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The Journal of Immunology, 2001, 166: 473-480.
Copyright © 2001 by The American Association of Immunologists

Borrelia burgdorferi and Other Bacterial Products Induce Expression and Release of the Urokinase Receptor (CD87)1

James L. Coleman2,*, Joseph A. Gebbia{dagger} and Jorge L. Benach{ddagger}

* State of New York Department of Health and Departments of {dagger} Pathology and {ddagger} Molecular Genetics and Microbiology, State University of New York, Stony Brook, NY 11794


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The urokinase-type plasminogen activator receptor (uPAR, CD87) is a highly glycosylated 55- to 60-kDa protein anchored to the cell membrane through a glycosylphosphatidylinositol moiety that promotes the acquisition of plasmin on the surface of cells and subsequent cell movement and migration by binding urokinase-type plasminogen activator. uPAR also occurs in a soluble form in body fluids and tumor extracts, and both membrane and soluble uPAR are overexpressed in patients with tumors. uPAR may be a factor in inflammatory disorders as well. We investigated whether Borrelia burgdorferi could stimulate up-regulation of cell membrane uPAR in vitro. B. burgdorferi, purified native outer surface protein A, and a synthetic outer surface protein A hexalipopeptide stimulated human monocytes to up-regulate membrane uPAR as measured by immunofluorescence/FACS and Western blot. The presence of soluble uPAR in culture supernatants, measured by Ag capture ELISA, was also observed. LPS from Salmonella typhimurium and lipotechoic acid from Streptococcus pyogenes also induced the up-regulation of both membrane and soluble uPAR protein by monocytes. Up-regulation of uPAR was induced by conditioned medium from B. burgdorferi/monocyte cocultures. The up-regulation of uPAR by B. burgdorferi was concomitant with an increase in uPAR mRNA, indicating that synthesis was de novo. The expression and release of uPAR in response to B. burgdorferi and other bacterial components suggests a role in the pathogenesis of Lyme disease as well as in other bacterial infections.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The spirochete Borrelia burgdorferi, the etiologic agent of Lyme disease (1), is transmitted by the feeding of hard ticks of the genus Ixodes and is the most prevalent arthropod-borne pathogen in North America. Following deposition in the skin, the organism subsequently disseminates hematogenously to distant tissues and organ systems (2, 3). This organism can use host plasmin, acquired through colocalization of plasmin(ogen) and urokinase-type plasminogen activator (urokinase)3 on the spirochete surface to promote its dissemination (4, 5, 6, 7, 8). The generation of proteolytically active plasmin on its surface endows B. burgdorferi with several invasive properties, including the ability to degrade extracellular matrix (ECM) components (4, 9) and penetrate endothelial cell monolayers in vitro (5), and enhanced invasiveness in mice (7) and ticks (10). Relapsing fever Borrelia also use host plasmin to enhance organ invasion in mice (11). Use of borrowed host proteolytic activity to enhance invasiveness is not limited to Borrelia, but also has been demonstrated in a wide variety of pathogenic Gram-positive and Gram-negative bacteria (12, 13) and also in viruses (14, 15). Acquisition of plasmin activity by microorganisms may be the initial stage of a proteolytic cascade leading ultimately to the direct degradation of ECM and other barriers. The apparent universality of this strategy underscores its potential importance as a mechanism for microbial invasion.

