Extracellular ATP and adenosine are important regulators of immune responses; however, contribution of purinergic signaling to host defense during persistent microbial infections remains obscure. Lyme borreliosis is a common arthropod-borne infection caused by Borrelia burgdorferi sensu lato. In this study, we investigated whether lymphoid purinergic signaling contributes to the mechanisms by which borreliae species evade the immune system and trigger joint inflammation. Intracutaneous inoculation of Borrelia garinii to C3H/He mice induced symptomatic infection manifested in elevated levels of borrelia-specific IgG Abs, persistent spirochete dissemination into the tissues and joint swelling, as well as ∼2- to 2.5-fold enlargement of draining lymph nodes with hyperplasia of B cell follicle area and L-selectin shedding from activated T lymphocytes. Purine catabolism was also activated in lymph nodes but not spleen and blood of infected C3H/He mice within the first 4 postinfection weeks, particularly manifested in transient upregulations of adenosine triphosphatase/ectonucleoside triphosphate diphosphohydrolase and ecto-5′-nucleotidase/CD73 on CD4+CD8+ T lymphocytes and adenosine deaminase activity on B220+ B lymphocytes. Compared with borrelia-susceptible C3H/He strain, lymphocytes from C57BL/6 mice displayed markedly enhanced adenosine-generating capability due to approximately three times higher ratio of ecto-5′-nucleotidase to adenosine deaminase. Borrelia-infected C57BL/6 mice efficiently eradicated the inoculated spirochetes at more chronic stage without any signs of arthritis. Strikingly, deletion of key adenosine-generating enzyme, ecto-5′-nucleotidase/CD73, was accompanied by significantly enhanced joint swelling in borrelia-infected CD73-deficient C57BL/6 mice. Collectively, these data suggest that insufficient basal adenosine level and/or pathogen-induced disordered lymphoid purine homeostasis may serve as important prerequisite for promotion of inflammatory responses and further host’s commitment to persistence of bacterial infection and arthritis development.

The immune system is constantly challenged by the surrounding microbes (bacteria, viruses, and parasites), which enter our body via the epithelial surfaces and try to invade, survive, and replicate in the mammalian host to establish persistent infection. A growing number of studies have demonstrated that such cosignaling molecules as ATP and adenosine may be involved in the dynamic processes that take place during inflammation and infection. An important role of ATP was inferred from the findings that it elicits the processing and release of proinflammatory cytokines, including IL-1β, IL-6, TNF-α, and IFN-γ, from activated macrophages, dendritic cells, and other immune cells (13), facilitates leukocyte trafficking to sites of infection and inflammation (4, 5), and generally functions as a “danger sensor” alerting the immune system to the presence of pathogen-associated molecular patterns (2). These effects are mediated through a series of ligand-gated (P2X) and G protein-coupled (P2Y) receptors (2, 6). In turn, adenosine generated in the course of ATP hydrolysis has a nonredundant counteracting role in attenuating the inflammation and tissue damage. Specific immunosuppressive functions of adenosine include inhibition of TNF-α, IL-1, IL-6, and IL-12 synthesis by lymphoid cells (2, 7, 8), maintenance of vascular endothelial barrier integrity, and control of lymphocyte trafficking between the blood and tissues (5, 9, 10). These effects are mediated through G protein-coupled adenosine receptors, which function either by activating (A2A and A2B) or inhibiting (A1 and A3) adenylate cyclase (6, 8).

The duration and magnitude of purinergic signaling is governed by a network of membrane-bound and soluble enzymes. General schemes of purine metabolism have included a role for purine-inactivating enzymes of the ectonucleoside triphosphate diphosphohydrolase (NTPDase) family, the ectonucleotide pyrophosphatase/phosphodiesterase family, ecto-5′-nucleotidase/CD73, and adenosine deaminase (ADA), whereas the elements of counteracting ATP-regenerating pathway are composed of adenylate kinase and NDP kinase (11). Studies with gene-modified mice lacking certain enzymes, such as E-NTPDase-1/CD39 (12, 13), ecto-5′-nucleotidase/CD73 (9, 10), or ADA (14), confirmed the importance of coordinated purine-inactivating cascade for proper immune and cardiovascular functions at various inflammatory, prothrombotic, and hypoxic states. Moreover, by using CD73−/− mice, it was shown recently that CD73-generated adenosine restricts lymphocyte migration into LPS-induced draining lymph nodes (LNs) (10) and prevents persistent gastritis and bacterial colonization during infection with Helicobacter species (15). Directional manipulations of adenosine levels via activation or inhibition of ADA activity in the intestinal environment had also strong effects on the outcome of enteropathogenic Escherichia coli infection in the ligated rabbit intestinal loop model by regulating the bacterial growth, fluid secretion, and expression of important virulence genes (16).

Lyme borreliosis is a common arthropod-borne infection caused by the spirochete Borrelia burgdorferi sensu lato, which circulates in endemic areas between Ixodes ticks and a large number of vertebrate hosts upon which ticks feed. Currently, 14 different borrelia species belonging to the B. burgdorferi sensu lato complex have been identified, with three genospecies, Borrelia garinii, Borrelia afzelii, and B. burgdorferi sensu stricto, being recognized as major human pathogens (1719). Infection of mammals with this extracellular pathogen requires the spirochete to evade clearance by the host’s immune defenses without compromising host viability, thus providing a unique insight into the host–pathogen interactions. Joint inflammation and dysfunction are consequences of bacterial invasion into the joint, and chronic Lyme arthritis has been considered a hallmark of late-stage disease (17). Various mouse strains develop mild or severe symptoms of inflammation and joint swelling several weeks after borrelia inoculation, thus providing a good experimental model for studying the pathogenesis of persistent infection and subacute Lyme arthritis most commonly seen in humans (2023).

Because previous studies have demonstrated a role for ATP and adenosine in the regulation of immune system (2, 8), development of arthritis (24, 25), and host defense during microbial intervention (26, 27), we determined whether purinergic signaling contributes to the mechanisms by which borreliae species evade the immune system to disseminate the infection. The obtained results demonstrate that key purine-inactivating ectoenzymes are selectively upregulated in the peripheral LN of acutely infected C3H/He mice, and this disordered purine homeostasis may be implicated in the pathogenesis of persistent bacterial infection and arthritis development.

