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Departments of
* Cell Biology and
Immunology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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when Th cells were treated with PMA
and ionomycin. However, both proteins attenuated ionomycin-induced
cytosolic free calcium ([Ca2+ ]i), but not
thapsigargin-induced changes in [Ca2+]i. In
summary, inhibition of T cell proliferation by SP-A and SP-D occurs via
two mechanisms, an IL-2-dependent mechanism observed with accessory
cell-dependent T cell mitogens and specific Ag, as well as an
IL-2-independent mechanism of suppression that potentially involves
attenuation of
[Ca2+]i. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pulmonary surfactant is comprised of phospholipids and proteins, including four surfactant proteins (SP),3 SP-A, SP-B, SP-C, and SP-D (5). SP-A is the most abundant of the surfactant-associated proteins, while SP-D is present at about 1/10th the concentration of SP-A (3). SP-A and SP-D are both members of the collectin family of proteins, characterized by their N-terminal collagen-like domains and C-terminal carbohydrate recognition (lectin) domains (6). The collectins, which include the serum mannose binding lectin, function as soluble scavenger receptors by interacting via their lectin domains with sugars and glycolipids on pathogens. This interaction often results in enhanced phagocytosis and clearance of the pathogen in vitro (7). Recent in vivo studies with mice made deficient in SP-A by homologous recombination (8) demonstrate a role for the lung collectins in the innate immune response, since SP-A-deficient mice had a diminished capacity to fully recover from intratracheal injections of bacteria and viruses (9, 10, 11, 12) and increased susceptibility to LPS-induced lung inflammation (13).
In addition to participating in the innate immune response, SP-A and SP-D regulate the functions of cells of the adaptive immune system, including lymphocytes (14, 15, 16, 17). It has been known for >20 yr that lymphocytes obtained from lung lavage and activated with T cell mitogens are less proliferative than lymphocytes isolated from peripheral blood (18). Because alveolar lymphocytes are exposed to surfactant, Kaltreider (18) and others (19, 20) proposed that surfactant may suppress lymphocyte functions. They found that whole surfactant, containing lipids and proteins, as well as individual surfactant lipids inhibited mitogen-induced lymphocyte proliferation (18, 19, 20). We investigated whether SP-A and SP-D could also contribute to the observed inhibitory effects of surfactant on lymphocyte proliferation and showed that SP-A and SP-D inhibit T cell proliferation and IL-2 production in mixed cultures of PBMC stimulated with TCR/accessory cell (AC)-dependent T cell mitogens (14, 15, 16). SP-A and SP-D have also been shown to suppress the allergen-specific proliferative response of lymphocytes in mixed populations of PBMC from asthmatic patients (17). We propose that surfactant-mediated inhibition of T cell activation would help protect the delicate alveolar epithelium from inflammation-mediated damage that could occur if the T cells were constantly activated in the airspaces.
Because previous studies showing that SP-A and SP-D inhibit lymphocyte proliferation were conducted with mixed populations of lymphocytes and monocytes, it was not known whether SP-A and SP-D were acting directly on T lymphocytes or indirectly via a paracrine effect. The present study sought to address whether SP-A and SP-D act directly on T cells independent of accessory cells as well as to determine where these proteins act during the process of T cell activation and cytokine production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood was obtained from healthy volunteers
using a study protocol approved by Duke University institutional
review board. Alternatively, buffy coats were purchased from the
American Red Cross (Charlotte, NC). PBMC were isolated by a
one-step gradient centrifugation using Lymphoprep (Life Technologies,
Gaithersburg, MD). Washed cells were resuspended at a concentration of
2 x 106/ml in RPMI 1640 containing 10% FBS
(HyClone, Logan, UT) and antibiotic-antimycotic solution plus
gentamicin (Life Technologies). Cells were then added to either T-75 or
T-150 flasks (Nalge Nunc International, Rochester, NY) for 1 h at
37°C in 5% CO2. Plastic nonadherent cells were
recovered and transferred to new flasks for an additional 1-h
incubation. Initially, the Dynabead and Detach-a-Bead systems (Dynal,
Lake Success, NY) were used according to the manufacturer’s
specifications to positively select for CD4+
lymphocytes. These cells were cultured in RPMI 1640 medium (Life
Technologies) containing 10% FBS and antibiotic-antimycotic solution.
In later experiments the MACS (Miltenyi Biotec, Auburn, CA) cell
separation system was used to positively select (without plastic
adherence steps) for CD4+ cells. We routinely
obtained CD4+/CD3+ cells
with a purity of 
98% using either isolation procedure. mAbs,
anti-CD4-R-PE (clone 13B8.2), anti-CD8-PE (clone B9.11),
anti-CD3-PC5 (PE-cyanin-5, clone UCHT1), and appropriate isotype
controls were purchased from Immunotech (IO Test; Marseilles, France)
and were used to confirm cell purity by flow cytometry at Duke
University Flow Cytometry Facility.