In both physiologic and pathologic conditions, extravascular plasmin acquisition by cells is thought to be due to cell-associated urokinase activation of plasminogen. A key event in this process is the association of urokinase with its high affinity cellular receptor, the urokinase receptor (uPAR, CD87) (16, 17, 18). uPAR was first identified on monocytes and cells of monocytic origin (16, 19, 20), and is a highly glycosylated 55- to 60-kDa protein (21) coupled to the plasma membrane through a GPI anchor (22). The uPAR molecule has three extracellular domains (D1-D3) and binds urokinase with high affinity via its receptor-binding domain, D1, which is located in the amino-terminal portion of the molecule. The functions of D2 and D3 are incompletely understood. The concentration of urokinase and subsequent generation of plasmin at the leading edge of the cell is the mechanism by which inflammatory and neoplastic cells degrade ECM and migrate from one anatomic site to another. In vitro, a number of proinflammatory agents and growth factors such as PMA (23), TGF-{beta} and TNF-{alpha} (24), and nerve growth factor (25) have an enhancing effect on cellular uPAR expression, and uPAR is overexpressed in a number of types of cancerous tumors (26). In the context of infection, LPS in vitro (27, 28) and in vivo (29, 30), and muramyl dipeptide in vitro (27), can cause an increase in expression of uPAR by monocytes and neoplastic cells. Lyme disease is characterized by inflammatory manifestations in skin, heart, CNS, and joints (2), resulting in tissue damage due to infiltration of inflammatory cells, primarily monocytes/macrophages. Therefore, we investigated the levels of uPAR expression by monocytic cells after coculture with B. burgdorferi and several of its purified components in vitro. We report that exposure to B. burgdorferi results in an increase in both uPAR mRNA and protein. Furthermore, uPAR protein was detected on the monocyte cell membrane as well as in a soluble isoform in conditioned medium (CM) from cocultures.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Rabbit anti-human uPAR Ab, FITC-conjugated murine mAb (IgG2a) against uPAR, and the IMUBIND Total uPAR Strip-well ELISA Kit were purchased from American Diagnostica (Greenwich, CT). FITC-conjugated mouse IgG2a (UPC-10) (the isotype-matched control for FITC-conjugated murine mAb against human uPAR) was purchased from Sigma (St. Louis, MO). Alkaline phosphatase-conjugated goat anti-rabbit secondary Ab and 5-bromo-4-chloro-3-indoyle-phosphate/nitroblue tetrazolium phosphatase substrate used in Western blots, was purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). FITC-conjugated murine mAbs against human CD14 and human CD45 were purchased from Becton Dickinson (San Jose, CA). LPS from Salmonella typhimurium, lipotechoic acid (LTA) from Streptococcus pyogenes, BSA (fraction V, low endotoxin), Barbour-Stoenner-Kelley (BSK-H) medium (with and without rabbit serum), dextran T-500, normal mouse serum (NMS), and tissue culture grade water were purchased from Sigma. The BSK-H medium and tissue culture grade water are certified by the manufacturer to be endotoxin-free. RPMI 1640, sodium pyruvate, penicillin/streptomycin, and Trizol reagent were purchased from Life Technologies (Grand Island, NY). FBS (low endotoxin) was purchased from HyClone (Logan, UT). Accudenz A.G. was purchased from Accurate Chemical and Scientific (Westbury, NY). Tissue culture plates (24-well) were purchased from Costar (Cambridge, MA). The Titan One Tube RT-PCR System was purchased from Roche (Indianapolis, IN). B. burgdorferi native outer surface protein A (N-OspA) (31) and lipidated and nonlipidated OspA hexapeptide (32, 33) were gifts from Dr. Justin Radolf of the University of Connecticut (Storrs, CT). N-OspA was affinity purified from cultured B. burgdorferi. These reagents were tested for endotoxin activity using the QCL-1000 quantitative chromogenic Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). N-OspA and the lipidated and nonlipidated hexapeptide preparations had activities <= 12 pg LPS/µg and <= 1 pg LPS/µg of total protein, respectively.

Bacteria

The infectious N40 strain of B. burgdorferi (34) was used, except where otherwise specified, in all experiments. To ensure infectivity, N40 were passaged in vivo by injecting 2 x 103 organisms intradermally into the shaved back of C3H/Hen mice. At 16–21 days post injection, the mice were sacrificed, and N40 was cultured from skin and organs in BSK-H medium supplemented with 6% rabbit serum as previously described (10). Only N40 derived from mouse skin cultures were used as a source of infectious B. burgdorferi. In certain experiments we used the noninfectious high passage B31 strain (1). All B. burgdorferi were cultured at 33°C in BSK-H medium in the absence of serum.

Eukaryotic cells

The human monocyte-like cell line U937 was cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). Human peripheral blood monocytes (hPBM) were isolated from healthy volunteers using Accudenz cell separation medium according to a method previously described (35).