Female C3H/He mice were obtained from M&B A/S (Ry, Denmark). Ecto-5′-nucleotidase/CD73−/− mice were generated and provided to us by Prof. L. Thompson (Oklahoma University, Norman, OK) (9). Female and male CD73−/− mice, which were backcrossed to C57BL/6 background for eight generations, and C57BL/6 wild-type mice were used in the experiments. Mice were maintained in Central Animal Laboratory in the University of Turku (Turku, Finland), raised in pathogen-free conditions, and provided with food and water ad libitum. At the end of the experiment, the mice were killed with carbon dioxide. All animal studies were approved by the County Administrative Board (permission number 1656/06).

A low-passage, complement-sensitive strain of B. garinii Å218 (a Finnish tick isolate) was used to infect the mice. The spirochetes were cultivated in modified Barbour-Stoenner-Kelly II medium at 34°C without antibiotics and enumerated by using phase contrast or dark-field microscopy. All mice were 4–5 wk of age at the time of infection. Mice were infected by intracutaneous syringe inoculation of 106 spirochetes in 100 μl PBS in the lower back, whereas mock-infected animals received an equal volume of PBS only.

In study 1, B. garinii-infected and mock-treated C3H/He mice (three animals from each group) were killed at 1, 2, 3, 4, 5–7, 9–11, and 15 wk postinfection (p.i.). Peripheral LN and spleen were dissected and used for preparation of single-cell suspensions and histochemical staining. Blood was also collected to prepare serum specimens for measurement of soluble enzymatic activities and chemokine levels. The whole experimental setting was repeated independently three times. Collectively, 54 mice were infected with B. garinii and another 60 served as noninfected controls (6–12 animals per each time point).

In study 2, CD73−/− and wild-type mice of C57BL/6 background and C3H/He mice were inoculated with B. garinii or received PBS only (5–14 animals/group). The development of joint manifestations was monitored by periodical measuring the mediolateral diameter of the hind tibiotarsal joints using a metric caliper. At the end of the experiment, the mice were sacrificed, and their infection status was assessed by measuring serum borrelia-specific IgG Abs and culturing samples from the ear lobe, urinary bladder, and hind tibiotarsal joints for spirochete recovery, as described earlier (23).

The dissected spleen and pooled peripheral (auxiliary and inguinal) LNs were placed into cold RPMI 1640 medium supplemented with 10% (v/v) FCS. Single-cell suspensions were prepared by mechanical teasing of lymphoid tissues and consecutive passing them through a steel mesh and 77-μm nylon filters. Splenocytes were additionally purified from erythrocytes by a brief hypotonic lysis. The obtained cell suspensions were divided into two parts. One was washed and resuspended in serum-free RPMI 1640 for determination of cellular ATP and purinergic activities. The other part was directly used for flow cytometric analysis, as specified below. In some experiments, the LN cells were additionally divided onto T and B subsets by using colloidal superparamagnetic MACS MicroBeads conjugated to monoclonal rat anti-mouse CD45R (B220) or CD4 (L3T4) Abs and VarioMACS AS depletion columns (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. Because of substantial cell losses in the course of MACS separation, purified lymphocytes from three to four mice were pooled to achieve cell yields sufficient for subsequent analyses. Cell counts were done by microscopic counting of the trypan blue-stained cells in Neubauer chambers.

LN cells and splenocytes were analyzed for expression of major adhesion molecules by using a panel of anti-mouse FITC-conjugated CD3, CD8, and L-selectin (CD62L) mAbs (BD Biosciences, San Jose, CA), in combination with R-PE–conjugated CD4 (Caltag Laboratories, Burlingame, CA) or biotinylated anti-B220 (TIB146) Ab (American Type Culture Collection, Manassas, VA). Cells were stained with saturating concentrations of the Abs, and B220-labeled cells were additionally incubated with PE-streptavidin (BD Biosciences) as a second stage reagent. Data were collected on 10,000 living cells as determined by forward and side scatter intensity on a FACSCalibur cytometer and analyzed using CellQuest software (BD Biosciences). The expression of CD4+CD25+Foxp3+ cells was determined using mouse T regulatory (Treg) cell staining kit (eBioscience, San Diego, CA). Cells were stained with allophycocyanin-conjugated CD25 (PC61.5) and FITC-CD4 (RM4-5) anti-mouse Abs, fixed using provided fixation/permeabilization buffer, and then incubated with PE-conjugated Foxp3 (FJK-16s) or isotype control anti-mouse/rat Ab. Flow cytometry acquisition and analysis were performed using LSR II cytometer and FACSDiva software (BD Biosciences).

Formaldehyde-fixed, paraffin-embedded LN sections from the control and B. garinii-infected mice were immunostained with 2 μg/ml rat anti-mouse Ab TIB-146 (marker for B lymphocytes) or negative control Ab 9B5 (rat anti-human CD44) by using Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). For visualization of spirochetes, LN sections were also stained histochemically by impregnation with silver, according to standard techniques. Images of the sections were generated using an Olympus BX60 microscope with bright-field illumination (Olympus Optical, Tokyo, Japan).

Lymphocytes (2 × 104 cells) were incubated for 10 min at 37°C in 40 μl RPMI 1640 containing 2 mmol/L β-glycerophosphate, 0–1 mmol/L of various unlabeled nucleotides, and either 200 μmol/L ATP with tracer [γ-32P]ATP (PerkinElmer, Boston, MA) or 100 μmol/L AMP plus [α-32P]AMP (Amersham Biosciences, Little Chalfont, U.K.). Some cells were also pretreated for 20 min with Ap5A (100 μmol/L) prior to addition of 32P-nucleotides. Mixture aliquots were spotted onto Polygram CEL-300 sheets (Macherey-Nagel, Duren, Germany) and separated by TLC with 0.75 mol/L KH2PO4 (pH 3.5) as solvent and developed by autoradiography.

