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*
Pulmonary Center and
Department of Pathology, Boston University School of Medicine, Boston, MA 02188; and Boston Veterans Affairs Medical Center, Boston, MA 02130
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Osteopontin (OPN)5 is a noncollagenous adhesive matrix protein normally found in bone and at epithelial surfaces that contains the arginine-glycine-aspartate (RGD)-binding motif common to many extracellular matrix (ECM) proteins (4, 5, 6). Unlike other matrix proteins, OPN contains a thrombin cleavage site adjacent to the RGD domain which, upon cleavage, alters the adhesive capacity of OPN (7, 8, 9). Studies have reported both the augmentation and inhibition of OPN-mediated adhesion by thrombin cleavage (7, 8, 9). In vitro, OPN promotes integrin- and CD44-mediated cell adhesion and chemotaxis in osteoclasts, smooth muscle cells, monocytes, and B lymphocytes (10, 11, 12, 13, 14, 15). These findings have led to research identifying a role for OPN in bone resorption, neoplastic transformation, atheromatous plaque formation, and dystrophic calcification of inflamed and damaged tissues (5, 16, 17, 18, 19).
Recent data suggest a role for OPN in the immune response. It is produced in a regulated fashion by macrophages, T cells, and NK cells (20, 21). OPN injected s.c. induces macrophage migration, and in vitro it supports monocyte adhesion and migration (13, 22). In addition, the expression of OPN by macrophages and T cells in heavily calcified prosthetic heart valves suggests that OPN may play a role in the cell-mediated process of dystrophic calcification (19).
OPN is also important in the genetic resistance to intracellular pathogens (20). In mice, the OPN gene maps to the same locus as the immune mediator of natural resistance to the intracellular pathogen Rickettsia tsutsugamushi. Alleles of this gene that result in deficient early production of OPN in response to R. tsutsugamushi are associated with disease susceptibility (20). We and others have recently described the expression of OPN at sites of granuloma formation in tuberculosis and silicosis and its up-regulation in alveolar macrophages infected with mycobacteria (23, 24). The function of OPN in granulomatous inflammation is unknown. In view of these data, we sought to characterize the in vivo expression of OPN in the granulomas of sarcoidosis and to define the in vitro effects of OPN on T cell function.
In this report, we demonstrate the expression of OPN in granulomas of pulmonary sarcoidosis and note a characteristic pattern of prominent lymphocyte staining. Based on these observations, we characterize the effects of OPN on human T lymphocyte function in vitro. OPN binds to T cells, supports T cell adhesion and chemotaxis, and costimulates resting T cell proliferation in response to ligation of the Ag receptor. Adhesion is modulated by thrombin cleavage of OPN. These data show that OPN is a T cell-associated, RGD-containing protein with cytokine/chemokine-like functions and that its T cell interactions are regulated by thrombin cleavage. The juxtaposition of OPN between the cell-mediated response and the coagulation cascade suggests a novel role by which matrix proteins regulate the immune response. In view of these findings it is possible that OPN may regulate the intensity of sarcoid granulomatous inflammation by recruitment and immobilization of T lymphocytes and by modulating T cell function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human PBMC from normal donors were separated by Ficoll-Hypaque density gradient centrifugation. Monocytes and B cells were removed on nylon wool columns as previously described (25). Resting CD3-positive T cells were isolated by immunomagnetic negative selection with Dynabeads M-450 (Dynal, Great Neck, NY) (26, 27). Negative selection using a mixture of mAbs including HLA DR on activated T cells, B cells, and monocytes; CD20 on B cells; CD16 on NK cells; and CD14 on monocytes resulted in cell populations that were >95% CD3+ and were completely depleted of monocytes as determined by FACS analysis and the lack of a proliferative response to optimal concentrations of PHA (PHA-L form; Sigma, St. Louis, MO) (28).
Abs and reagents
The following Abs were used. Anti-OPN Abs, OP148APA (kindly donated by D. Senger, Boston, MA), a polyclonal Ab directed to human milk OPN, MPIIIB10 (specific for rat and human OPN), and its isotype control, QH1 (specific for quail vascular endothelial cells), purchased from the Developmental Studies Hybridoma Bank (Iowa City, IA), were used for immunohistochemistry; polyclonal rabbit anti-fibronectin (FN) Ab (No. A0245; DAKO, Carpinteria, CA) for immunohistochemistry; anti-CD3 Abs, monoclonal anti-CD3 (Clone HIT3a, PharMingen, San Diego, CA) for T cell proliferation, and polyclonal anti-CD3 (No. A0452; DAKO) for immunohistochemistry.