Spirochete/eukaryotic cell cocultures

U937 or freshly isolated hPBM were washed two times with RPMI 1640 containing 10% FBS (RPMI/FBS) and enumerated. The cells were dispensed into 24-well polystyrene tissue culture plates (1 ml per well) at a density of 2 x 105 per ml for U937 and 1 x 106 per ml for hPBM. B. burgdorferi strain N40 were washed two times in RPMI/FBS (20 min at 7000 x g at 250°C), enumerated, and added to the cells at ratios of 1, 10, and 100 spirochetes per cell. The final volume of each well was 2 ml. Control additions were RPMI/FBS alone and a BSK-H medium sham preparation to control for nonspecific activity derived from the BSK-H medium. The sham control was prepared at the same time as the spirochete preparation and consisted of an equal volume of uninoculated BSK-H medium that was subjected to the same manipulations. In some experiments, eukaryotic cells were incubated with LPS, LTA, B. burgdorferi N-OspA, or lipidated and nonlipidated B. burgdorferi OspA hexapeptide in a range of concentrations. Eukaryotic cell cultures containing LPS and LTA were prepared from 1 mg/ml stock solutions of LPS and LTA in endotoxin-free tissue culture grade water. Eukaryotic cell cultures containing lipidated and nonlipidated B. burgdorferi OspA hexapeptide were prepared from 100x stock solutions in endotoxin-free tissue culture grade water. In experiments where CM was used to stimulate hPBM, the CM was first centrifuged at 14,000 x g for 15 min and then filter sterilized (0.2 µm) before being added to freshly isolated hPBM.

FACS

At the appropriate timepoint, hPBM or U937 (1 x 106) were centrifuged at 400 x g. The cells were washed two times with RPMI containing 3% NMS (RPMI/NMS), resuspended in 25 µl of a saturating concentration of FITC-conjugated murine mAb to uPAR in RPMI/NMS, and incubated for 1 h on ice. Unbound mAb was removed by two washes in RPMI/NMS. The cells were finally resuspended in 0.5 ml of Dulbecco’s PBS containing 1% formalin. An isotype-matched mAb (FITC-conjugated murine monoclonal IgG2a) was used as a negative control. Fluorescence intensity was measured with a FACSScan Flow Cytometer (Becton Dickinson) in which the cells were gated with forward vs side scatter.

RT-PCR of uPAR RNA

U937 cells were cocultured with B. burgdorferi as described above, with the exception that 100-mm tissue culture dishes were used in place of 24-well plates. Total coculture volume was 15 ml. At each timepoint, the U937 cells were pelleted, and total RNA was extracted using Trizol according to the manufacturer’s instructions. RNase-free reagents, pipet tips, and disposable plasticware were used throughout this procedure to minimize the possibility of RNA degradation. RNA was quantified by spectrophotometry at 260 and 280 nm, and all samples had an absorbance ratio between 1.7 and 1.8. The cDNA reaction and the PCR were conducted in the same tube using 1 µg total RNA and the Titan One Tube RT-PCR System following manufacturer’s guidelines. A 530-bp uPAR fragment was amplified from the cDNA using the oligomers 5'-CAT CAG ACA TGA GCT GTG AGA GGG-3' (forward) and 5'-CCA GGT CTG GGT GGT TAC AGC C-3' (reverse), which targeted two exons separated by an intron (36). An internal region of {beta}-actin was also amplified as an induction control and used the oligomers 5'-CCA AGG CCA ACC GCG AGA A-3' (forward) and 5'-AGG GTA CAT GGT GGT GCC GCC AGA C-3' 9 (reverse).

Agarose gel electrophoresis

The samples were electrophoresed on 1% agarose gels and stained as previously described (37). Gels were photographed with a Polaroid DS34 Direct Screen Instant Camera (Hoefer Scientific, San Francisco, CA).

Detection of soluble uPAR by quantitative ELISA

Soluble uPAR in CM was measured by a quantitative ELISA using the IMUBIND Total uPAR Strip-well ELISA Kit according to the manufacturer’s instructions. The mAb supplied with the kit was specific for D2 of uPAR. The CM was routinely centrifuged for 15 min at 14,000 x g before use. In some instances, the CM was centrifuged at 100,000 x g for 1 h. Results were expressed as the mean uPAR concentration ± SD of duplicate wells for each experimental group tested. Pooled experiments subjected to nonparametric statistical analysis were expressed as medians with maximum and minimum values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Coincubation with B. burgdorferi triggers an increase in expression of cell membrane uPAR in human monocytes