Specific lymphoid purinergic activities were determined by using [2,8-3H]ATP (PerkinElmer), [2-3H]AMP (Amersham Biosciences), and [2-3H]adenosine (Amersham Biosciences) as appropriate tracer substrates. The assays were performed at 37°C in a final volume of 80 μl RPMI 1640 supplemented with 4 mmol/L β-glycerophosphate in the following ways: 1) for adenosine triphosphatase (ATPase) assay, 5 × 104 cells were incubated for 40 min with 500 μmol/L [3H]ATP; 2) ecto-5′-nucleotidase was assayed by incubating 105 cells for 60 min with 300 μmol/L [3H]AMP; 3) adenylate kinase activity was determined by incubating 105 cells for 40 min with 400 μmol/L [3H]AMP plus 800 μmol/L γ-phosphate–donating ATP; and 4) ADA was assayed by incubating 105 cells for 60 min with 300 μmol/L [3H]adenosine. Incubation times were chosen so that the amount of metabolites did not exceed 10–15% of initially introduced substrate. Mixture aliquots were applied onto Alugram G/UV254 sheets (Macherey-Nagel) and separated by TLC. Enzymatic activities were quantified by scintillation beta counting, as described elsewhere (28), and expressed as nanomoles of 3H-substrate metabolized per hour by 106 cells.

The results are presented as mean ± SEM, unless otherwise stated. In the case when SEM is not indicated, it does not exceed the size of symbols. For statistical analyses, the Student t test or Mann-Whitney U test was used (GraphPad Prism 4.03 software; GraphPad, San Diego, CA). The significance level was set at p < 0.05.

By using C3H/He mice infected with B. garinii as an appropriate experimental model for murine borreliosis (23), these studies were undertaken to identify a link between immune responses and purinergic signaling in the course of bacterial infection. For this purpose, single-cell suspensions were prepared from lymphoid tissues of control and B. garinii-infected mice, and their adhesion phenotype and major purine-converting activities were simultaneously evaluated at acute (1–4 wk) and chronic (5–15 wk) stages of infection. First, the cellularity of peripheral LN and spleen was determined by using a panel of FITC- and PE-conjugated Abs (Fig. 1A–C). Percentage of B220+ B cells in the infected LN was increased by ∼2-fold at 1–7 wk p.i. (Fig. 1D), whereas the fractions of CD8+ and especially CD4+ T cells decreased accordingly (Fig. 1E). Contrary to the LN, only moderate shifts in splenocyte subpopulations were observed following B. garinii infection, manifested in slightly decreased number of CD8+ cells during first 3 wk p.i. (Fig. 1F). To substantiate these flow cytometry data, formalin-fixed and paraffin-embedded LN sections were stained for B cell marker B220. Immunohistochemical analysis confirmed the increased number of B cells in the infected LN, accompanied by enlargement of the whole lymphoid body and hyperplasia of B cell follicles in the cortical area (Fig. 2A). Moreover, by using the silver impregnation technique, we were able to directly visualize spirochetes in acutely infected LN, which were not eradicated even at more chronic stage (Fig. 2B).

FIGURE 1.

Flow cytometric analysis of lymphoid tissues from B. garinii-infected C3H/He mice. LN cells (A, B) and splenocytes (C) were costained with PE-B220 and FITC-CD3 (A) or PE-CD4 and FITC-CD8 (B, C) Abs, and two-dimensional representative dot blots are shown. The graphs represent the quantitative data from the obtained scattergrams and show B220+ (D) and CD4+CD8+ (E, F) expression profiles in the LN (D, E) and spleen (F) from control (co) and B. garinii-infected (inf) mice at various intervals p.i. (mean ± SE; n = 6–12). *p < 0.05 as compared with noninfected controls.

FIGURE 1.

Flow cytometric analysis of lymphoid tissues from B. garinii-infected C3H/He mice. LN cells (A, B) and splenocytes (C) were costained with PE-B220 and FITC-CD3 (A) or PE-CD4 and FITC-CD8 (B, C) Abs, and two-dimensional representative dot blots are shown. The graphs represent the quantitative data from the obtained scattergrams and show B220+ (D) and CD4+CD8+ (E, F) expression profiles in the LN (D, E) and spleen (F) from control (co) and B. garinii-infected (inf) mice at various intervals p.i. (mean ± SE; n = 6–12). *p < 0.05 as compared with noninfected controls.

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FIGURE 2.

Histological analysis of LN architecture in B. garinii-infected C3H/He mice. A, Paraffin-embedded sections were prepared from the LN of control and B. garinii-infected mice at acute (3 wk) and chronic (11 wk) stages of infection and stained for B cell marker B220 (TIB-146) or negative control Ab, as indicated. B, Silver stainings show that, along with fine reticular architecture of the LN body, which appears brown in color, darker silver impregnated spirochete-like elements can be visualized in the infected LN sections (as pointed out by arrows at higher magnification on inset). Scale bar, 200 μm (A, original magnification ×40) and 20 μm (B, original magnification ×1000).

FIGURE 2.

Histological analysis of LN architecture in B. garinii-infected C3H/He mice. A, Paraffin-embedded sections were prepared from the LN of control and B. garinii-infected mice at acute (3 wk) and chronic (11 wk) stages of infection and stained for B cell marker B220 (TIB-146) or negative control Ab, as indicated. B, Silver stainings show that, along with fine reticular architecture of the LN body, which appears brown in color, darker silver impregnated spirochete-like elements can be visualized in the infected LN sections (as pointed out by arrows at higher magnification on inset). Scale bar, 200 μm (A, original magnification ×40) and 20 μm (B, original magnification ×1000).