To confirm that results obtained were not influenced by the method of T cell isolation, the MACS negative selection kit for isolation of CD4+ cells was employed. Harvested cells were then cultured in AIM V lymphocyte activation medium (Life Technologies) at a concentration of 1 x 105 cells/well in a flat-bottom, 96-well plate in a final volume of 100 µl. The purity of recovered Th cells was routinely >98% as determined by coexpression of CD3/CD4 and the absence of CD8, CD11b (Mac-1, R-PE, clone ICRF44) (4), and CD-19 (FITC, clone HIB19; all purchased from BD PharMingen, San Diego, CA). Activated Th cells were analyzed for IL-2R expression using anti-CD-25-CyChrome-conjugated Ab (BD PharMingen, clone M-A51).
Murine T cell hybridomas specific for the OVA peptide 258–276 on I-Ab were generated as previously described (21). The OVA peptide (IINFEKLTEWTSSNVMEER) was synthesized in the laboratory of Dr. D. Kappler (University of North Carolina, Chapel Hill, NC). Peptide-specific hybridomas were grown in DMEM (Life Technologies) containing 10% FBS and antibiotic-antimycotic solution. Th hybridomas were routinely analyzed for high expression of both CD3 and CD4 by FACS using anti-mouse CD3 R-PE (clone 29B) and anti-mouse CD4 FITC (clone H129.19; Life Technologies). Splenocytes, which were neither irradiated nor treated with mitomycin, were used as a source of APC for Ag presentation assays. Healthy C57BL/6 mice were euthanized by CO2 inhalation. All surgical and experimental procedures followed institutional animal care guidelines. A single-cell suspension of splenocytes was prepared by aseptically removing spleens and pressing the tissue through a 40-µm sterile nylon mesh. Cells were washed twice in serum-free medium before use in Ag presentation assays.
Flow cytometry
Cells (3 x 105) were resuspended in FACS staining buffer consisting of HBSS (Life Technologies), 1.0% (w/v) BSA (fraction V; Sigma, St. Louis, MO), and 0.1% (w/v) NaN3 (Sigma) in a final volume of 100 µl. Nonspecific binding was blocked by preincubation for 15 min on ice with mouse IgG (Sigma; 50 µg/ml). PBMC or purified Th cells were analyzed with labeled mAbs or isotype controls. These Abs were incubated with cells for 30 min on ice using amounts recommended by the manufacturer. Cells were washed twice and fixed in 1.5% buffered formalin.
Gates for FACS analyses were set through use of forward and side scatter parameters. Background fluorescence was determined by analyzing the appropriate isotype controls in tandem with samples. Samples were analyzed at least in duplicate, and a minimum of 10,000 gated events were analyzed per sample. An annexin V-FITC apoptosis detection kit (BD PharMingen) was used to measure cell viability and apoptosis of PBMC and CD3+/CD4+ lymphocytes. Analysis was conducted using a FACStar Plus flow cytometer (BD Biosciences, Mountain View, CA) and the CellQuest software package (BD Biosciences). Analysis of data from cytosolic free calcium ([Ca2+]i) assays was performed using the FlowJo software package (Tree Star, San Carlos CA).
[3H]thymidine incorporation assay
As a test of cell function, lymphocyte proliferation was measured using the [3H]thymidine incorporation assay. CD3+/CD4+ T cells were activated by treatment with PMA (1 ng/ml) and ionomycin (1 µM; Sigma). Activated cells (1 x 105 cells/well) were cultured in 96-well, flat-bottom plates (Nalge Nunc International) and treated with varying concentrations of human C1q; human, cow, or rat SP-A; and recombinant or native rat SP-D. To investigate the role of IL-2 in this culture system, some CD3+/CD4+ cultures were treated with a final concentration of 1 or 10 ng/ml of recombinant human (rh) IL-2 (R&D Systems, Minneapolis, MN). After 60 h of incubation, 1 µCi/well of [3H]thymidine was added to each well (sp. act., 6.7 Ci/mmol; ICN, Costa Mesa, CA). DNA from each well was harvested with a semiautomated cell harvester (Skatron Instruments, Lier, Norway). [3H]thymidine incorporated into DNA was measured by liquid scintillation spectrophotometry. All conditions were repeated at least three times and tested in a minimum of three independent experiments.