We measured the in vitro response of human monocytes to exposure to B. burgdorferi in the context of the cell membrane expression of uPAR. The N40 strain of B. burgdorferi was chosen for this study because of its infectious nature. Human monocyte-like U937 cells as well as freshly isolated hPBM were incubated for 48 h either alone or with increasing amounts of B. burgdorferi and stained with a FITC-conjugated murine mAb to human uPAR. A representative experiment showing FACS analysis of the relative uPAR expression of the experimental groups is presented in Fig. 1Go. In Table IGo, the statistical relevance is demonstrated. The anti-uPAR mAb was previously determined to be nonreactive toward B. burgdorferi. An isotype-matched irrelevant FITC-conjugated mAb was used as a negative control. Although there was a pronounced difference in the levels of increased uPAR expression between the two cell types, with the hPBM being the more vigorous, there was a clear spirochete concentration-dependent response by the cells. The basal level of uPAR expression (cells incubated in the absence of B. burgdorferi) was higher for the hPBM than for the U937 cells. Incubation of the monocytes with a sham BSK-H medium preparation did not induce an increase in cell membrane uPAR.



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  		FIGURE 1. FACS analysis of up-regulation of uPAR in U937 and hPBM after coculture with B. burgdorferi. Cells were incubated at a density of 2 x 105 (U937) or 106 (hPBM) per well at 37°C, 5% CO2 for 48 h with varying concentrations of B. burgdorferi. Filled histograms, cells that received no B. burgdorferi. Unfilled histograms, cells that received either 1, 10, or 100 B. burgdorferi per cell or a sham B. burgdorferi medium control (SH).

 

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  		Table I. FACS analysis of up-regulation of cell membrane uPAR by U937 and hPBM exposed to B. burgdorferi

 
Epifluorescent microscopic examination of both U937 and hPBM cocultured with B. burgdorferi was conducted to further characterize the up-regulation of uPAR by these cells after 48 h of exposure (Fig. 2Go). Cells exposed to B. burgdorferi as well as unexposed cells were not reactive to an irrelevant FITC-conjugated isotype-matched murine mAb (data not shown). Results for cells that were stained with mAb to uPAR closely paralleled those obtained by FACS. Both U937 and hPBM exposed to B. burgdorferi showed an increase in cell membrane uPAR expression compared with cells that received either no B. burgdorferi or a medium sham. The most intense fluorescence occurred at the cell periphery for each cell type. As in the experiments analyzed by FACS, the basal level of surface uPAR expression (obtained from controls that received no B. burgdorferi) was higher for hPBM than for U937.



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  		FIGURE 2. Immunofluorescent characterization of up-regulation of uPAR in U937 and hPBM after coculture with B. burgdorferi. U937 were incubated at a density of 2 x 105 cells per well in the absence of B. burgdorferi (A), with a sham B. burgdorferi medium control (B) or B. burgdorferi at a ratio of 10 spirochetes per cell (C). In addition, hPBM were incubated at a density of 1 x 106 cells per well in the absence of B. burgdorferi (D) with a sham B. burgdorferi medium control (E) or B. burgdorferi at a ratio of 10 spirochetes per cell (F). Cocultures were incubated for 48 h at 37°C, 5% CO2. Cells were stained for 30 min on ice with murine anti-uPAR mAb, and examined by epifluorescence microscopy.

 
The Western blot banding pattern of uPAR expressed by U937 cells exposed to varying concentrations of B. burgdorferi over time is shown in Fig. 3Go. U937 cells were cocultured with 0, 1, 10, 50, and 100 B. burgdorferi per cell. At 72 h, the U937 cells expressed a diffuse band ranging from ~40 to ~60 kDa. The banding pattern was more focused at 24 and 48 h but in all cases the dose- and time-dependent nature of the response was observable. A medium sham preparation did not induce up-regulation of uPAR.