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We also examined whether B. garinii affects the level of CD4+CD25+Foxp3+ Treg cells, a population of naturally arising suppressor T cells known to play a crucial role in autoimmune responses (8, 29). No significant shifts in Treg cell level were detected in peripheral LN and spleen of acutely infected mice (Fig. 3A). Notwithstanding, despite the unchanged percentage of CD4+ cells coexpressing CD25/Foxp3, calculation of the relative number of Treg cells with reference to the total LN cells demonstrated its decrease by ∼30–40% following B. garinii infection, thus reflecting the lower proportion of whole population of CD4+ cells in the infected LN (Fig. 1E). To further evaluate the immunomodulatory effects of B. garinii, CD4+ T cells were costained for CD62L expression. Flow cytometric analysis revealed significant shedding of CD62L from T splenocytes during the first 3 wk p.i., whereas the release of CD62L from infected LN T cells was detectable starting at week 2 and persisted up to week 7 p.i. (Fig. 3B). Enzymatic cleavage of CD62L generally serves as important indicator of T cell activation, and this process can be particularly mediated via release of extracellular ATP and subsequent activation of P2X7 receptors (30) or through depletion of intracellular ATP pool with respective reduction in the energy capacity of the cell (31). However, direct measurements of cellular ATP levels did not reveal significant differences between control and B. garinii-infected LN cells (Supplemental Fig. 1), thus excluding the latter possibility of CD62L shedding via potential disturbance in purine salvage and energy consumption. Taking into account the potential contribution of IFN-γ and IL-4 in spirochete clearance and arthritis development in B. burgdorferi-infected C3H/He mice (20, 32), we also determined serum concentrations of these proinflammatory cytokines. Circulating levels of IFN-γ, but not IL-4, were increased upon infection of C3H/He mice with B. garinii, especially within the first week p.i. (Supplemental Fig. 2), although this did not reach statistical significance. These findings are consistent with previous observations that CD4+ T lymphocytes from this arthritis-susceptible strain may be skewed toward production of Th1 cytokines following infection with borrelia (32).

FIGURE 3.

Effect of B. garinii on Treg cell expression and CD62L shedding in C3H/He mice. A, Cells were costained with allophycocyanin-conjugated CD25 (CD25-allophyc), FITC-CD4, and PE-Foxp3 Abs and analyzed by flow cytometry. Representative dot blots of CD4+CD25+ LN T cells gated and plotted versus Foxp3+ are depicted. B, Lymphocytes were also stained for PE-CD4 and FITC-CD62L, and representative histogram generated for the gated CD4+ LN T cells is shown. The curves on the right show the expression of CD4+CD25+Foxp3+ Treg cells (A) and CD4+CD62Llow (B) LN cells and splenocytes (Spl) in control (co) and B. garinii-infected (inf) mice, all expressed as percentage of CD4+ cells (mean ± SEM; n = 3–5). *p < 0.05 as compared with noninfected controls.

FIGURE 3.

Effect of B. garinii on Treg cell expression and CD62L shedding in C3H/He mice. A, Cells were costained with allophycocyanin-conjugated CD25 (CD25-allophyc), FITC-CD4, and PE-Foxp3 Abs and analyzed by flow cytometry. Representative dot blots of CD4+CD25+ LN T cells gated and plotted versus Foxp3+ are depicted. B, Lymphocytes were also stained for PE-CD4 and FITC-CD62L, and representative histogram generated for the gated CD4+ LN T cells is shown. The curves on the right show the expression of CD4+CD25+Foxp3+ Treg cells (A) and CD4+CD62Llow (B) LN cells and splenocytes (Spl) in control (co) and B. garinii-infected (inf) mice, all expressed as percentage of CD4+ cells (mean ± SEM; n = 3–5). *p < 0.05 as compared with noninfected controls.

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Next, we took advantage of TLC for evaluating the lymphoid purine-converting pathways. Because none of the previous studies attempted to characterize the whole pattern of purine metabolism in murine lymphocytes, we first performed an autoradiographic analysis of major exchange activities in control LN cells using 32P-labeled and unlabeled nucleotide substrates. As illustrated in Fig. 4A, mouse lymphocytes converted [γ-32P]ATP into ADP and [32P]i without formation of [32P]Pi (Fig. 4A, lane 1), thus indicating that ATP hydrolysis is mediated via NTPDase rather than nucleotide pyrophosphatase/phosphodiesterase reaction. Lymphocyte coincubation with [32P]ATP and AMP stimulated their transphosphorylation into [32P]ADP (Fig. 4A, lane 2), whereas adenylate kinase inhibitor diadenosine pentaphosphate (Ap5A) prevented this [32P]i transfer (Fig. 4A, lane 3). Joint addition of [32P]ATP and UDP caused generation of [32P]UTP via NDP kinase reaction (Fig. 4A, lane4). Likewise, studies with another tracer substrate [α-32P]AMP revealed its breakdown into adenosine and [32P]i via ecto-5′-nucleotidase reaction (Fig. 4B, lane 2). Unlabeled ATP activated sequential [32P]AMP phosphorylation via [32P]ADP into [32P]ATP (Fig. 4B, lane 3), which can be prevented by pretreatment the cells with Ap5A (lane4). These data, together with our previous kinetic and competitive studies on human lymphocytes (33), demonstrate the presence of extensive network of purinergic ectoenzymes on lymphoid cell surfaces (Fig. 4C).

FIGURE 4.

Effect of B. garinii on purinergic activities in peripheral LN from C3H/He mice. LN cells were incubated with 200 μmol/L [γ-32P]ATP (A) or 100 μmol/L [α-32P]AMP (B) in the absence (Contr) and presence of 1 mmol/L AMP, UDP, and ATP and 100 μmol/L Ap5A, as indicated. Mixture aliquots were separated by TLC and developed by autoradiography. Blanks show the radiochemical purity of 32P-nucleotides after incubation without lymphocytes. Arrows indicate the positions of nucleotide standards, inorganic phosphorus (Pi) and pyrophosphate (PPi). C, Scheme of major purine-converting pathways on lymphoid surface. The elements of inactivating cascade are composed of NTPDase (1, 2), ecto-5′-nucleotidase (3), and ADA (4), whereas the backward ATP-generating pathway is represented by adenylate kinase (5) and NDP kinase (6). LN cells from control and B. garinii-infected mice were assayed for NTPDase/ATPase (D), ecto-5′-nucleotidase (E), ADA (F), and adenylate kinase (G) activities by radio-TLC, as specified in 1Materials and Methods (mean ± SEM; n = 6–12). Note, numbers in the shadowed circles specify the particular catalytic reaction within the larger network of purinergic ectoenzymes, as shown in C. *p < 0.05 as compared with controls.

