA expression in CD4+ T cells, but not in
CD8+ T cells (data not shown). Therefore, both
CD4+ Th and CD8+ T cells
are sources of IFN- within the lung at 14 days after infection,
whereas CD4+ Th cells also expressed
IL-4.
Mycoplasma-specific IL-4 and IFN- cytokine
production by pulmonary lymphocytes
To monitor mycoplasma-specific T cell responses, IFN- and IL-4
production by lymphocytes was measured after stimulation with
mycoplasma Ag. Lung, LRN, and spleen cells were isolated from mice on
0, 7, and 14 days after infection and cultured in the presence or
absence of mycoplasma Ag in vitro. Four days later, supernatants were
collected, and the levels of IFN- and IL-4 cytokine were determined.
In response to stimulation with mycoplasma Ag, little, if any, IL-4 or
IFN- was detected within supernatants from cultures of LRN, lung,
and splenic cell collected from mice at day 7 after infection (Fig. 3). However, mycoplasma-specific cytokine
responses were readily detected in cells obtained from mice infected
for 14 days. IL-4, but not IFN-, responses were present in LRN. In
contrast, only IFN- responses were found in Ag-stimulated spleen
cell cultures. Interestingly, lung cells produced both IL-4 and IFN-
in response to mycoplasma Ag. At this time point (14 days), the levels
of IFN- cytokine production were significantly higher than IL-4
levels within all lymphocyte cultures. Stimulation of lymphocytes from
naive (0 day) mice with mycoplasma Ag did not result in IFN- or IL-4
production. Also, there was little or no cytokine production in cell
cultures not stimulated with mycoplasma Ag.
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10% of that produced by whole (undepleted) pulmonary lymphocytes
(Table II). Depletion of either lung
CD4+ Th cells or CD8+ T
cells significantly reduced IFN- levels to 50% of the levels
produced by mycoplasma Ag-stimulated whole lymphocytes. IL-4 and
IFN- production was diminished to low levels within mycoplasma
Ag-stimulated cultures consisting of cells depleted of both
CD4+ and CD8+ T cells from
infected mice. In spleen, CD8+ T cells were the
major source of IFN-, although CD4+ T cells
contributed a smaller, but significant portion of the IFN-
response.
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To determine the impact of T cell populations, mice were
treated withanti-CD4 or anti-CD8 Abs. One group of
mice was treated with a mixture of both Abs. Three days later, mice
were infected with mycoplasma. Ab treatments were continued throughout
the course of disease. At 14 days after infection, the severity of
mycoplasma lung disease was determined. In addition, in vivo Ab
depletion was confirmed by examining spleen cells from Ab-treated mice
for CD8+ and CD4+ T cells
using immunofluorescent staining and flow cytometry. All mice were
shown to have 98% depletion of the appropriate cell population(s).
For example, in mice treated with anti-CD4 Ab, 2% of the
CD3+ cells were CD4 positive and 98% were CD8
positive.
No clinical signs of disease were observed between 0 and 10 days after
infection in all groups as compared with sham-infected mice. On day 11,
mice treated with anti-CD8 Ab showed signs of lethargy and ruffled
fur. At this time point, clinical signs were absent in mice treated
with anti-CD4 Ab alone or mice treated with a mixture of
anti-CD4 and anti-CD8 Abs. By day 14, anti-CD8 Ab-treated
mice were very ill. At this time, control-infected mice were showing
signs of illness, but these signs were not seen in mice treated with
anti-CD4 Ab or the mixture of anti-CD4 and anti-CD8 Abs. In
addition, mice treated with anti-CD8 Ab had significantly greater
(p 0.05) weight loss than any other group of
mice (Fig. 4a). There was no
significant difference in weight loss between the other groups of mice.
Thus, depletion of CD8+ T cells resulted in
increased disease severity, as indicated by clinical signs.
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b). In contrast, mycoplasma-infected mice
developed dramatically more severe pulmonary lesions after
CD8+ T cells were depleted. However, few lesions
were found in infected mice depleted of not only
CD8+ T cells, but also CD4+
T cells. Importantly, Ab treatment had no effect on mice that were not
infected with mycoplasma (data not shown). Thus, depletion of
CD8+ T cells resulted in more severe mycoplasma
pulmonary disease, while simultaneous depletion of
CD4+ T cells reversed this effect.