For all experiments we used endotoxin-free OPN, purified from human milk over an Ab column (kindly donated by D. Senger, Boston MA) (29). Human FN and thrombin (3000 National Institute of Health units/mg protein) were purchased from Sigma, and rat tail collagen was purchased from Life Technologies (Grand Island, NY).
Thrombin cleavage of OPN
In the indicated experiments, OPN was cleaved in the manner described by Senger et al. (29). Purified OPN was incubated with purified human thrombin at a concentration of 18 units thrombin/ml for 60 min at 37°C. Protein cleavage was confirmed by SDS-PAGE and Western blot with OP148APA Ab.
Immunohistochemical staining
Lung biopsies from six patients with pulmonary sarcoidosis were obtained from the Gaensler Lung Archive at the Boston University Pulmonary Center and the Boston Veterans Affairs Hospital. These specimens were paraffin embedded using standard techniques. Immunohistochemical staining was facilitated with a Ventana ES automated stainer (Ventana, Tuscon, AZ). Serial sections 5 µm thick were cut and baked for 1 h at 60°C onto positively charged slides and then deparaffinized with xylene and hydrated in graded alcohol washes to water. Slides were then processed with Ventana’s protease I reagent for 4 min and incubated with primary Abs for 32 min at 42°C. Staining was detected with a diaminobenzidine kit. Images were photographed with Kodak Ektachrome film and scanned into Adobe Photoshop to create composite figures.
Primary Abs, anti-OPN mAb, MPIIIB10, and isotype control QH1 were used at 1:150 dilution of hybridoma supernatant (500 and 625 ng/ml, respectively). Polyclonal rabbit anti-human FN and anti-human CD3 Abs were used at a concentration of 1:200 and 1:150, respectively. Isotype control rabbit anti-IgG polyclonal Ab (Organo Teknika, Durham, NC) was used at similar concentrations. The above concentrations were selected based on characteristic and selective staining patterns of positive control paraffin-embedded tissue samples (human kidney for OPN and human tonsil for FN and CD3). All slides were read by a blinded pathologist who graded the intensity of staining as follows: 0 = no staining; 1 = weak staining; 2 = strong staining.
Migration assay
T cell migration was assayed using a modified 48-well Boyden chemotaxis chamber (30). Fifty microliters of a T lymphocyte suspension containing 10 x 106 cells/ml of M199-HPS with 0.4% BSA was added to the upper chambers, separated from various concentrations of human milk OPN by an 8-µm pore size nitrocellulose filter, and incubated at 37°C for 3 h. The filters were fixed, stained, dehydrated, and mounted using standard histologic methods. Lymphocyte migration was quantitated by counting the total number of cells migrating beyond a certain depth, with that depth set to give a baseline migration under control conditions of 5–15 cells per high power field (hpf). Five hpf were counted for each of duplicate wells, and results were calculated as mean cells/hpf ± SD; for purposes of comparison data are expressed as mean percentage of control migration ± SEM. Gradient dependence was determined by a checkerboard technique (30). OPN was placed only below the filter (chemoattractant present with concentration gradient), both above and below the filter (chemoattractant present without concentration gradient), or only above the filter (reverse gradient).
Adhesion assay
The adhesion of T cells to OPN was assessed utilizing a fluorometric adhesion assay (31, 32). Briefly, 96-well, non-tissue culture-treated plates were incubated overnight with various concentrations of either OPN, FN, or rat collagen in PBS at 4°C. OPN adhesion was confirmed by Ab detection ELISA using OP148APA mAb. The plates were washed, and the nonspecific binding sites were blocked by incubation with 2.5% BSA/PBS for 2 h at 37°C. T cells were labeled with the acetoxymethyl fluorescein ester Calcein AM (Molecular Probes, Eugene, OR), by incubation at room temperature for 30 min. The cells were washed, activated with PMA (Sigma) at a concentration of 5ng/ml, and then 75,000 cells were added to each well. The cells were allowed to settle at 4°C for 1 h and then were rapidly warmed in a water bath to 37°C for 10 min, a time previously determined to give maximal adhesion. The wells were washed five times, and the fluorescence of the remaining adherent cells was counted on a plate fluorometer (Flostar, BMG, Offenburg, Germany). The percentage of adherent cells was calculated using a standard curve derived for each adhesion assay. The results are expressed as the mean ± SD percentage of cells binding from triplicate wells.
Proliferation assay
Proliferation assays to demonstrate costimulation were performed as previously described (26). Ninety-six-well, non-tissue culture-treated microtiter plates were prepared by incubating the indicated concentrations of anti-CD3 mAb overnight at 4°C in PBS. Unbound Ab was removed, and various concentrations of ECM proteins were incubated overnight at 4°C. OPN adhesion was confirmed by ELISA. Human T cells, 50,000 in 200 µl of RPMI 1640 supplemented with glutamine, 10% heat inactivated FCS, penicillin, and streptomycin, were added to each well and cultured for 72 h at 37°C. [3H]thymidine (1 µCi/well) was added for the last 12 h of the assay, and the cells were harvested onto filters using an automated cell harvester. Where indicated, PMA was added at a concentration of 1 ng/ml of cells and incubated either with immobilized anti-CD3 or alone in a similar manner as outlined. Results are expressed as the arithmetic mean cpm ± SD of triplicate cultures.