Ag presentation assay
OVA peptide (50 µg/ml) was added to 105 T cell hybridomas and 105 murine splenocytes in a final volume of 100 µl in the presence or the absence of SP-A, SP-D, or C1q (25 µg/ml) for 24 h. As an index of Ag presentation, IL-2 was quantified in cell-free culture supernatants using a commercially available ELISA kit (Endogen, Woburn, MA).
Cell cycle analysis
Th cells were cultured as outlined for the 3H incorporation assay. At 72 h cells were harvested by gentle aspiration with calcium/magnesium-free Dulbecco’s PBS, fixed in ice-cold 70% ethanol, and stained with propidium iodide (Sigma) (22). Data were collected by FACS and analyzed using the FlowJo software package.
Isolation of proteins
SP-A was isolated from lung lavage of silica-treated rats, normal bovine lungs, or lungs of patients with alveolar proteinosis (23) (24, 25). A non-butanol method of isolation was used to obtain SP-A from all sources. The method used was a modification of the version reported by Suwabe et al. (26). SP-A was eluted from the surfactant pellet with 5 mM Tris water (pH 7.4) containing 2 mM EGTA and 1 mM MgCl2. SP-D was isolated from the lung lavage of rats given intratracheal injections of silica 3 wk previously. Ultracentrifugation of the lavage was followed by maltose affinity chromatography and gel filtration chromatography (27). Recombinant rat SP-D expressed in CHO cells was also used for these studies. Human C1q was purchased from Advanced Research Technologies (San Diego, CA).
Cytokine assays
Th cells (2 x 105 cells/well) were
cultured in 96-well, flat-bottom, tissue culture plates in 200 µl
medium. Cultures were activated with PMA (1 ng/ml) and ionomycin (1
µM) in the presence or the absence of SP-A, SP-D, or C1q (12.5 and 25
µg/ml). Supernatants were harvested 24 and 48 later and assayed for
IL-2, IL-4, and IFN- using commercially available kits (R&D Systems
and Endogen (Woburn, MA)). Samples from each well were assayed in
duplicate.
Calcium release assay
Th cells were resuspended in dye loading buffer (HBSS, 1 mM calcium, 1 mM magnesium, and 0.5% (w/v) BSA (fraction V; Sigma)) at a concentration of 2 x 106 cells/ml. Indo-1/AM (Molecular Probes, Eugene, OR) was reconstituted in sterile DMSO (Sigma) at a concentration of 1 mg/ml; 2 µg of this suspension was added per milliliter of cells. Th cells were incubated at 37°C in the dark for 30 min. After washing away extracellular Indo-1/AM, the CD3+/CD4+ lymphocytes (2 x 106 cells/ml) were preincubated for 2 h at room temperature in the presence or the absence of SP-A, SP-D, or C1q (25 µg/ml; final volume, 1 ml). Capacitance calcium entry was induced by ionomycin (100 ng/ml) or thapsigargin (2 µM; Sigma) and measured by ratiometric analysis of [Ca2+]i by FACS. Data were analyzed with the FlowJo software package (Tree Star). Each response was quantified by measuring fluorescence at the time of the peak response. The kinetics of each response were analyzed by calculating the area under the curve during the entire response after addition of the intracellular calcium agonist. A 1 min baseline reading was also taken before activation of the cells.
An alternative method of performing calcium release assays used a ratiometric assay performed on a SPEX FluoroMax spectrophotometer (SPEX, Edison, NJ) with an excitation wavelength of 350 nm and emission wavelengths of 405 and 485 nm. CD3+/CD4+ cells were activated in a silica microcell (ISA/JY, Spex) containing 200 µl of a suspension of CD3+/CD4+ cells (2 x 106 cells/ml) and a magnetic stir bar. Again, a 1 min baseline reading was obtained, at which time cells were activated with thapsigargin (4 µM) alone or immediately after addition of EGTA (5 mM; Sigma). All data obtained using the spectrofluorometer were confirmed using FACS analysis.
Statistics
Statistical analysis was performed with the Primer for Biostatistics computer program and manual (28). Data from individual experiments were expressed as a percentage of the positive control tested in that experiment. To analyze differences between protein-treated cells (single concentration) and untreated cells, Student’s t test was used. ANOVA was used to determine differences among experimental doses. More specifically, a multiple comparison procedure, the Student-Newman-Keuls test, was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To address the question of whether accessory cells were required
for SP-A and SP-D to suppress lymphocyte proliferation, PBMC and cells
from each step of the CD4+ cell isolation
procedure were cocultured with SP-A, SP-D, or C1q and stimulated with
PMA and ionomycin. SP-A preparations isolated from a variety of sources
were tested in individual experiments, and the data were combined.