To determine whether depletion of either population of T lymphocytes
had an effect on disease resistance, the above experiments were
repeated 14 days after infection, the lungs were removed, and
mycoplasma CFU were determined in the lung. There were no significant
differences between any of the treatment groups in the numbers of
mycoplasma CFU in the lungs (Fig. 4c). Thus, depletion of T
cell populations did not affect the level of pulmonary infection by
mycoplasma.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Increases in T cell population numbers were indeed associated with the development of mycoplasma respiratory disease. Initial changes in lymphocyte numbers were found in LRN. There was a 5-fold increase in Th cells, whereas the numbers of B and CD8+ T cell populations did not vary significantly. This indicates that Th responses are the initial lymphocyte population responding after infection, and that in the lower respiratory tract, these responses first appear in the draining lymph nodes. However, by day 14, changes in lymphocyte numbers are readily apparent in the lungs, corresponding with the presence of peribronchial and perivascular mononuclear cell infiltrates. At this time point (data not shown), T cells were the major lymphocyte population recovered from the lungs of infected mice, and the number of T cells was 10 times greater than those recovered from naive mice. Although B cell numbers also increased in lungs, their increases were significantly smaller. Furthermore, Th cells were the major T cell population in lung, and although both pulmonary Th and CD8+ T cells increased after mycoplasma infection, there was a preferential expansion of Th cells. Importantly, the large increases in Th cell numbers are consistent with Th cells being the dominant lymphocyte population in the inflammatory infiltrate associated with mycoplasma respiratory disease, suggesting a major role for Th cell activity in disease pathogenesis. In support, we demonstrated that in vivo depletion of CD4+ T cells results in less severe mycoplasma respiratory disease in both control and most dramatically CD8 cell-depleted mice, without having an effect on mycoplasma numbers in lungs. Thus, Th cells are a major component in the lymphoid responses generated in the lower respiratory tract after mycoplasma infection and are likely to contribute to immune-mediated inflammatory responses in mycoplasma respiratory disease.
Mycoplasma-specific Th1 and Th2 responses were generated in the lower
respiratory tract after infection with M. pulmonis, but
there were distinct differences in the Th cell responses in each of the
tissues examined with the appearance of Th1 responses associated with
inflammatory lesions in the lung. At 14 days after infection,
mycoplasma-specific IL-4 production by LRN lymphocytes was readily
found, while IFN- production was low. In contrast, splenic
lymphocytes produced high levels of IFN-, but not IL-4, after
stimulation with mycoplasma Ag. Lung lymphocytes produced both IFN-
and IL-4 in response to mycoplasma Ag. CD4+ Th
cells were the major source of IL-4, as their depletion eliminated IL-4
production. We similarly showed that Th cells in spleens and lungs were
a source of IFN-, although CD8+ T cells also
contributed to IFN- production, particularly in spleens. These
results demonstrate that mycoplasma-specific Th2 responses were
dominant in LRN, while Th1 responses predominated in spleen. However,
both Th1 and Th2 cytokine responses were present in the lungs, but
IFN- levels were greater, suggesting that Th1 cell activation was a
major component of the Th cell response in lungs. The association of
IL-4 responses with respiratory tissues, e.g., lungs and LRN, is
consistent with our studies demonstrating that resident Th cells in the
lung are Th2, which influences immune responses to intranasally given
Ag (49). The Th2 dominance of pulmonary immune responses
is also consistent with the work of others (54). Thus, the
appearance of Th1, as well as Th2, responses in the lung coincides with
the development of CD4+ cell-mediated chronic
inflammatory lesions in mycoplasma respiratory disease.
Mycoplasma-specific Th1 responses in lungs are likely to contribute to the immune-mediated inflammatory reactions in the lungs of mycoplasma-infected mice. Th1 cells promote delayed-type hypersensitivity responses, which are characterized by mononuclear cell infiltration (26), similar to that found in mycoplasma disease (6, 7, 8, 24, 25). Although Th2 cell activation can also lead to inflammatory responses, eosinophils are a characteristic cell type within these reactions, but eosinophils are not a major component of mycoplasma respiratory disease. While Th1 cells may be critical in the pathogenesis mycoplasma respiratory disease, Th2 cell activation may also be important. Although Th2 cell responses alone may not be responsible for inflammatory lesions in mycoplasma, interactions between Th1 and Th2 activities have been implicated in increased severity inflammatory reactions associated with asthma (55) and colitis (56). Furthermore, Th2 responses may also contribute to complications associated with mycoplasma respiratory diseases. For example, there are several studies suggesting that mycoplasma respiratory infections in humans can increase the severity of asthma (4, 5). IL-4-mediated Th2 responses promote IgE production that can lead to allergies and asthma (26, 55, 57, 58, 59), and we have shown the generation of mycoplasma-specific IgE responses during the pathogenesis of M. pulmonis disease (23). Thus, Th1 cell activity is likely to directly contribute to the inflammatory lesions in mycoplasma respiratory; however, regulatory and effector functions of Th2 cells may lead to increased disease severity and/or complications associated with mycoplasma respiratory diseases. Nevertheless, further studies are needed to uncover the contributions of Th1 and Th2 cell responses to mycoplasma respiratory disease pathogenesis.