FIGURE 4.

Effect of B. garinii on purinergic activities in peripheral LN from C3H/He mice. LN cells were incubated with 200 μmol/L [γ-32P]ATP (A) or 100 μmol/L [α-32P]AMP (B) in the absence (Contr) and presence of 1 mmol/L AMP, UDP, and ATP and 100 μmol/L Ap5A, as indicated. Mixture aliquots were separated by TLC and developed by autoradiography. Blanks show the radiochemical purity of 32P-nucleotides after incubation without lymphocytes. Arrows indicate the positions of nucleotide standards, inorganic phosphorus (Pi) and pyrophosphate (PPi). C, Scheme of major purine-converting pathways on lymphoid surface. The elements of inactivating cascade are composed of NTPDase (1, 2), ecto-5′-nucleotidase (3), and ADA (4), whereas the backward ATP-generating pathway is represented by adenylate kinase (5) and NDP kinase (6). LN cells from control and B. garinii-infected mice were assayed for NTPDase/ATPase (D), ecto-5′-nucleotidase (E), ADA (F), and adenylate kinase (G) activities by radio-TLC, as specified in 1Materials and Methods (mean ± SEM; n = 6–12). Note, numbers in the shadowed circles specify the particular catalytic reaction within the larger network of purinergic ectoenzymes, as shown in C. *p < 0.05 as compared with controls.

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Specific lymphoid purinergic activities were then quantified by TLC using saturating concentrations of 3H-labeled nucleotide/nucleoside substrates. In particular, ATPase was evaluated as the rate of [3H]ATP dephosphorylation into 3H-metabolites that were quantified as pooled ADP, AMP, and nucleoside fractions, whereas ecto-5′-nucleotidase activity of the cells was determined by their ability to hydrolyze [3H]AMP into [3H]adenosine. ATPase activity of the infected LN was increased at 1 wk p.i. and then decayed to control levels (Fig. 4D). B. garinii infection also induced two-directional shifts in lymphoid ecto-5′-nucleotidase, the activity of which was decreased at week 1 and then significantly upregulated at week 3 p.i. (Fig. 4E). Measurement of ADA activity revealed transient ∼2-fold increase in the rate of [3H]adenosine deamination by infected LN cells at 2–4 wk p.i. (Fig. 4F). Adenylate kinase was also determined by extent of ATP-induced phosphorylation of [3H]AMP into 3H-phosphoryls ADP/ATP, but its activity remained unchanged during B. garinii infection (Fig. 4G). Likewise, no changes in other nucleotide-phosphorylating ectoenzyme, NDP kinase, were observed in the LN from B. garinii-infected mice (data not shown). Noteworthy, control activities for each particular ectoenzyme were maintained at certain steady and characteristic levels throughout the whole period of the study (1–15 wk).

Concurrent TLC analysis of purine metabolism in splenic cells did not reveal any differences between the control and infected groups (Supplemental Fig. 3). Knowing that various purinergic enzymes freely circulate in the bloodstream of humans, rodents, and other species (11, 28), we also tested the possibility that intravascular purine turnover is affected by infecting the mice with B. garinii. However, except for acute (presumably, tissue stress-mediated) decreases in soluble 5′-nucleotidase and adenylate kinase at week 1, the activities of serum 5′-nucleotidase, ADA, and adenylate kinase remained unchanged at all subsequent monitored intervals, 2–15 wk p.i. (Supplemental Fig. 4). It also has to be emphasized that B. garinii itself does not display detectable purinergic activities, as ascertained by direct incubation of ∼106 spirochetes with 3H-labeled nucleotide and adenosine substrates (data not shown).

The observation that borrelia infection induces marked abnormalities in the LN cellularity (Fig. 1) prompted us to further investigate whether the above shifts in lymphoid purine metabolism might be due to disparate enzymatic activities among various lymphocyte subsets. For this purpose, control and infected LN cells were separated using MACS columns, followed by TLC analysis of purine-converting activities in the purified B and T cell subsets (Fig. 5). In fact, taking into account relatively low ecto-5′-nucleotidase activity on B versus T lymphocytes (Fig. 5C), we do not exclude that diminished AMP hydrolysis in infected LN at week 1 p.i. (Fig. 4E) might be simply due to increased percentage of B220+ B cells. However, in all other cases, the enhanced purine catabolism by B. garinii-infected LN is proven to be associated with upregulation of ectoenzymes on certain cell subsets. Specifically, the infected CD3+ (CD4+CD8+) T lymphocytes displayed markedly increased ATPase/NTPDase (Fig. 5B) and ecto-5′-nucleotidase (Fig. 5C) activities, which occurred at 1 and 3 wk p.i., respectively. Probably, these drastic shifts in ATPase and ecto-5′-nucleotidase on surface of T lymphocytes were sufficient for increasing the nucleotide-inactivating capacity of the whole LN (Fig. 4D, 4E). Likewise, the enhanced adenosine-deaminating capacity of the infected LN also occurred because of cell subtype-specific activation of ADA; however, this increase was associated with B220+ B rather than CD4+CD8+ T lymphocytes (Fig. 5D). As in the case of unchanged adenylate kinase in total LN cells, B. garinii also did not affect the activity of this phosphorylating ectoenzyme in the purified cell subpopulations (Fig. 5E).

FIGURE 5.

Effect of B. garinii on purine metabolism by purified T and B lymphocytes. Single-cell LN suspensions were divided onto B220+ B cell and CD3+, CD4+, and CD8+ T cell subsets by using MACS separation columns. A, Cell purity was verified by flow cytometry to be >95%, and representative dot blots are shown. T and B lymphocytes from the control and B. garinii-infected mice were assayed by TLC for ATPase/NTPDase (B), ecto-5′-nucleotidase (C), ADA (D), and adenylate kinase (E) activities. Because of the low yield, the purified lymphocytes from three to four mice were pooled to obtain one specimen. Data show mean ± SEM from three independent experiments. *p < 0.05 as compared with mock-infected controls.