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  		FIGURE 3. Western blot characterization of uPAR up-regulation in U937 cells after coculture with varying concentrations of B. burgdorferi over time. Cells were recovered at the indicated time points, washed with Dulbecco’s PBS, and normalized for cell density by enumeration in a hemacytometer. Whole-cell lysates (the equivalent of 2 x 105 cells) were separated by SDS-PAGE (12.5% polyacrylamide) under nonreducing conditions. Following electrophoretic transfer of the separated whole cell lysates to nitrocellulose, the membranes were stained with a polyclonal rabbit Ab to uPAR that had previously been determined to be nonreactive to B. burgdorferi, followed by alkaline phosphatase-conjugated goat anti-rabbit Ab and 5-bromo-4-chloro-3-indoyle-phosphate/nitroblue tetrazolium phosphatase substrate.

 
B. burgdorferi induces the up-regulation of uPAR expression at the transcriptional level

We used RT-PCR to determine whether up-regulation of uPAR in our cocultures was concomitant with an increase in uPAR mRNA. Total RNA was extracted after 48 h from U937 cells exposed to 0, 1, 10, or 100 B. burgdorferi per cell. Generation of cDNA and subsequent PCR of a 530-bp uPAR fragment was conducted in the same tube. At the same time, reverse transcription and PCR for an internal region of {beta} actin was conducted for use as an internal induction control. The PCR of target cDNA from cells that had been incubated with B. burgdorferi revealed a spirochete-dose-dependent increase in uPAR mRNA that was maximized at 100 spirochetes per cell, resulting in a total increase of ~4-fold over cells that had received no B. burgdorferi. A representative experiment is shown in Fig. 4Go, A and B. These results paralleled those for protein expression at 48 h (Fig. 3Go). OD values obtained from densitometric scans of the {beta} actin bands were similar (data not shown). This experiment was repeated several times with consistent results.



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  		FIGURE 4. Up-regulation of uPAR in U937 cocultured with B. burgdorferi is transcriptionally regulated. Figure shows the RT-PCR analysis of total RNA extracted from U937 cells exposed to B. burgdorferi for 48 h. A, Agarose gel electrophoresis; top, dose dependence of uPAR up-regulation by U937 in response to increasing concentrations of B. burgdorferi. Lanes show amplification of an internal fragment of uPAR that targeted two exons separated by an intron. Bottom, An internal region of {beta}-actin was also amplified and served as an induction control. B, Densitometric scans of individual lanes of agarose gel. Bars represent the relative band densities of each lane (OD x mm) expressed as a percentage of {beta}-actin OD for the same sample.

 
Bacterial components induce the up-regulation of cell membrane uPAR expression

The overexpression of uPAR in monocytes cocultured with viable B. burgdorferi ( Figs. 1–4GoGoGoGo) led us to explore whether a similar response could be elicited by spirochete components (i.e., outer surface lipoproteins) or by components of other Gram-positive and Gram-negative bacteria alone. For this experiment, hPBM were chosen because of their more vigorous response in previous experiments. B. burgdorferi codes for a large number of outer surface lipoproteins, one of the major examples being outer surface protein A (OspA). Therefore, we tried purified N-OspA and synthetic lipidated and nonlipidated hexapeptides corresponding to the N terminus of OspA. The use of synthetic peptide analogs of OspA was appealing because the products are endotoxin free, and the biologically active domains of the lipoprotein remains intact. We also tested LPS from S. typhimurium, and LTA from S. pyogenes. The cell membrane expression of uPAR after exposure to these substances is shown in Fig. 5Go. Incubation of hPBM with B. burgdorferi OspA lipopeptide at a concentration of 5 µg/ml induced expression of membrane uPAR, where incubation with the nonlipidated OspA peptide (at a concentration of 15 µg/ml) did not. N-OspA induced the expression of uPAR at a concentration of 62 ng/ml. LPS from S. typhimurium and LTA from S. pyogenes (0.6 µg/ml) induced similar responses.



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  		FIGURE 5. FACS analysis of up-regulation of uPAR in hPBM following coculture with various bacterial components. Cells were incubated at a density of 1 x 106 per well at 37°C, 5% CO2 with nonlipidated B. burgdorferi OspA hexapeptide (OspA Pept, 15 µg/ml), B. burgdorferi hexalipopeptide (L-Pept, 5 µg/ml), B. burgdorferi N-OspA (N-OspA, 62.5 ng/ml), LTA from S. pyogenes (0.6 µg/ml), and LPS from S. typhimurium (0.6 µg/ml). Filled histograms represent cells that received no B. burgdorferi. Unfilled histograms represent cells that received the additions indicated.