These sources were rat, human, or bovine. Recombinant rat SP-D was
tested, as was SP-D obtained from silica-treated rat lungs. Both SP-A
and SP-D inhibited [3H]thymidine incorporation
into whole PBMC treated with PMA and ionomycin (Table I
). Similar results were obtained with a
subpopulation of nonadherent cells obtained from the same donors. SP-A
and SP-D also suppressed [3H]thymidine
incorporation by a subset of the nonadherent cells that were depleted
of CD4+ cells during CD4+
cell purification. The results presented in Table I show that SP-A and
SP-D inhibited [3H]thymidine incorporation of
these cell populations in a dose-dependent manner.
CD3+/CD4+ cells were
isolated as described in Materials and Methods and were
activated with PMA and ionomycin. Both SP-A and SP-D inhibited
incorporation of [3H]thymidine by these highly
purified (98%) cells in a dose-dependent manner (Fig. 1). Apoptosis and necrotic cell death
were quantified at 24 and 72 h. No differences were found among
experimental groups at either time point (25 µg/ml protein) compared
with the untreated control group (data not shown).
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PMA- and ionomycin-treated CD4+ lymphocytes
were cultured under the same conditions as those outlined for the
proliferation assays, stained with propidium iodide, and used for cell
cycle analysis. Fig. 2 shows that SP-A
and SP-D, but not C1q, inhibited the number of Th cells that progressed
into the G2 + M phase of the cell cycle.
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Previously we had shown that T cell proliferation stimulated by
TCR/AC-dependent mitogens as well as IL-2 secretion was inhibited by
SP-A and SP-D and that addition of recombinant human (rh) IL-2 restored
proliferative activity to control values (14, 15, 16). In
contrast, SP-A, SP-D, or C1q (25 µg/ml) did not significantly alter
IL-2 secretion by Th cells stimulated with PMA and ionomycin measured
at 24 or 48 h (Fig. 3). Similar data
were obtained when IL-4 and IFN- were measured in these supernatants
(data not shown). We simultaneously performed proliferation assays to
compare the observed anti-proliferative effect with cytokine
production and found no correlation.
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show that when
CD3+/CD4+ T cells were
activated with a TCR/AC-independent mitogen (PMA and ionomycin), SP-A-
and SP-D-mediated inhibition of proliferation was not altered by
addition of rhIL-2 to a final concentration of 1 or 10 ng/ml.
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Because of the surprising nature of our results, showing that both
proteins could inhibit proliferation in an IL-2-independent manner, we
sought to reconfirm that the protein samples being tested did, in fact,
attenuate IL-2 production when activated in an AC/TCR-dependent manner.
Rather than rely on mitogens, we sought to assess the effect of these
proteins using the most physiologically appropriate stimulus possible.
This was accomplished by coculturing OVA-specific T cell hybridomas
with whole splenocytes and OVA peptide. Fig. 5 reveals that both SP-A (1159 ±
490 pg/ml) and SP-D (2511 ± 693 pg/ml) inhibited IL-2 production
in this system compared with the control group (7286 ± 2147
pg/ml). Doubling the concentration of OVA peptide (100 µg/ml) did not
have an impact on SP-A- and SP-D-mediated inhibition of IL-2 secretion
(data not shown).
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An increase in
[Ca2+]i is an essential
early event in the stimulation of lymphocytes by a variety of agents
(29). To test the possibility that SP-A and SP-D inhibit
increases in [Ca2+]i,
CD3+/CD4+ lymphocytes
loaded with Indo-1/AM were preincubated with SP-A, SP-D, or C1q. A 1
min baseline reading was obtained for each condition before treating
the cells with ionomycin (100 ng/ml). A representative experiment is
shown in Fig. 6. The data presented in
Fig. 6 demonstrate that SP-A and SP-D reduced the amount of
[Ca2+]i measured in the
CD3+/CD4+ lymphocytes after
ionomycin treatment. The use of a 4-fold lower concentration of
proteins (6.25 µg/ml) did not produce a significant difference
(n = 3; data not shown). SP-A- or SP-D-treated cells
activated with a 10-fold higher concentration of ionomycin (100 ng/ml)
did not respond differently from untreated cells (n =
2; data not shown).
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summarizes mean
(1-s interval) peak fluorescence data from three experiments. Treatment
of Th cells with either SP-A or SP-D (25 µg/ml) exerted a significant
inhibitory effect on the magnitude of the peak fluorescence in response
to ionomycin (33 and 22%, respectively). Conversely, C1q had a minimal
effect on peak fluorescence (6.4%). We did not observe differences in
peak fluorescence when
[Ca2+]i was measured in
Th cells activated by thapsigargin (data not shown).