FIGURE 5.

Effect of B. garinii on purine metabolism by purified T and B lymphocytes. Single-cell LN suspensions were divided onto B220+ B cell and CD3+, CD4+, and CD8+ T cell subsets by using MACS separation columns. A, Cell purity was verified by flow cytometry to be >95%, and representative dot blots are shown. T and B lymphocytes from the control and B. garinii-infected mice were assayed by TLC for ATPase/NTPDase (B), ecto-5′-nucleotidase (C), ADA (D), and adenylate kinase (E) activities. Because of the low yield, the purified lymphocytes from three to four mice were pooled to obtain one specimen. Data show mean ± SEM from three independent experiments. *p < 0.05 as compared with mock-infected controls.

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Previous studies with B. burgdorferi have indicated that susceptibility to infection is influenced by the genetic composition of the host: particularly, the C3H/He mice are characterized by more severe disease than the C57BL/6 strain (21, 22). To further extend our understanding of the role of purinergic signaling in microbial infection, we performed comparative analysis of major purinergic activities in C57BL/6 versus C3H/He mice. Relative to C3H/He background, T lymphocytes from C57BL/6 mice display higher ATP- and AMP-inactivating activities, whereas both LN and spleen from these mice are characterized by lower ADA activity, primarily because of the diminished enzyme level on surface of B cells (Fig. 6A). Because of such inverse strain-dependent distinctions in the key purine-inactivating ectoenzymes, the ratio of ecto-5′-nucleotidase to ADA activity in peripheral LN from C57BL/6 mice exceeds those in C3H/He mice by ∼3-fold, thus indicating higher production of extracellular adenosine in the former mice.

FIGURE 6.

Comparative analyses of lymphoid purine metabolism and B. garinii-induced joint swelling in different mouse strains. A, Cells were isolated from spleens and peripheral LNs of C57BL/6 and C3H/He mice, and LN cells were additionally separated using MACS selection onto CD3+ (T-lymph) and B220+ (B-lymph) subsets. ATPase/NTPDase, ecto-5′-nucleotidase, ADA, and adenylate kinase were assayed by radio-TLC in lymphoid cells from at least four to six mice per each strain. Bars show the average activities in C57BL/6 relative to C3H/He mice, and asterisks indicate significant differences between the strains (*p < 0.05). Mean lateral diameters of tibiotarsal joints were monitored in C3H/He mice (B) as well as CD73−/− mice and their C57BL/6 wild-type littermates (C), inoculated either with B. garinii (inf) or PBS only (co). Joint swelling data were obtained by subtracting the joint diameter measured at various intervals p.i. from the preinfection diameter (mean ± SEM). The numbers of studied animals are specified in the parentheses. Because of the absence of any differences in joint diameters of mock-infected C57BL/6 and syngenic CD73−/− mice, data from these control mice with similar parental background were pooled and presented as “C57 co.” *p < 0.05 as compared with controls.

FIGURE 6.

Comparative analyses of lymphoid purine metabolism and B. garinii-induced joint swelling in different mouse strains. A, Cells were isolated from spleens and peripheral LNs of C57BL/6 and C3H/He mice, and LN cells were additionally separated using MACS selection onto CD3+ (T-lymph) and B220+ (B-lymph) subsets. ATPase/NTPDase, ecto-5′-nucleotidase, ADA, and adenylate kinase were assayed by radio-TLC in lymphoid cells from at least four to six mice per each strain. Bars show the average activities in C57BL/6 relative to C3H/He mice, and asterisks indicate significant differences between the strains (*p < 0.05). Mean lateral diameters of tibiotarsal joints were monitored in C3H/He mice (B) as well as CD73−/− mice and their C57BL/6 wild-type littermates (C), inoculated either with B. garinii (inf) or PBS only (co). Joint swelling data were obtained by subtracting the joint diameter measured at various intervals p.i. from the preinfection diameter (mean ± SEM). The numbers of studied animals are specified in the parentheses. Because of the absence of any differences in joint diameters of mock-infected C57BL/6 and syngenic CD73−/− mice, data from these control mice with similar parental background were pooled and presented as “C57 co.” *p < 0.05 as compared with controls.

Close modal

The severity of borrelia infection was then determined in these mouse strains by measuring the diameters of tibiotarsal joints as an appropriate assessment of arthritis severity reflecting local tissue edema. Clear joint swelling was observed several weeks p.i. of C3H/He mice with B. garinii, and it persisted without regression until 12 wk (Fig. 6B). Note, because the mice were 4–5 wk of age at the time of infection, the diameter of mock-infected joints was also increased during the first 3 wk because of the natural growth of the animal. Contrary to C3H/He mice, no additional increment in the joint diameter was seen in B. garinii-infected C57BL/6 mice (Fig. 6C). It may be reasonably suggested that higher resistance of C57BL/6 mice to arthritis development is defined, at least in part, by more efficient production of adenosine. To verify this hypothesis, we used C57BL/6 mice lacking the key enzyme of nucleotide-inactivating chain, ecto-5′-nucleotidase/CD73, and therefore having the impaired adenosinergic signaling (9, 10). Strikingly, unlike their wild-type littermates, B. garinii-infected CD73−/− mice developed mild but significant joint swelling at 9–12 wk p.i. (Fig. 6C), thus confirming the important role of endogenous adenosine in the pathogenesis of infection.

The development of symptomatic infection was also verified by measuring serum levels of borrelia-specific IgG Abs, which were maintained at highly positive levels in all B. garinii-infected mice (Fig. 7A) and remained nearly undetectable in control mock-infected animals (data not shown). However, evaluation of the infection status by culturing ear punch, tibiotarsal joint, and bladder samples revealed clear-cut differences between studied mice (Fig. 7B–D). Specifically, most of the tissues from C3H/He mice remain culture positive throughout the whole period of examination, indicating the development of persistent infection in this borrelia-prone mouse strain. By contrast, both wild-type and CD73−/− mice of C57BL/6 background were only transiently infected with B. garinii and able to efficiently eradicate the cultivable spirochetes at more chronic stage of infection.