Statistical analysis
Data are expressed as mean ± SD. Results were compared for
significance using Student’s paired two-tailed t test.
P values 
0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OPN expression was studied in open lung biopsies from six patients
with pulmonary sarcoidosis (Table I
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These patients varied in demographics, clinical indices of disease
activity, and chest x-ray findings and represent stages in the spectrum
of pulmonary sarcoidosis from early active to advanced end-stage
disease. Tissue sections were stained with anti-OPN (clone
MPIIIB10) or isotype control (clone QH1) mAb as previously described
(21, 23). Widespread OPN expression was seen in sarcoid granulomas
(Fig. 1, a and b).
A pattern showing predominant staining of the peripheral lymphocyte
rich component of granulomas was a characteristic observation in all
patients (Fig. 1b). Using serial sections, we demonstrated
intense OPN staining of CD3-positive cells (T lymphocytes) (Fig. 1, e and f). The central core of the granulomas
containing macrophages, epithelioid cells, and multinucleated giant
cells also exhibited OPN staining; however, this was less than that
seen in lymphocytes (Fig. 1b). The ECM did not stain for OPN
(Fig. 1a). No staining was seen with the isotype control Ab
(Fig. 1c). To determine the specificity of OPN expression in
inflammatory lung disease, we stained lung biopsies from four patients
with acute respiratory distress syndrome (ARDS), a neutrophil-mediated
disease. In contrast to the widespread expression of OPN in
sarcoidosis, OPN was seen only in airway epithelium and scattered
macrophages in ARDS (Fig. 1d). As we have previously
reported, this staining of airway epithelium is seen in inflamed but
not normal lung (23). In summary, sarcoid granulomas exhibit a
characteristic pattern of strong lymphocyte-associated OPN staining.
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OPN expression was determined in granulomas of varying cellularity
and maturity (Table I
; Fig. 2,
a–f). The early cellular granulomas (Fig. 2a) and intermediate granulomas expressing both cellular and
matrix elements (Fig. 2b) showed OPN staining (Fig. 2, d and e) of lymphocytes and histiocytes but not
matrix. The late fibrotic granulomas (Fig. 2c) exhibited
significantly less OPN expression, which was limited to multinucleated
giant cells (Fig. 2f). This pattern was in contrast to that
seen with staining for FN, another matrix protein with immunomodulatory
functions that is expressed in sarcoidosis (Table I; Fig. 2, g–i) (26, 33). FN was present in cellular and
stromal elements within and surrounding granulomas with relative
sparing of lymphocyte rich areas (Fig 2, g–i).
In addition, diffuse matrix-associated staining for FN was seen in all
granulomas, irrespective of cellularity and maturity (Table I; Fig. 2, g–i). Again, no staining was seen with isotype
control (Fig 2, j–l). These data demonstrate
that OPN expression is more pronounced in the early cellular granulomas
of sarcoidosis and that this pattern of expression is distinct from
that seen with FN.
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Matrix proteins can modulate lymphocyte function (34). Based on
OPN immunoreactivity in T cell areas of granulomas and its known
ability to induce migration in monocytes and a murine T cell hybridoma,
we postulated that OPN would modulate human T cell migration (12, 13).
The ability of OPN to induce T cell chemotaxis was analyzed using a
modified Boyden chamber. OPN induced chemotaxis in monocyte-depleted
nylon wool nonadherent T cells (NWNTs) at OPN concentrations between
150 ng/ml and 16 µg/ml (2.5–250 nM). The experiment was repeated
three times with similar results, and a representative experiment is
shown in Fig. 3. In all experiments, peak
migration was seen between 2–4 µg/ml (30–60 nM), although in two
experiments significant migration was noted at concentrations as low as
150 ng/ml. Although it is possible that this represents chemotaxis
mediated by different receptors, it is more likely due to intrinsic
variation in the bioassay. The magnitude of chemotaxis seen with OPN
(200–300% over migration under control conditions), was similar to
that seen in this assay with other lymphocyte chemoattractants such as
RANTES and IL-16.