Although a smaller component of the T cell response,
CD8+ T cell numbers also increased in lungs after
infection and have a significant impact on the progression of
mycoplasma respiratory disease. In addition to cell numbers, we found
that mycoplasma-specific CD8+ T cell responses
were generated in the lungs of M. pulmonis-infected mice.
This was shown by an increased expression of IFN- mRNA in purified
CD8+ cells, and through our studies demonstrating
that depletion of CD8+ T cells reduced
Mycoplasma-specific IFN- production in vitro.
Importantly, we demonstrated that in vivo depletion of
CD8+ T cells resulted in dramatically more severe
mycoplasma respiratory disease, while depletion of
CD4+ T cells reduced the severity of these
reactions, but there was no change in mycoplasma numbers after
depletion. These results suggest that CD8+ T
cells dampen CD4+ T cell-mediated inflammatory
reactions in response to mycoplasma infection. There is increasing
evidence supporting a similar role for CD8+ T
cells in pulmonary inflammatory reactions (36, 37, 60).
Although not elucidated in the current study, several potential
mechanisms could be responsible for the effect
CD8+ T cells have in regulation of pulmonary
inflammatory reactions. There is evidence that
CD8+ T cells, through IFN- cytokine
production, have a regulatory role in eosinophilia, neutrophilia, and
eotaxin activation observed in a murine model of asthma (37, 61). Additionally, elevated IgE Ab levels occur in mice after in
vivo CD8+ T cell depletion and immunization with
OVA Ag (62, 63). It is likely that the effect of
CD8+ T cells in regulation of Th2-mediated
pulmonary inflammatory responses observed in the above studies is a
result of interactions with CD4+ T cells. There
was, however, no evidence in our in vitro studies that depletion of
CD8+ cells resulted in an enhancement of IL-4
responses, suggesting that in our in vitro studies,
CD8+ cells had a major regulatory effect on
existing Th2 responses. Additional studies are needed to examine
whether CD8+ T cell cytokines have an in vivo
effect during Th cell differentiation, although preliminary studies do
not support a major shift in IL-4 and IFN- mRNA expression (data not
shown). Alternatively, CD8+ T cells have been
shown to act as veto cells in peripheral tolerance against graft
rejection through TGF-1 production (64, 65), and it is
possible that a similar mechanism is involved in the modulation of
mycoplasma respiratory disease. An additional mechanism may be through
the reduction of APC numbers due to CD8+ T
cell-mediated cytolysis (66), thereby indirectly
decreasing Th cell activation. Interestingly, the expansion of
CD8+ T cells is not found in all inflammatory
responses in the lung, as nasal immunization using cholera toxin
results in a perivascular and peribronchial mononuclear cell infiltrate
that is comprised of CD4+ T cells, but not
CD8+ T cells (49). Thus,
CD8+ T cells are a significant component of the
chronic inflammatory infiltrate, and they play a significant role in
mycoplasma respiratory disease pathogenesis through the dampening of
CD4+ T cell-mediated inflammation. Further
studies are needed to determine the mechanisms through which
CD8+ T cells modulate these responses.
In summary, the present study provides insight to the mechanisms of immunity involved in the pathogenesis of mycoplasma respiratory disease. We delineated the changes in T cell populations and their activation after Mycoplasma infection. We also determined their impact on the pathogenesis of murine mycoplasma respiratory disease. Based on our results, mycoplasma-specific Th cell responses in the lung are linked to the development of immunopathologic reactions. Of particular interest is the observation of the shift from a Th2 pulmonary environment to a mixed Th1 and Th2 response and its correspondence with disease. As we have shown in a previous study, a similar change in pulmonary T cell responses occurs after intense immune stimulation (49); future studies need to determine the mechanisms through which this occurs and its impact on resistance and protection against respiratory diseases. Although a smaller component of the T cell response, mycoplasma-specific CD8+ T cells are a significant component of the chronic inflammatory infiltrate, and their activation leads to a dampening of the inflammatory reaction in mycoplasma respiratory disease. To our knowledge, this is the first study to examine the development of T cell subset responses and their role in any mycoplasma disease. We have shown that the major T cell subsets, Th and CD8+ T cells, have opposing regulatory roles in progression of mycoplasma inflammatory disease of the lung. We anticipate these studies will provide a strong foundation for further studies examining the role of each T cell component in murine mycoplasma respiratory disease. This will undoubtedly benefit our understanding of the detrimental effects of immune-mediated M. pneumoniae infection in humans. Additionally, as similar mechanisms may be present in other chronic respiratory diseases, we believe these studies will also yield insights to a greater understanding of respiratory disease pathogenesis as a whole.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jerry W. Simecka, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107. E-mail address: jsimecka{at}hsc.unt.edu
3 Abbreviations used in this paper: LRN, lower respiratory lymph node; 2m, 2-microglobulin.
Received for publication August 24, 2001. Accepted for publication January 29, 2002.
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