FIGURE 7.

B. garinii induces persistent infection in C3H/He but not in C57BL/6 and CD73−/− mice. C3H/He, as well as wild-type (C57BL/6) and CD73−/− mice, of C57BL/6 background were infected with B. garinii and sacrificed at the indicated intervals p.i. A, The sera of the mice were tested for borrelia-specific IgG Abs, and the results are expressed as ODs at 492-nm wavelength (mean ± SEM). Ear lobe (B), tibiotarsal joint (C), and urinary bladder (D) samples were cultured for spirochete recovery. The graphs show scatter plots of borrelia-positive (defined as “1”) and negative (“0”) cultures with a horizontal line at the mean. Numbers of positive cultures per total numbers of studied mice are specified above the symbols. *One culture sample was contaminated.

FIGURE 7.

B. garinii induces persistent infection in C3H/He but not in C57BL/6 and CD73−/− mice. C3H/He, as well as wild-type (C57BL/6) and CD73−/− mice, of C57BL/6 background were infected with B. garinii and sacrificed at the indicated intervals p.i. A, The sera of the mice were tested for borrelia-specific IgG Abs, and the results are expressed as ODs at 492-nm wavelength (mean ± SEM). Ear lobe (B), tibiotarsal joint (C), and urinary bladder (D) samples were cultured for spirochete recovery. The graphs show scatter plots of borrelia-positive (defined as “1”) and negative (“0”) cultures with a horizontal line at the mean. Numbers of positive cultures per total numbers of studied mice are specified above the symbols. *One culture sample was contaminated.

Close modal

By investigating the combined features of the immune responses and lymphoid purine metabolism in B. garinii-infected mice, we have identified a link between these different but apparently interrelated processes and further demonstrated the involvement of purinergic signaling in the development of persistent infection. Consistent with our earlier observations (23), inoculation of B. garinii to C3H/He mice caused a disseminated infection, joint swelling, and elevated levels of borrelia-specific IgG Abs. The microenvironment of peripheral LN was also deeply modified during infection, as ascertained by ∼2- to 2.5-fold enlargement of the tissue, hyperplasia of B cell follicle area, and drastic increase in ratio of B to T lymphocytes. These findings corroborate previous observations that B. burgdorferi infection of animals and humans generally results in marked LN lymphoproliferation, possibly because of strong B and, to less extent, T cell mitogenic activity of borrelia lipoproteins (34, 35). Collectively, this experimental model of murine borreliosis with “classical” symptoms of infection and inflammation provides sufficient background for further comprehensive investigation of the role of purinergic signaling in the pathogenesis of disease.

The principal finding of this work is that B. garinii selectively enhances purine catabolism in peripheral LN but not spleen and blood of acutely infected C3H/He mice, which is particularly manifested in upregulations of ATPase/NTPDase and ecto-5′-nucleotidase activities on T lymphocytes as well as ADA activity on B lymphocytes. No significant B. garinii-mediated shifts were determined in studies on yet another ectoenzyme adenylate kinase. Along with clear evidence for an inability of spirochetes to affect the backward ATP-regenerating pathway, such unchanged activity may additionally serve as suitable “internal control” for comparable assay conditions among the control and infected lymphocytes. Although the exact mechanisms behind these transient shifts in lymphoid ectoenzymes remain unknown, it is worth mentioning that p.i. period of 2–4 wk corresponds to the maximal host’s efforts to clear borrelia infection, accompanied by release of proinflammatory cytokines and common induction of genes affiliated with host defense mechanisms (22).

The lymphoid cells usually possess ATP-generating/adenosine-eliminating phenotype with relatively low ectonucleotidase activities that is consistent with their overall repertoire of proinflammatory functions (11, 33). For instance, dendritic cells remove adenosine from their environment via high intrinsic ADA activity, which allows cutting them off from the suppression and mediating T cell activation and other inflammatory responses (7). Despite this general trend, NTPDase1/CD39 and ecto-5′-nucleotidase/CD73 can be selectively upregulated on a certain population of activated CD4+CD25+Foxp3+ Treg cell lymphocytes, thereby protecting these immunosuppressive cells from cytotoxic effects of extracellular ATP (30) and contributing to the control of inflammatory autoimmune diseases (8, 29). Therefore, transient upregulation of ATP- and AMP-hydrolyzing enzymes on T lymphocytes in our study may reflect the increased suppressive activity of Treg cells during B. garinii infection, even despite the unchanged percentage of this T cell population.

Changes in lymphoid nucleotide homeostasis can also be involved in other immune responses, specifically via ATP-mediated shedding of CD62L from the activated T cells (30, 31) and control of lymphocyte trafficking into inflamed tissues (10). Interestingly, although splenic CD4+ T cells became activated immediately upon B. garinii infection, significant shedding of CD62L from LN cells occurred only at 2 wk p.i. (Fig. 3B). It may be reasonably speculated that such delayed activation results from coincidentally increased ATP-scavenging capability of T lymphocytes in the infected animals, which makes the LN cells unresponsive to the initial surge of proinflammatory ATP. In this context, it is also relevant to mention that ATP-hydrolyzing capability of various murine T cell clones and hybridomas was upregulated upon antigenic activation of naive T cells, thereby attenuating the ATP-mediated secretion of proinflammatory cytokines IFN-γ, IL-2, but not IL-4 (1).