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) was
employed to determine whether this migration was chemotactic (gradient
dependent) or chemokinetic in nature (random migration induced by
chemoattractants in zero gradient, obtained by adding equal amounts of
OPN to the upper and lower chambers) (30). By this analysis, the
response of T cells to OPN was present solely when a gradient existed
(OPN in the bottom well only). Chemokinetic activity was absent, and,
in fact, high concentrations of OPN in the upper chamber significantly
reduced migration below that seen with control. Identical results were
obtained in two separate experiments. Thus, OPN induces chemotactic
migration in human T cells.
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Coordinated migration and adhesion is essential to the recruitment
of T cells to inflammatory sites. Having demonstrated OPN-induced T
cell chemotaxis, we tested the ability of OPN to support T cell
adhesion using a fluorometric adhesion assay. Although resting T cells
did not adhere to immobilized OPN, PMA activation resulted in
significant T cell:OPN adhesion. Results from three experiments showed
that, at a concentration of 5 µg/ml of OPN, 27 ± 6% (mean
± SD) of PMA-activated T lymphocytes adhered to OPN. This was
significantly (p < 0.05) greater than
lymphocyte adhesion to control wells (mean ± SD, 11 ± 1%)
but less than adhesion to FN (mean ± SD, 57 ± 15%). A
representative experiment is shown in Fig. 4. The requirement for PMA activation is
a recognized phenomenon common to integrin receptors and facilitates
adhesion by clustering or altering the affinity of cell surface
adhesion molecules (35, 36).
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In view of the ability of T cells to adhere to OPN, and the
known modulatory effect of thrombin cleavage on tumor cell adhesion to
OPN, we tested the hypothesis that thrombin-cleaved OPN would support
increased T cell adhesion compared with uncleaved OPN. OPN was cleaved
by thrombin and immobilized on microtiter plates, and adhesion was
compared with uncleaved OPN (Fig. 5).
Results from four experiments demonstrated that the cleaved fragments
of OPN supported adhesion of a greater percentage of T cells (mean
± SD, 60 ± 15%, p < 0.05) than uncleaved OPN
(mean ± SD, 35 ± 5%) and a similar percentage to that seen
with FN (mean ± SD, 70 ± 20%). T cell adhesion to
thrombin-treated rat collagen did not significantly increase.
Therefore, OPN is a potent substrate for T cell adhesion, and this
adhesion is positively modulated by thrombin cleavage.
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Having demonstrated that OPN is widely expressed in T cells of
sarcoidosis, a disease characterized by chronic T cell activation and
proliferation, we postulated that OPN would modulate T cell
proliferation. Immobilized anti-CD3 mAb ligates the TCR and
provides a partial, but submaximal, signal for T cell proliferation
that is enhanced by an appropriate costimulus. We observed that OPN
markedly amplified this CD3-mediated proliferative response in
monocyte-depleted NWNTs (Fig. 6).
Although high concentrations of anti-CD3 mAb engaged the TCR
sufficiently to trigger some T cell proliferation, at lower or
suboptimal doses the proliferation was negligible (Fig. 6). At all
concentrations of anti-CD3 mAb, the presence of immobilized OPN
resulted in significant T cell proliferation (Fig. 6). Despite
significant donor variation in the absolute degree of proliferation as
measured by [3H]thymidine incorporation, over several
experiments the T cell proliferative response in the presence of
coimmobilized OPN was two- to sevenfold greater than that seen with
anti-CD3 mAb alone (n = 10, mean ± SD,
4.5 ± 2.5-fold). Although variability in the magnitude of
proliferation between experiments makes it impossible to compare the
potency of various costimuli in vitro, OPN-mediated costimulation was
similar to the response observed with anti-CD3 mAb combined with
either FN or PMA, both known costimulants (Fig. 7) (26, 28).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In sarcoidosis the initial granulomatous response involves the coordinated recruitment and focused adhesion of both macrophages and activated T cells (38). Later, possibly as the Ag inducing the response is cleared or contained, granulomas become acellular and fibrotic. The coordinated expression, both in time and space, of different components of the ECM may influence and regulate this response. In this regard, we identified OPN predominantly in the T cell-rich areas of sarcoid granulomas and noted that its expression was limited or absent in acellular fibrotic granulomas. Since both macrophages and activated T cells can produce OPN, it is unclear whether this lymphocyte-associated staining represents T cell adhesion to macrophage-derived OPN or de novo expression of OPN by T lymphocytes (20, 21). These findings suggest that OPN may interact with and modulate the function of T cells in sarcoidosis. Furthermore, the more pronounced expression of OPN in active cellular granulomas supports a potential role for OPN early in granuloma formation.
The expression of OPN in granulomas differed from that of FN, another matrix protein known to support T cell adhesion and proliferation (26). In contrast to the cell-associated expression of OPN, FN was predominantly associated with the ECM. Furthermore, unlike OPN, FN was found in the matrix of both highly cellular inflammatory granulomas as well as acellular fibrotic granulomas. This dissociation of FN and OPN expression suggests that OPN may function differently from FN in the granulomatous response.