 
A soluble isoform of uPAR is shed into the medium upon coincubation of hPBM with B. burgdorferi

Membrane receptors can have soluble counterparts that may play a significant role in human disease progression. Our finding that monocyte membrane uPAR can be induced by exposure to B. burgdorferi and/or bacterial, components led us to investigate the possibility that a soluble isoform of uPAR could be released into the medium under the same conditions. For this purpose, a specific, quantitative enzyme immunoassay kit suitable for the detection of soluble, ligand-free uPAR as well as uPAR/urokinase complexes was used. The results of a representative assay demonstrating the presence of soluble uPAR in the tissue culture medium of spirochete/hPBM cocultures is presented in Fig. 6Go. Incubation of hPBM for 48 h with increasing densities of B. burgdorferi resulted in a time- and dose-related response peaking at ~1.5 ng/ml for wells that received 100 spirochetes per cell (a ~6-fold increase over control hPBM, which received no B. burgdorferi), although uPAR release was detected with as little as 1 spirochete/cell. hPBM exposed to a medium sham control did not release uPAR. The results of two independent experiments were pooled to demonstrate the statistical relevance of the data (Table IIGo). Prior ultracentrifugation ofthe samples did not result in a change of the ELISA signal (data not shown).



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  		FIGURE 6. Demonstration of presence of soluble uPAR in CM of cocultures in which hPBM were exposed to B. burgdorferi. hPBM were plated at a density of 1 x 106 per well at 37°C, 5% CO2. A, At the 48-h timepoint, hPBM exposed to varying amounts of B. burgdorferi were centrifuged, and the amount of soluble uPAR present in the CM was determined by quantitative ELISA. B, Time-dependent release of soluble uPAR by hPBM exposed to a ratio of 100 B. burgdorferi per cell as determined by quantitative ELISA.

 

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  		Table II. Release of soluble uPAR by hPBM exposed to B. burgdorferi

 
Bacterial components induce the release of soluble uPAR

hPBM were tested by quantitative ELISA to determine whether they produce soluble uPAR in response to lipidated B. burgdorferi OspA hexapeptide, LPS from S. typhimurium, and LTA from S. pyogenes. Exposure to lipidated B. burgdorferi OspA hexapeptide in concentrations ranging between 1.7 and 45 µg/ml resulted in a dose-dependent response after 48 h of coincubation (Fig. 7GoA). As with cell membrane uPAR, incubation of hPBM with nonlipidated B. burgdorferi OspA hexapeptide (at the highest concentration used for the lipidated hexapeptide) resulted in little or no release of soluble uPAR. The LPS response (Fig. 7GoB) was saturated even at the lowest concentration used, whereas LTA (Fig. 7GoC) induced a dose response at a concentration range similar to that of the lipidated hexapeptide.



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  		FIGURE 7. Presence of soluble uPAR in CM of hPBM cocultured with several bacteria components (48 h, 37°C, 5% CO2). Dose-dependent response of hPBM to coculture with nonlipidated B. burgdorferi OspA hexapeptide (OspA Peptide) and varying amounts of B. burgdorferi hexalipopeptide (LP) (A), varying amounts of LPS from S. typhimurium (B), or varying amounts of LTA from S. pyogenes (C).

 
CM induces the expression of membrane uPAR and the release of soluble uPAR

CM from a B. burgdorferi/hPBM coculture was tested for its ability to induce the expression of membrane uPAR and the release of soluble uPAR from freshly isolated hPBM. Three different CM preparations were used: 1) CM from hPBM that had received no B. burgdorferi; 2) CM from B. burgdorferi that had received no hPBM; and 3) CM from hPBM that had received B. burgdorferi at a ratio of 100 spirochetes per cell. The CM, obtained after 48 h of incubation, was centrifuged at 14,000 x g to rid the preparation of cells and filter sterilized before its addition to fresh hPBM. As measured by FACS, CM from hPBM cultures that had received B. burgdorferi caused an increase in expression of cell membrane uPAR in comparison to CM from hPBM cultures that had received no B. burgdorferi (Fig. 8GoA). CM from cultures that had received B. burgdorferi but no hPBM did not induce expression of cell membrane uPAR (data not shown). We also investigated the possibility that CM from hPBM cultures that had received B. burgdorferi could induce the release of soluble uPAR from freshly isolated hPBM. To correct for the presence of soluble uPAR already present in the CM, some coincubation wells received CM alone, but no hPBM. Fresh coincubation medium was added to these wells to compensate for the absence of the hPBM inoculum. Following recovery of the supernatant and subsequent ELISA, their absorbance values were subtracted from those of corresponding samples that had received both CM and hPBM. CM from hPBM cultures that had received B. burgdorferi induced the release of soluble uPAR (p < 0.05) (Fig. 8GoB). As was found with cell membrane uPAR, CM from cultures that had received B. burgdorferi but no hPBM did not induce the expression of soluble uPAR (data not shown).