Altogether, these directional and apparently interrelated shifts in lymphoid purine-converting activities and expression of adhesion receptors presumably comprise an important constituent of innate immune response attempted to prevent the dissemination of spirochetes during the acute phase of borrelia infection and tissue stress. However, such initial proinflammatory burst seems not to be sufficient and advantageous to the host in terms of efficient spirochete eradication. In contrast, from the standpoint of strong immunosuppressive activity of adenosine (2, 8), shifting of purine metabolism toward the diminished adenosine level may provide positive feedback signals to continue overrecruitment and sustained activation of potentially harmful cytolytic lymphocytes, NK cells, macrophages, and other immune cells. Our data on clear-cut differences in the lymphoid purinergic activities and infection status of C3H/He versus C57BL/6 mice count in favor of this hypothesis. Specifically, in comparison with C57BL/6 background, the lymphocytes from C3H/He mice possess diminished adenosine-generating capability because of a low ratio of ecto-5′-nucleotidase to ADA, which can be further misbalanced by invaded pathogen via upregulation of ADA activity on B lymphocytes. At the same time, B. garinii-infected C57BL/6 but not C3H/He mice were able to efficiently eradicate spirochetes and did not develop any signs of joint swelling. Most strikingly, targeted deletion of the key adenosine-generating enzyme, ecto-5′-nucleotidase/CD73, triggered mild but significant joint swelling in B. garinii-infected CD73−/− mice (Fig. 6C). Collectively, these data suggest that insufficient basal adenosine level and/or disordered lymphoid purine metabolism during bacterial infection may serve as important prerequisite for ongoing promotion of local inflammatory responses and development of persistent infection. We do not exclude that impaired adenosinergic signaling could also contribute to the previously reported ability of borrelia to break normal tolerance mechanisms via autoreactive activation of B lymphocytes and overproduction of rheumatoid factor (35).

These findings reinforce the general view of adenosine as important anti-inflammatory and antirheumatic molecule. Particularly, selective A3 receptor agonists were shown to suppress TNF-α production and ameliorate the clinical and histological features of arthritis both in rheumatoid arthritis patients and rat models of adjuvant-induced arthritis (24). The anti-inflammatory effects of methotrexate, one of the most effective antirheumatic drugs, are reportedly due in large part to its capacity to enhance adenosine levels at sites of inflammation, which can be achieved via activation of endothelial ecto-5′-nucleotidase (36) and/or downregulation of lymphoid ADA (37). This conclusion is further supported by studies with gene-modified mice, where methotrexate reduced the leukocyte accumulation and TNF-α concentration and simultaneously increased adenosine concentration in the air-pouch exudates of wild-type but not A2A and A3 receptor knockout (38) or CD73-deficient (39) mice. Data on significantly elevated ADA activity in the synovial fluid but not in serum of rheumatoid arthritis patients (40) also lend credence to the role of adenosine in pathogenesis of joint inflammation. We believe that our study gains another novel insight into these concepts by demonstrating the important role of adenosine signaling as a link between the infection and immunity.

Previous studies have also demonstrated that certain components of purinergic signaling cascade can be modulated during microbial infection, either by the host cell or by the invaded pathogen. The inflammatory pathways activated by extracellular ATP at the early steps of microbial infection are particularly pertinent for enhanced production of proinflammatory cytokines, formation of chemotactic fields, and facilitating the ability of phagocytic cells to localize to sites of inflammation (2, 4). For instance, ATP release by Mycobacterium tuberculosis-infected macrophages enhances their ability to kill bacteria via production of NO and reactive oxygen intermediates (41). In turn, various microbes have also evolved different mechanisms to activate ATP-releasing pathways, which should thus be viewed as virulence factors for these pathogens. For instance, Shigella flexneri triggers massive ATP release by host epithelial cells via opening of connexin-26 hemichannels, which favors further bacterial invasion and spreading (42). ATP derived from intestinal commensal bacteria may activate various P2 receptors on lamina propria CD11c+ APCs to induce TGF-β, IL-6, and IL-23 production, thereby leading to local differentiation of “pathogenic” Th17 cells, induction of intestinal inflammation, and deterioration of colitis (3).

Some parasites and bacteria (like Leishmania, Mycobacterium, apicomplexan, and trypanosome species) can express and secrete certain purinergic enzymes, including NTPDases, 5′-nucleotidase, NDP kinase, and in this way, maintain essential nutrient salvage pathways and interfere with signaling pathways triggered by the host (26, 43). Interestingly, Staphylococcus aureus, Bacillus antracis, and some other bacterial pathogens express adenosine synthase, a cell wall-anchored enzyme with 5′-nucleotidase signature sequences, and via generation of adenosine promote their escape from phagocytic clearance during host infection and subsequent survival and replication in organ tissues (27). Our data suggest, however, that B. garinii does not express purine-converting enzymes but instead has the capacity to modify the host’s metabolism, thus allowing the bacterium to evade the host defense mechanisms. Interestingly, recent data have shown that an intracellular protozoan Leishmania remains viable following transmission to the skin by sand flies and subsequent capturing by invading neutrophils, and this “Trojan horse” phagocytic mechanism is crucial for silent entry, survival, and spreading of the parasite (44). Because ATP also plays a role in leishmaniasis, presumably by decreasing Leishmania survival (43), it might be an attractive idea to test whether this parasite can directly modulate purinergic cascade, thus protecting itself and the infected immune cells from the death.

In conclusion, in this study, we show that local nucleotide/nucleoside concentrations are selectively and transiently misbalanced in peripheral LN from B. garinii-infected C3H/He mice and further suggest a new role for purine-converting ectoenzymes in the control of immune responses and pathology of Lyme disease. Further elucidation of the involvement of purinergic signaling, in conjunction with other multiple cellular and genetic components, in the development of arthritis and other disease manifestations of borrelia infection might have broader implications on our understanding of host defense mechanisms and inflammatory responses to various types of bacteria.

We thank Sari Mäki, Marju Niskala, and Perttu Terho for excellent technical assistance and Anne Sovikoski-Georgieva for secretarial help. We also thank Drs. Linda F. Thompson for providing us with CD73−/− mice and Marko Salmi for critical reading the manuscript.

Disclosures The authors have no financial conflict of interest.

This work was supported by grants from the Academy of Finland and Sigrid Juselius Foundation.

The online version of this article contains supplemental material.

Abbreviations used in this paper:

ADA

adenosine deaminase

ATPase

adenosine triphosphatase

CD25-allophyc

allophycocyanin-conjugated CD25

CD62L

L-selectin

inf

B. garinii-infected

co

control

LN

lymph node

NTPDase

ectonucleoside triphosphate diphosphohydrolase

p.i.

postinfection

Pi

inorganic phosphorus

PPi

pyrophosphate

Spl

splenocytes

Treg

T regulatory.

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