Our immunohistochemical findings differ from those of Carlson et al., who found OPN immunoreactivity restricted to histiocytes in granulomas of diverse etiology (24). Using Carlson’s Ab, a mAb against human OPN, we found a similar result in granulomas of sarcoidosis. However, strong lymphocyte-associated OPN immunoreactivity, in a pattern identical to that of MPBIII10 mAb, was detected with a second Ab directed against an epitope adjacent to the thrombin cleavage site on human OPN (not shown). Whether this disparity is due to differences in Ab affinity or expression of variant epitopes or forms of OPN is not yet clear. Based on the concordance of our findings using two distinct Abs and the extensive prior use of MPBIII10 mAb for immunohistochemistry in human tissue, our data demonstrate widespread association of OPN with T cells in sarcoidosis (21, 23).
While OPN may be beneficial to the host early in the granulomatous response, persistent OPN expression may contribute to disease morbidity and mortality. OPN, along with other bone matrix proteins, is known to regulate skeletal mineralization (16). In fact, OPN has been reported to exert both positive and negative influences on tissue calcification (19). Dystrophic calcification, the process by which inflamed or damaged tissues become calcified, is thought to be a coordinated cell-mediated response regulated by noncollagenous ECM components (19). It has been shown that, in this response, OPN expression correlates with macrophage and T cell infiltration and subsequent tissue calcification (19). Dystrophic calcification occurs in some chronic granulomatous lung diseases and is associated with significant tissue dysfunction. Although dystrophic calcification is uncommon in sarcoidosis, it is possible that persisting OPN expression may be a determinant of lung tissue mineralization in granulomatous inflammation. An improved understanding of the role of OPN in this response may offer a therapeutic approach to inhibit an aggressive and persistent host granulomatous response that is ultimately detrimental to disease outcome.
We have shown that OPN supports the chemotaxis and adhesion of human T cells. Taken with the known adhesive and chemotactic effects of OPN for monocytes and a murine T cell hybridoma, our data suggest that OPN could act in the recruitment and focused adhesion of T cells and monocytes in early granuloma formation and may promote cell-cell interactions important to T cell activation (13, 14, 22). Our immunohistochemical findings suggest that OPN may be important early in granuloma formation. This is further supported by other studies that show that lack of an early OPN response is associated with host susceptibility to R. tsutsugamushi in mice, that macrophages respond to Mycobacterium tuberculosis infection by prominent early production of OPN, and that OPN (or early T lymphocyte activation 1 (eta-1) protein ) is a major early protein produced by activated T cells (20, 23, 37).
The adhesion of T cells to OPN is increased by thrombin cleavage of OPN. A number of previous studies evaluating the regulation of OPN activity by thrombin cleavage have reported conflicting results. One report demonstrated that thrombin treatment reduced RGD-mediated adhesion to recombinant human and native bovine OPN (9). Conversely, it has been shown that cleavage of native human OPN augments adhesion to a variety of human cell lines including fibroblasts, smooth muscle cells, and tumor cells (7). In addition, by generating peptides corresponding to the cleaved fragments of OPN, it was shown that the N-terminal fragment, which contains the RGD domain, supports adhesion of a human melanoma cell line that is unable to adhere to the C-terminal fragment or native OPN (8). Our data concur with the latter two studies and support a role for thrombin in the augmentation of T cell adhesion to OPN. These conflicting studies do not represent mutually exclusive results. OPN contains another potential thrombin cleavage site within the RGD domain, and it is possible that different thrombin cleavage conditions or variable access to this thrombin cleavage site could disrupt the RGD sequence and RGD-mediated adhesion (8). Furthermore, since OPN can bind a number of receptors that are differentially expressed on different cells and cell lines, it is possible that some interactions are augmented while others are inhibited by thrombin cleavage, depending on the cell tested. Based on our data, thrombin serves to up-regulate T cell adhesion to OPN in vitro.
The expression of OPN in granulomatous diseases and the role of thrombin in regulating OPN adhesion have interesting implications in the pathogenesis of granuloma formation. There is ample data suggesting that active thrombin and "procoagulant activity" follow granuloma formation. Increased bronchoalveolar lavage procoagulant activity and the presence of circulating D-dimers have been demonstrated in pulmonary sarcoidosis (39, 40). Moreover, Perez reported that mice susceptible to granuloma-inducing Ags from M. tuberculosis have increased procoagulant activity while nonsusceptible mice have increased plasminogen activator activity (41). Taken together with our data, this suggests that activation of the T cell-adhesive properties of OPN represents a novel role for thrombin in granuloma formation and that the immunomodulatory effects of the coagulation system may, in part, be delivered through its interaction with OPN. The impact of inhibition of coagulation on granulomatous inflammation is unknown, although heparin treatment has been shown to inhibit delayed-type hypersensitivity reactions (42).