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  		FIGURE 8. Up-regulation of uPAR in hPBM is induced by exposure to CM. This figure shows an experiment in which CM, obtained from a previous experiment where hPBM were cocultured with B. burgdorferi (48 h, 37°C, 5% CO2) at a ratio of 100 spirochetes per cell was used to induce expression of uPAR in freshly isolated hPBM. A, Histograms represent up-regulation of cell membrane uPAR as measured by FACS. Filled histogram, uPAR expression by hPBM exposed to CM obtained from a culture that received no B. burgdorferi. Unfilled histogram, uPAR expression by hPBM exposed to CM obtained from a culture that received 100 B. burgdorferi per cell. B, Bars represent up-regulation of soluble uPAR as measured by quantitative ELISA. (0 Bb/cell), uPAR expression by hPBM exposed to CM obtained from a culture that received no B. burgdorferi. (100 Bb/cell), uPAR expression by hPBM exposed to CM obtained from a culture that received 100 B. burgdorferi per cell. *, Significantly different from control, p < 0.05 as determined by Student’s t test, where p < 0.05 was used as the {alpha} value to determine statistical significance.

 
Induction of uPAR is not dependent upon viability or invasiveness of B. burgdorferi

To investigate whether viable B. burgdorferi were necessary to promote increased expression of uPAR, we exposed hPBM to viable or heat-treated spirochetes. There were no significant differences in expression of cell membrane or soluble uPAR between the two groups (data not shown). We also compared the use of the high passage B31 laboratory strain of B. burgdorferi with the infectious N40 strain used for the majority of the experiments. There were no significant differences between the abilities of either strain to promote the up-regulated expression of cell membrane or the release of soluble uPAR in hPBM (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have presented evidence for up-regulation of cell membrane and soluble uPAR in human monocytes as a result of exposure to the Lyme disease agent, B. burgdorferi in vitro. Three independent methods (immunofluorescence, Western blot, and RT-PCR) were used to measure membrane uPAR. Cell membrane up-regulation of uPAR protein after exposure to B. burgdorferi in vitro was observed in both hPBM and the human monocyte-like cell line U937, using a murine mAb to human uPAR. This increase in membrane uPAR was accompanied by an increase in mRNA, an indication that the uPAR synthesis was de novo. The electrophoretic mobility of the uPAR band detected by Western blot was similar to that previously reported for the cell line U937. Although the deduced molecular mass of uPAR is ~35-kDa (36), the Mr can vary according to the level of glycosylation for a given cell type, with the reported Mr range of uPAR in U937 being 55–60 kDa (20). In our Western blot experiments using whole cell lysates from U937 cells, a diffuse band ranging between 40 and 60 kDa was observed; however, the most intense staining was in the area ranging between 45 and 55 kDa.

A soluble isoform of uPAR is present in body fluids of healthy individuals but is elevated in patients with various cancers (38). This observation has led to speculation that the monitoring of soluble uPAR levels in these patients may be of prognostic value. In addition, it has recently been observed that soluble uPAR is elevated in the plasma of patients with rheumatoid arthritis, a chronic inflammatory disorder, with levels being directly related to severity of disease (39). Because of the chronic inflammatory nature of Lyme disease, and our results with cell membrane uPAR, we determined whether soluble uPAR is elicited by monocytes during their exposure to B. burgdorferi. By use of a specific Ag capture ELISA technique, we demonstrated an increase in soluble uPAR concentrations in the supernatants of B. burgdorferi/monocyte cocultures. Increases in soluble uPAR levels were also detected in culture supernatants after exposure of monocytes to a B. burgdorferi outer surface lipoprotein analog, as well as the bacterial components LPS and LTA.