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). SP-A and SP-D reduced the ionomycin-induced rise in
[Ca2+]i by 34 and 28%,
respectively. Conversely, C1q reduced Th cell responsiveness by <7%.
Similar negative results were obtained when cells were treated with
thapsigargin (data not shown). Importantly, the mean total fluorescence
of baseline readings were not significantly different from that of the
control group regardless of the agonist used. Furthermore,
CD3+/CD4+ cells that had
been pretreated with various proteins and then pulsed with EGTA (5 mM
final concentration) just before activation with thapsigargin
demonstrated that the
[Ca2+]i was not altered
by incubation with the proteins. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown here that SP-A and SP-D from a variety of sources
inhibited the proliferation of PBMC, plastic-nonadherent PBMC, and
CD4+-depleted plastic-nonadherent cells that had
been activated with PMA and ionomycin.
CD3+/CD4+ cell
proliferation was also inhibited by SP-A or SP-D. The inhibition of
phorbol ester/calcium ionophore-induced proliferation of
CD3+/CD4+ cells was not
associated with a decrease in IL-2, IL-4, or IFN- secretion, nor
could exogenous IL-2 rescue proliferation. SP-A and SP-D, but not C1q,
inhibited the number of Th cells that progressed into the
G2 + M phase of the cell cycle. Conversely, we
demonstrated that these same preparations of SP-A and SP-D suppressed
Ag-driven IL-2 production by OVA peptide-specific T cell hybridomas
when splenocytes were used as a source of APC. The mechanism by which
SP-A and SP-D suppress Th cell proliferation was investigated by
measuring one parameter important in the signaling capacity of Th
cells, [Ca2+]i. SP-A and
SP-D both readily attenuated the release of large amounts of
[Ca2+]i generated by
ionomycin, but had no effect on thapsigargin-induced capacitance
calcium entry (CCE). Overall, these results imply that SP-A and SP-D
share at least two common mechanisms for inhibiting Th cell function:
an IL-2-independent and an IL-2-dependent mechanism.
SP-A and SP-D directly inhibit lymphocyte proliferation independently of AC
Previous studies with whole PBMC in cultures and AC-dependent T cell mitogens provided evidence that SP-A and SP-D inhibit T cell proliferation, but raised the question of whether SP-A and SP-D could act directly on lymphocytes in isolation (14, 15, 16). We found that neither the presence of AC nor the removal of AC by plastic adherence and magnetic bead technology influenced the responses of PMA and ionomycin-stimulated cells to SP-A and SP-D. These results suggest that SP-A or SP-D can directly suppress the proliferation of Th cells. Requirements for T cell activation, proliferation, and cytokine secretion are highly dependent on the differentiation state of the cells, the method of isolation, and the activating stimulus. Moreover, the inhibitory effects of SP-A and SP-D could be acting at one or more steps in the activation cascade. Further studies with other lymphocyte subsets will be required to determine whether SP-A or SP-D acts similarly on other cell types and to determine the precise mechanism(s) by which SP-A and SP-D mediated the observed inhibition.
We focused our study on isolated highly purified CD3+/CD4+ cells to determine the effects of SP-A and SP-D on proliferation and cytokine production induced by PMA and ionomycin. Decreased proliferation did not coincide with alterations in cell death or apoptosis (24 and 72 h; data not shown). FACS analysis of CD4 and CD25 expression did not differ among experimental groups (SP-A, SP-D, C1q, or no protein) on the basis of percentage of positive cells or mean fluorescence (data not shown).
Both SP-A and SP-D inhibit T lymphocyte proliferation in an IL-2-dependent and an IL-2-independent manner
We have previously demonstrated that SP-A and SP-D inhibit T
lymphocyte proliferation in an IL-2-dependent manner when AC are
present. In those studies proliferation was stimulated by a variety of
agonists that signal through the TCR/CD3 complex and are AC dependent
(14, 15, 16). In those studies we found that SP-A and SP-D
reduced levels of IL-2 in the culture medium and that addition of
exogenous IL-2 restored the proliferative response. In addition, in
recent studies (data not shown) we have found that SP-A and SP-D both
inhibited the proliferation of highly purified
CD3/CD4+ cells that were activated via the TCR
complex with soluble anti-CD3 and anti-rat
(Fab')2. In this case, rhL-2 also rescued the
proliferative response (data not shown). In contrast, when cells were
activated with immobilized anti-CD3, proliferation was inhibited by
SP-A and SP-D, but IL-2 was not decreased (data not shown). In the
current study in which AC were not present, SP-A and SP-D inhibited the
proliferation of highly purified CD3/CD4+ cells
activated with PMA and ionomycin, but IL-2 production was not
inhibited. Furthermore, these same preparations of SP-A and SP-D both
dampened IL-2 production by Ag-exposed T cell hybridomas (Fig. 5), yet
had no effect on the high basal proliferative activity of the T cell
hybridomas or Jurkat cells (data not shown). Taken together, these data
suggest that both SP-A and SP-D can inhibit lymphocyte proliferation in
both an IL-2-dependent and an IL-2-independent manner. We cannot
exclude the possibility that the IL-2-dependent inhibition seen in the
presence of AC involves an interaction of SP-A or SP-D with the AC;
further studies will address this possibility. Studies by Carreno and
co-workers (30) provided a precedent for multiple levels
of regulation. They reported that CTLA-4 (CD-152) can either induce a
down-regulating signal directly or disrupt CD-28/B7-mediated signaling
by competitive binding (30). In addition, studies by
Brunner et al. (31) concluded that CTLA-4 can suppress T
cell activation at different stages of the activation process, and that
these events may be both IL-2 dependent and IL-2 independent. Finally,
it has been shown that T cells proliferate in an IL-2-dependent and an
IL-2-independent manner in vivo (32, 33).