Costimulation of proliferation by OPN is a property shared with other matrix proteins (34). The ability of FN, laminin, and hyaluronic acid to costimulate T cell proliferation through distinct receptors, both RGD-dependent and -independent, suggests that different components of the ECM may have specific immune modulatory effects (26, 43). Chronic T cell activation and proliferation is an essential component of sarcoid granulomatous inflammation (38). The lymphocyte-associated expression of OPN in sarcoidosis and its ability to costimulate T cells suggest another mechanism by which T cell proliferation is augmented within the sarcoid granuloma.
In the sarcoid lung and in models of sarcoid granuloma formation, the earliest pathologic finding is a mononuclear infiltration of T lymphocytes and monocyte-macrophages followed by the formation of distinct granulomas (38). Elaboration of specific granulomagenic factors is thought to coordinate this process (38). While granuloma formation likely reflects a complex interaction of cytokines, cell-cell, and cell-matrix interactions, an ideal granulomagenic factor would be produced in large amounts early in granuloma formation, persist during inflammation, and be cleared when inflammation wanes; and it would support the chemotaxis and subsequent adhesion, activation, and proliferation of recruited inflammatory cells. Our data show that OPN possesses many of the properties of an ideal granuloma-forming matrix. In addition, the known association of genetically determined OPN expression and susceptibility to intracellular infection in mice suggests that different alleles of the OPN gene may determine genetic susceptibility and pathogenesis of granulomatous immune responses, including sarcoidosis. Finally, the link between the coagulation cascade and OPN activity suggests a novel therapeutic approach to sarcoidosis using anticoagulant inhibitors of thrombin generation and activation.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to the work.
3 Present address: Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510-8040.
4 Address correspondence and reprint requests to Dr. Jeffrey S. Berman, Pulmonary Center, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118-2394. E-mail address:
5 Abbreviations used in this paper: OPN, osteopontin; ECM, extracellular matrix; RGD, arginine-glycine-aspartate; FN, fibronectin; hpf, high power field; ARDS, acute respiratory distress syndrome; NWNT, nylon wool nonadherent T cell(s).
Received for publication July 9, 1998. Accepted for publication September 25, 1998.
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 C. H. Polman, P. W. O'Connor, E. Havrdova, M. Hutchinson, L. Kappos, D. H. Miller, J. T. Phillips, F. D. Lublin, G. Giovannoni, A. Wajgt, et al. A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N. Engl. J. Med., March 2, 2006; 354(9): 899 - 910. [Abstract] [Full Text] [PDF]
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 C. Agostini, A. Cabrelle, F. Calabrese, M. Bortoli, E. Scquizzato, S. Carraro, M. Miorin, B. Beghe, L. Trentin, R. Zambello, et al. Role for CXCR6 and Its Ligand CXCL16 in the Pathogenesis of T-Cell Alveolitis in Sarcoidosis Am. J. Respir. Crit. Care Med., November 15, 2005; 172(10): 1290 - 1298. [Abstract] [Full Text] [PDF]
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 T Sato, T Nakai, N Tamura, S Okamoto, K Matsuoka, A Sakuraba, T Fukushima, T Uede, and T Hibi Osteopontin/Eta-1 upregulated in Crohn's disease regulates the Th1 immune response Gut, September 1, 2005; 54(9): 1254 - 1262. [Abstract] [Full Text] [PDF]
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 A. C. Renkl, J. Wussler, T. Ahrens, K. Thoma, S. Kon, T. Uede, S. F. Martin, J. C. Simon, and J. M. Weiss Osteopontin functionally activates dendritic cells and induces their differentiation toward a Th1-polarizing phenotype Blood, August 1, 2005; 106(3): 946 - 955. [Abstract] [Full Text] [PDF]
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 S. Stier, Y. Ko, R. Forkert, C. Lutz, T. Neuhaus, E. Grunewald, T. Cheng, D. Dombkowski, L. M. Calvi, S. R. Rittling, et al. Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size J. Exp. Med., June 6, 2005; 201(11): 1781 - 1791. [Abstract] [Full Text] [PDF]