The exact mechanism of release of the soluble uPAR detected in our experiments is unknown, as is its function. Initially, we considered the possibility that the assay could be detecting membrane uPAR on small fragments of disrupted monocytes not sedimented by low speed centrifugation. To determine whether this was the case, we split the CM samples into two groups, one of which was centrifuged at 14,000 x g for 15 min, whereas the other was subjected to ultracentrifugation at 100,000 x g for 1 h. The ELISA was conducted and the absorbance values were compared. Ultracentrifugation of the supernatant before the assay did not reduce the ELISA signal. Therefore, this possibility was not considered further. Another possibility is that the soluble uPAR may be the result of the proteolytic degradation of membrane uPAR as it is down-regulated from the cell surface. In this case it need not have any biological function. However, the ongoing accumulation of information in the literature, particularly in the field of neoplasia, in which soluble uPAR is present in a large and mixed group of tumors, argues against it being merely a nonfunctional degradation product. A convincing argument, based on the literature, can be made that the release of the soluble uPAR from the hPBM cell surface was the result of an enzymatic process, where its general structure would be predetermined, in large part, by the specific type of proteolysis through which it is cleaved from the cell membrane. It is believed that the GPI-specific phospholipases (22, 40) can cleave the GPI anchor at the cell surface, thus releasing a fragment containing D1, D2, and D3, but lacking the hydrophobic moiety. Alternatively, uPAR can be cleaved by its own ligand, urokinase, to release D1, leaving D2 and D3 anchored to the cell, but without the capacity to bind ligand (41, 42). In either case, the released component would contain the ligand-binding domain, and could have a biological function as a competitive inhibitor of the cell-bound receptor. The mAb used in our assay was specific for D2. Its reactivity eliminates the possibility that the uPAR we detected is composed of D1 alone. Therefore, it is likely that the soluble uPAR detected in our assay is composed of D1, D2, and D3.

Direct contact between hPBM and B. burgdorferi was not necessary for induction of up-regulation of membrane uPAR or release of soluble uPAR, as CM was also capable of eliciting these responses. Neither was spirochete viability a requirement. Taken together, this evidence suggests that more complex regulatory mechanisms may be involved.

The stimulatory activity of B. burgdorferi is most likely due to its outer surface lipoproteins (43). We have demonstrated that N-OspA and an OspA lipopeptide containing only the six N-terminal amino acid residues are capable of uPAR induction, with N-OspA being considerably more potent. A similar difference was observed in the ability of full-length N-OspA and OspA lipopeptide to stimulate cultured endothelium (44). A probable reason for this apparent difference in stimulatory activity is the absence of important regions of the full-length molecule required for maximal effect.

We have provided evidence for the up-regulation of cell membrane and the release of soluble uPAR in response to a bacterial organism and other common bacterial components. These results make it reasonable to speculate that uPAR overexpression and release is an important response in infection. Although some of the known biological features of uPAR could be used to support a potential function for monocyte-bound uPAR (increased proteolytic activity for monocytes leading to enhanced extravasation and accumulation in inflammatory sites), the role of soluble uPAR cannot yet be gleaned from the present state of knowledge.


   Footnotes
 
1 This work was supported by a grant from the National Institutes of Health (AR-40445) and a grant from the Mathers Foundation (to J.L.B.). Back

2 Address correspondence and reprint requests to James L. Coleman, State of New York Department of Health, 5120 Center for Infectious Diseases, State University of New York at Stony Brook, Stony Brook, NY 11794-5120. Back

3 Abbreviations used in this paper: urokinase, urokinase-type plasminogen activator; ECM, extracellular matrix; uPAR, the urokinase receptor; N-OspA, native outer surface protein A; hPBM, human peripheral blood monocytes; LTA, lipotechoic acid; NMS, normal mouse serum; CM, conditioned medium; BSK-H, Barbour-Stoenner-Kelley; RPMI/FBS, RPMI 1640 containing 10% FBS; RPMI/NMS, RPMI containing 3% NMS; D, domain. Back

Received for publication July 20, 2000. Accepted for publication September 29, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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