SP-A and SP-D inhibit ionomycin-induced [Ca2+]i
In an attempt to better understand the mechanisms by which SP-A
and SP-D inhibit T cell proliferation, we examined
[Ca2+]i as a measurement
of the signaling capacity of Th cells (29). Both ionomycin
and thapsigargin initiate the process known as CCE (34).
CCE is the influx of Ca2+ across the plasma
membrane by store-operated Ca2+ channels in
response to depletion of intracellular stores of
Ca2+ (34). The type of
store-operated Ca2+ channels expressed in T cells
are Ca2+ release-activated
Ca2+ channels and are considered to be essential
for Ca2+ influx resulting in activation of T
cells (34), but not the sole source of incoming
Ca2+ (34). Novel,
Ca2+ release-activated nonselective cation
channels have been identified in human PBMC and are functionally
different from Ca2+ release-activated
Ca2+ channels (35).
Ca2+ release-activated nonselective cation
channels can also be activated by chemicals such as ionomycin and
thapsigargin (35). Our results showed that SP-A- or
SP-D-treated lymphocytes had a decreased response to ionomycin-induced
changes in [Ca2+]i, but
surprisingly neither SP-A nor SP-D altered thapsigargin-induced
increases in [Ca2+]i.
This suggests that SP-A and SP-D can act on
CD3+/CD4+ cells by means of
ion channels employed exclusively or to a much larger extent in
ionomycin-induced
[Ca2+]i, but not
thapsigargin-induced
[Ca2+]i. Other studies
support our hypothesis that SP-A and SP-D regulate cell signaling. For
example, SP-A inhibited ionomycin-induced production and release of
IL-8 from human peripheral blood eosinophils (36) and
attenuated surfactant release from type II pneumocytes
(37) as well as TNF-
release from LPS-treated alveolar
macrophages (24). These findings suggest that SP-A and
SP-D exert an inhibitory effect by a very basic component of cell
signaling.
Physiological significance
Pulmonary surfactant is the physiological medium in which the
pulmonary immune system first encounters Ag. Ample opportunity exists
for delivery of Ag to the lung through inhalation of 
10,000 liters
of non-sterile air/day. In vitro and in vivo experiments suggest that
SP-A and SP-D function as soluble scavenger receptors to maintain
immunological homeostasis within the lung by several different means.
First, SP-A and SP-D act as opsonins that enhance pathogen clearance.
Second, both proteins modulate the magnitude of the pulmonary immune
response to impede the conversion of areas of gas exchange to scar
tissue.
It is important to note that the estimated number of lymphocytes
(5 x 108) comprise 10% of all leukocytes
in the bronchoalveolar space of healthy human lungs and that 60% of
these lymphocytes are CD4+ cells, representing
5% of the total circulating blood pool of lymphocytes
(38). Because of their continuous exposure to non-sterile
air while residing in mechanically fragile structures such as the
alveoli, it is our contention that pulmonary surfactant and SP-A and
SP-D are important elements in attenuating lymphocyte function to
preserve effective gas exchange. This possibility is also supported by
our previous studies showing that SP-A and SP-D inhibit IL-2 production
and lymphocyte proliferation in the presence of AC (14, 16) and the study of Wang et al. (17) showing that
SP-A and SP-D suppressed lymphocyte proliferation of PHA-treated human
PBMC as well as allergen-induced proliferation of PBMC obtained from
asthmatic children (17). SP-A and SP-D were also shown to
dampen allergen-induced histamine release from these cells
(17). Our current study extends these previous
observations to show that SP-A and SP-D can directly inhibit the
proliferation of T cells and thus provides evidence that there are
multiple pathways for regulating this important process.