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 C. K. Wong, L. C. W. Lit, L. S. Tam, E. K. Li, and C. W. K. Lam Elevation of plasma osteopontin concentration is correlated with disease activity in patients with systemic lupus erythematosus Rheumatology, May 1, 2005; 44(5): 602 - 606. [Abstract] [Full Text] [PDF]
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G. P.A. Rice, H.-P. Hartung, and P. A. Calabresi Anti-{alpha}4 integrin therapy for multiple sclerosis: Mechanisms and rationale Neurology, April 26, 2005; 64(8): 1336 - 1342. [Abstract] [Full Text] [PDF]
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 K. Kawamura, K. Iyonaga, H. Ichiyasu, J. Nagano, M. Suga, and Y. Sasaki Differentiation, Maturation, and Survival of Dendritic Cells by Osteopontin Regulation Clin. Vaccine Immunol., January 1, 2005; 12(1): 206 - 212. [Abstract] [Full Text] [PDF]
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D. Bruder, A. M. Westendorf, R. Geffers, A. D. Gruber, M. Gereke, R. I. Enelow, and J. Buer CD4 T Lymphocyte-mediated Lung Disease: Steady State between Pathological and Tolerogenic Immune Reactions Am. J. Respir. Crit. Care Med., December 1, 2004; 170(11): 1145 - 1152. [Abstract] [Full Text] [PDF]
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M. Gattorno, A. Gregorio, F. Ferlito, V. Gerloni, A. Parafioriti, E. Felici, E. Sala, C. Gambini, P. Picco, and A. Martini Synovial expression of osteopontin correlates with angiogenesis in juvenile idiopathic arthritis Rheumatology, September 1, 2004; 43(9): 1091 - 1096. [Abstract] [Full Text] [PDF]
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 A. Sahai, P. Malladi, H. Melin-Aldana, R. M. Green, and P. F. Whitington Upregulation of osteopontin expression is involved in the development of nonalcoholic steatohepatitis in a dietary murine model Am J Physiol Gastrointest Liver Physiol, July 1, 2004; 287(1): G264 - G273. [Abstract] [Full Text] [PDF]
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 J. S. Berman, D. Serlin, X. Li, G. Whitley, J. Hayes, D. C. Rishikof, D. A. Ricupero, L. Liaw, M. Goetschkes, and A. W. O'Regan Altered bleomycin-induced lung fibrosis in osteopontin-deficient mice Am J Physiol Lung Cell Mol Physiol, June 1, 2004; 286(6): L1311 - L1318. [Abstract] [Full Text] [PDF]
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 J. Morimoto, M. Inobe, C. Kimura, S. Kon, H. Diao, M. Aoki, T. Miyazaki, D. T. Denhardt, S. Rittling, and T. Uede Osteopontin affects the persistence of {beta}-glucan-induced hepatic granuloma formation and tissue injury through two distinct mechanisms Int. Immunol., March 1, 2004; 16(3): 477 - 488. [Abstract] [Full Text] [PDF]
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S. Kobayashi, Y. Kaneko, K.-i. Seino, Y. Yamada, S. Motohashi, J. Koike, K. Sugaya, T. Kuriyama, S. Asano, T. Tsuda, et al. Impaired IFN-{gamma} production of V{alpha}24 NKT cells in non-remitting sarcoidosis Int. Immunol., February 1, 2004; 16(2): 215 - 222. [Abstract] [Full Text] [PDF]
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K. Tanaka, J. Morimoto, S. Kon, C. Kimura, M. Inobe, H. Diao, G. Hirschfeld, J. M. Weiss, and T. Uede Effect of Osteopontin Alleles on {beta}-Glucan-Induced Granuloma Formation in the Mouse Liver Am. J. Pathol., February 1, 2004; 164(2): 567 - 575. [Abstract] [Full Text] [PDF]
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 D. A. Culver, B. P. Barna, B. Raychaudhuri, T. L. Bonfield, S. Abraham, A. Malur, C. F. Farver, M. S. Kavuru, and M. J. Thomassen Peroxisome Proliferator-Activated Receptor {gamma} Activity Is Deficient in Alveolar Macrophages in Pulmonary Sarcoidosis Am. J. Respir. Cell Mol. Biol., January 1, 2004; 30(1): 1 - 5. [Abstract] [Full Text] [PDF]
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 G. A. Johnson, R. C. Burghardt, F. W. Bazer, and T. E. Spencer Osteopontin: Roles in Implantation and Placentation Biol Reprod, November 1, 2003; 69(5): 1458 - 1471. [Abstract] [Full Text] [PDF]
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Y. Koguchi, K. Kawakami, K. Uezu, K. Fukushima, S. Kon, M. Maeda, A. Nakamoto, I. Owan, M. Kuba, N. Kudeken, et al. High Plasma Osteopontin Level and Its Relationship with Interleukin-12-mediated Type 1 T Helper Cell Response in Tuberculosis Am. J. Respir. Crit. Care Med., May 15, 2003; 167(10): 1355 - 1359. [Abstract] [Full Text] [PDF]
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 M. A. Chellaiah, N. Kizer, R. Biswas, U. Alvarez, J. Strauss-Schoenberger, L. Rifas, S. R. Rittling, D. T. Denhardt, and K. A. Hruska Osteopontin Deficiency Produces Osteoclast Dysfunction Due to Reduced CD44 Surface Expression Mol. Biol. Cell, January 1, 2003; 14(1): 173 - 189. [Abstract] [Full Text]
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 S. R. Rittling, Y. Chen, F. Feng, and Y. Wu Tumor-derived Osteopontin Is Soluble, Not Matrix Associated J. Biol. Chem., March 8, 2002; 277(11): 9175 - 9182. [Abstract] [Full Text] [PDF]
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 Y. Koguchi, K. Kawakami, S. Kon, T. Segawa, M. Maeda, T. Uede, and A. Saito Penicillium marneffei Causes Osteopontin-Mediated Production of Interleukin-12 by Peripheral Blood Mononuclear Cells Infect. Immun., March 1, 2002; 70(3): 1042 - 1048. [Abstract] [Full Text] [PDF]
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 M. Mazzali, T. Kipari, V. Ophascharoensuk, J.A. Wesson, R. Johnson, and J. Hughes Osteopontin--a molecule for all seasons QJM, January 1, 2002; 95(1): 3 - 13. [Full Text] [PDF]
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A. W. O'REGAN, J. M. HAYDEN, S. BODY, L. LIAW, N. MULLIGAN, M. GOETSCHKES, and J. S. BERMAN Abnormal Pulmonary Granuloma Formation in Osteopontin-deficient Mice Am. J. Respir. Crit. Care Med., December 15, 2001; 164(12): 2243 - 2247. [Abstract] [Full Text] [PDF]
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J.M. Weiss, A.C. Renkl, C.S. Maier, M. Kimmig, L. Liaw, T. Ahrens, S. Kon, M. Maeda, H. Hotta, T. Uede, et al. Osteopontin Is Involved in the Initiation of Cutaneous Contact Hypersensitivity by Inducing Langerhans and Dendritic Cell Migration to Lymph Nodes J. Exp. Med., October 29, 2001; 194(9): 1219 - 1230. [Abstract] [Full Text] [PDF]
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S. Nagai, S.-i. Hashimoto, T. Yamashita, N. Toyoda, T. Satoh, T. Suzuki, and K. Matsushima Comprehensive gene expression profile of human activated Th1- and Th2-polarized cells Int. Immunol., March 1, 2001; 13(3): 367 - 376. [Abstract] [Full Text] [PDF]
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 A. W. O’Regan, J. M Hayden, and J. S. Berman Osteopontin augments CD3-mediated interferon-{gamma} and CD40 ligand expression by T cells, which results in IL-12 production from peripheral blood mononuclear cells J. Leukoc. Biol., October 1, 2000; 68(4): 495 - 502. [Abstract] [Full Text]
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 J. M. BONVINI, U. SCHATZMANN, B. BECK-SCHIMMER, L. K. SUN, S. R. RITTLING, D. T. DENHARDT, M. LE HIR, and R. P. WÜTHRICH Lack of In Vivo Function of Osteopontin in Experimental Anti-GBM Nephritis J. Am. Soc. Nephrol., September 1, 2000; 11(9): 1647 - 1655. [Abstract] [Full Text]
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G. J. Nau, G. L. Chupp, J.-F. Emile, E. Jouanguy, J. S. Berman, J.-L. Casanova, and R. A. Young Osteopontin Expression Correlates with Clinical Outcome in Patients with Mycobacterial Infection Am. J. Pathol., July 1, 2000; 157(1): 37 - 42. [Abstract] [Full Text] [PDF]
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 J. Sodek, B. Ganss, and M.D. McKee Osteopontin Critical Reviews in Oral Biology & Medicine, January 1, 2000; 11(3): 279 - 303. [Abstract] [Full Text] [PDF]
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Y. Yokosaki, N. Matsuura, T. Sasaki, I. Murakami, H. Schneider, S. Higashiyama, Y. Saitoh, M. Yamakido, Y. Taooka, and D. Sheppard The Integrin alpha 9beta 1 Binds to a Novel Recognition Sequence (SVVYGLR) in the Thrombin-cleaved Amino-terminal Fragment of Osteopontin J. Biol. Chem., December 17, 1999; 274(51): 36328 - 36334. [Abstract] [Full Text] [PDF]
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R. Agnihotri, H. C. Crawford, H. Haro, L. M. Matrisian, M. C. Havrda, and L. Liaw Osteopontin, a Novel Substrate for Matrix Metalloproteinase-3 (Stromelysin-1) and Matrix Metalloproteinase-7 (Matrilysin) J. Biol. Chem., July 20, 2001; 276(30): 28261 - 28267. [Abstract] [Full Text] [PDF]
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