In vivo experiments with SP-A-deficient mice showed that SP-A has anti-inflammatory activity (13). Characterization of the SP-D knockout mouse under normal conditions showed a significant number of monocytic cells that infiltrated the peribronchiolar-perivascular regions (39). Furthermore, hypertrophic alveolar macrophages were also found. Macrophages from SP-D-deficient mice had a 10-fold increase in hydrogen peroxide production. Ultimately, SP-D knockout mice developed pulmonary emphysema and subpleural fibrosis in conjunction with chronic inflammation (39). Our data suggest that while SP-A and SP-D appear to suppress certain Th cell functions by a similar mechanism, the phenotypes of the respective deficient mice show that each protein also plays a unique role in maintaining immunological homeostasis within the lung. It is not fully known to what extent these proteins differ in their immunomodulatory repertoire, especially with respect to naive and memory cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jo Rae Wright, Department of Cell Biology, Duke University Medical Center, Box 3709, 438 Nanaline Duke Building, Durham, NC 27710. E-mail address: j.wright{at}cellbio.duke.edu
3 Abbreviations used in this paper: SP, surfactant protein; AC, accessory cell; [Ca2+ ]i, cytosolic free calcium; CCE, capacitance calcium entry; rh, recombinant human.
Received for publication May 14, 2002. Accepted for publication September 5, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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D. Jain, E. N. Atochina-Vasserman, Y. Tomer, H. Kadire, and M. F. Beers Surfactant Protein D Protects against Acute Hyperoxic Lung Injury Am. J. Respir. Crit. Care Med., October 15, 2008; 178(8): 805 - 813. [Abstract] [Full Text] [PDF]
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 L. Mahajan, T. Madan, N. Kamal, V. K. Singh, R. B. Sim, S. D. Telang, C. N. Ramchand, P. Waters, U. Kishore, and P. U. Sarma Recombinant surfactant protein-D selectively increases apoptosis in eosinophils of allergic asthmatics and enhances uptake of apoptotic eosinophils by macrophages Int. Immunol., August 1, 2008; 20(8): 993 - 1007. [Abstract] [Full Text] [PDF]
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 B. Samten, J. C. Townsend, Z. Sever-Chroneos, V. Pasquinelli, P. F. Barnes, and Z. C. Chroneos An antibody against the surfactant protein A (SP-A)-binding domain of the SP-A receptor inhibits T cell-mediated immune responses to Mycobacterium tuberculosis J. Leukoc. Biol., July 1, 2008; 84(1): 115 - 123. [Abstract] [Full Text] [PDF]
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 C. L. S. George, K. L. Goss, D. K. Meyerholz, F. S. Lamb, and J. M. Snyder Surfactant-Associated Protein A Provides Critical Immunoprotection in Neonatal Mice Infect. Immun., January 1, 2008; 76(1): 380 - 390. [Abstract] [Full Text] [PDF]
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 C.-H. Shen, Q. Ge, O. Talay, H. N. Eisen, A. Garcia-Sastre, and J. Chen Loss of IL-7R and IL-15R Expression Is Associated with Disappearance of Memory T Cells in Respiratory Tract following Influenza Infection J. Immunol., January 1, 2008; 180(1): 171 - 178. [Abstract] [Full Text] [PDF]
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 R. E. Oberley, K. L. Goss, D. S. Hoffmann, K. A. Ault, T. L. Neff, K. H. Ramsey, and J. M. Snyder Regulation of surfactant protein D in the mouse female reproductive tract in vivo Mol. Hum. Reprod., December 1, 2007; 13(12): 863 - 868. [Abstract] [Full Text] [PDF]
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 G. de Laurentiis, L. Vitiello, L. Racioppi, F. Perna, M. Galgani, G. Merola, P. Carratu, M. Maniscalco, S. Marsico, and M. Sofia CD8+ T-cell alveolitis in familial pulmonary alveolar microlithiasis Eur. Respir. J., July 1, 2007; 30(1): 165 - 171. [Abstract] [Full Text] [PDF]
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A. P. Senft, T. R. Korfhagen, J. A. Whitsett, and A. M. LeVine Surfactant protein D regulates the cell surface expression of alveolar macrophage beta2-integrins Am J Physiol Lung Cell Mol Physiol, February 1, 2007; 292(2): L469 - L475. [Abstract] [Full Text] [PDF]
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J. R. Wright The "wisdom" of lung surfactant: balancing host defense and surface tension-reducing functions Am J Physiol Lung Cell Mol Physiol, November 1, 2006; 291(5): L847 - L850. [Full Text] [PDF]
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S. Kunzmann, J. R. Wright, W. Steinhilber, B. W. Kramer, K. Blaser, C. P. Speer, and C. Schmidt-Weber TGF-beta1 in SP-A preparations influence immune suppressive properties of SP-A on human CD4+ T lymphocytes Am J Physiol Lung Cell Mol Physiol, October 1, 2006; 291(4): L747 - L756. [Abstract] [Full Text] [PDF]
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D. A. Fraser, S. S. Bohlson, N. Jasinskiene, N. Rawal, G. Palmarini, S. Ruiz, R. Rochford, and A. J. Tenner C1q and MBL, components of the innate immune system, influence monocyte cytokine expression J. Leukoc. Biol., July 1, 2006; 80(1): 107 - 116. [Abstract] [Full Text] [PDF]
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A. Haczku, Y. Cao, G. Vass, S. Kierstein, P. Nath, E. N. Atochina-Vasserman, S. T. Scanlon, L. Li, D. E. Griswold, K. F. Chung, et al. IL-4 and IL-13 Form a Negative Feedback Circuit with Surfactant Protein-D in the Allergic Airway Response J. Immunol., March 15, 2006; 176(6): 3557 - 3565. [Abstract] [Full Text] [PDF]
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T. L. Bonfield, C. M. Swaisgood, B. P. Barna, C. F. Farver, M. S. Kavuru, and M. J. Thomassen Elevated gelatinase activity in pulmonary alveolar proteinosis: role of macrophage-colony stimulating factor J. Leukoc. Biol., January 1, 2006; 79(1): 133 - 139. [Abstract] [Full Text] [PDF]
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 J. Meschi, E. C. Crouch, P. Skolnik, K. Yahya, U. Holmskov, R. Leth-Larsen, I. Tornoe, T. Tecle, M. R. White, and K. L. Hartshorn Surfactant protein D binds to human immunodeficiency virus (HIV) envelope protein gp120 and inhibits HIV replication J. Gen. Virol., November 1, 2005; 86(11): 3097 - 3107. [Abstract] [Full Text] [PDF]
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D. C. Malherbe, V. J. Erpenbeck, S. N. Abraham, E. C. Crouch, J. M. Hohlfeld, and J. R. Wright Surfactant protein D decreases pollen-induced IgE-dependent mast cell degranulation Am J Physiol Lung Cell Mol Physiol, November 1, 2005; 289(5): L856 - L866. [Abstract] [Full Text] [PDF]
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 C.-H. Yang, J. Szeliga, J. Jordan, S. Faske, Z. Sever-Chroneos, B. Dorsett, R. E. Christian, R. E. Settlage, J. Shabanowitz, D. F. Hunt, et al. Identification of the Surfactant Protein A Receptor 210 as the Unconventional Myosin 18A J. Biol. Chem., October 14, 2005; 280(41): 34447 - 34457. [Abstract] [Full Text] [PDF]
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B. Janic, T. M. Umstead, D. S. Phelps, and J. Floros Modulatory effects of ozone on THP-1 cells in response to SP-A stimulation Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L317 - L325. [Abstract] [Full Text] [PDF]
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R. E. Oberley, K. L. Goss, K. A. Ault, E. C. Crouch, and J. M. Snyder Surfactant protein D is present in the human female reproductive tract and inhibits Chlamydia trachomatis infection Mol. Hum. Reprod., December 1, 2004; 10(12): 861 - 870. [Abstract] [Full Text] [PDF]
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Y.-S. Hahn, C. Taube, N. Jin, K. Takeda, J.-W. Park, J. M. Wands, M. K. Aydintug, C. L. Roark, M. Lahn, R. L. O'Brien, et al. V{gamma}4+ {gamma}{delta} T Cells Regulate Airway Hyperreactivity to Methacholine in Ovalbumin-Sensitized and Challenged Mice J. Immunol., September 15, 2003; 171(6): 3170 - 3178. [Abstract] [Full Text] [PDF]
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I. Y. Haddad, C. Milla, S. Yang, A. Panoskaltsis-Mortari, S. Hawgood, D. L. Lacey, and B. R. Blazar Surfactant protein A is a required mediator of keratinocyte growth factor after experimental marrow transplantation Am J Physiol Lung Cell Mol Physiol, September 1, 2003; 285(3): L602 - L610. [Abstract] [Full Text] [PDF]
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Workshop on Lung Disease and the Environment: Where Do We Go from Here? Am. J. Respir. Crit. Care Med., July 15, 2003; 168(2): 250 - 254. [Full Text] [PDF]
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0.05 compared with cultures treated with
PMA and ionomycin alone.
