The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dame, T. M.
Right arrow Articles by Furie, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dame, T. M.
Right arrow Articles by Furie, M. B.
Right arrowPubmed/NCBI databases
*GEO DataSet
*Substance via MeSH
The Journal of Immunology, 2007, 178: 1172-1179.
Copyright © 2007 by The American Association of Immunologists, Inc.

IFN-{gamma} Alters the Response of Borrelia burgdorferi-Activated Endothelium to Favor Chronic Inflammation1

Tarah M. Dame*,{dagger}, Barbara L. Orenzoff{dagger}, Lance E. Palmer{dagger},{ddagger} and Martha B. Furie2,{dagger},{ddagger},§

* Graduate Program in Genetics, {dagger} Center for Infectious Diseases, {ddagger} Department of Molecular Genetics and Microbiology, and § Department of Pathology, School of Medicine, Stony Brook University, Stony Brook, NY 11794


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Borrelia burgdorferi, the agent of Lyme disease, promotes proinflammatory changes in the endothelium that lead to the recruitment of leukocytes. The host immune response to infection results in increased levels of IFN-{gamma} in the serum and lesions of Lyme disease patients that correlate with greater severity of disease. Therefore, the effect of IFN-{gamma} on the gene expression profile of primary human endothelial cells exposed to B. burgdorferi was determined. B. burgdorferi and IFN-{gamma} synergistically augmented the expression of 34 genes, 7 of which encode chemokines. Six of these (CCL7, CCL8, CX3CL1, CXCL9, CXCL10, and CXCL11) attract T lymphocytes, and one (CXCL2) is specific for neutrophils. Synergistic production of the attractants for T cells was confirmed at the protein level. IL-1beta, TNF-{alpha}, and LPS also cooperated with IFN-{gamma} to induce synergistic production of CXCL10 by the endothelium, indicating that IFN-{gamma} potentiates inflammation in concert with a variety of mediators. An in vitro model of the blood vessel wall revealed that an increased number of human T lymphocytes traversed the endothelium exposed to B. burgdorferi and IFN-{gamma}, as compared with unstimulated endothelial monolayers. In contrast, addition of IFN-{gamma} diminished the migration of neutrophils across the B. burgdorferi-activated endothelium. IFN-{gamma} thus alters gene expression by endothelia exposed to B. burgdorferi in a manner that promotes recruitment of T cells and suppresses that of neutrophils. This modulation may facilitate the development of chronic inflammatory lesions in Lyme disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lyme disease (LD)3 is the most common vector-borne disease in the United States. The etiologic agent of LD, Borrelia burgdorferi, is transmitted to its mammalian host via a bite from an infected Ixodes tick (1). Upon transmission to humans, the spirochetes spread in the skin and induce a local inflammatory response characterized by recruitment of neutrophils, macrophages, and lymphocytes. The spirochetes then disseminate through the blood and lymph to secondary sites of infection including the heart, joints, and nervous system. Dissemination results in a more chronic inflammatory response involving recruitment of lymphocytes and plasma cells (2).

The endothelial lining of the blood vessel wall is the first barrier encountered by migrating leukocytes and therefore plays an important role in their recruitment. B. burgdorferi causes proinflammatory changes in endothelia in vitro, including up-regulation of adhesion molecules for leukocytes, secretion of chemokines, and subsequent enhancement of the transmigration of several types of leukocytes (3, 4, 5, 6, 7, 8, 9). The development of Lyme arthritis in animal models is associated with the production of chemokines that attract neutrophils. Mice that carry a deficiency in CXCR2, a receptor for neutrophil-specific chemokines, develop less severe arthritis upon infection with B. burgdorferi (10). These data suggest that neutrophils are key mediators of murine Lyme arthritis. Despite their role in the murine model, neutrophils are much less abundant in the synovial tissues of human LD patients, where T lymphocytes and plasma cells predominate (11).

Several lines of evidence indicate a role for T lymphocytes in the pathogenesis of human LD. T lymphocytes that secrete IFN-{gamma} are selectively recruited by endothelia exposed to B. burgdorferi in vitro (9). Similarly, the synovial fluids of Lyme arthritis patients are enriched for Th1 cells, which secrete IFN-{gamma}, and their presence correlates with the severity of arthritis (12). There is also a high correlation between symptom score in LD and the serum levels of IFN-{gamma} (13). In addition, as compared with circulating T lymphocytes, a greater proportion of T cells in cutaneous lesions of LD patients express the chemokine receptors CXCR3 and CCR5, which are associated with a Th1 phenotype. Therefore, IFN-{gamma} and the T lymphocytes that secrete it appear to be critical mediators of the inflammatory response in LD. Moreover, CXCR3 is the receptor for the IFN-{gamma}-induced chemokines CXCL9, CXCL10, and CXCL11 (14) and the abundance of CXCR3+ T cells in LD lesions suggests that IFN-{gamma} may facilitate their recruitment.

In this study, we provide the first evidence that B. burgdorferi cooperates with IFN-{gamma} to synergistically augment transcription of 34 genes by primary human endothelial cells. Of these genes, 6 encoded chemokines that attract T lymphocytes. In contrast, levels of transcripts encoding chemoattractants for neutrophils remained mostly unaltered. Further analysis verified that concurrent exposure to IFN-{gamma} and B. burgdorferi drove endothelial cells to secrete high levels of chemokines that recruit T cells. Moreover, addition of IFN-{gamma} to endothelial monolayers activated by B. burgdorferi promoted transmigration of T cells but suppressed that of neutrophils. IFN-{gamma} thereby likely mediates the shift toward a chronic inflammatory response in human LD.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and cytokines

Recombinant human IFN-{gamma}, TNF-{alpha}, and IL-1beta, as well as mAb 51673 to CX3CL1, were provided by R&D Systems. Escherichia coli LPS 026:B6 was supplied by Sigma-Aldrich.

Culture of B. burgdorferi

B. burgdorferi strain HBD1 (1) (passages 40–56) was cultured at 33°C in Barbour-Stoenner-Kelly medium that was modified to minimize the presence of LPS (5) and contained 5% EX-CYTE Growth Enhancement Medium Supplement (Serologicals). For each experiment, the spirochetes were centrifuged, resuspended in assay medium, counted via dark-field microscopy, and then added to the cells at a ratio of 10 spirochetes per endothelial cell (sp/EC).

Isolation and culture of endothelial cells

HUVEC were isolated from umbilical cords by collagenase perfusion as previously reported (15). In brief, the umbilical vein was flushed with HEPES-buffered saline and then incubated at 37°C for 12 min with 0.35 mg/ml type II collagenase (Worthington), followed by a wash with medium 199 (Invitrogen Life Technologies) containing 5% FBS (HyClone Laboratories). The HUVEC were cultured for up to 5 days in medium 199 supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), amphotericin B (2 µg/ml), and 20% FBS (referred to as 20% medium), passaged once, and used for experiments upon reaching confluence.

Microarray analysis

HUVEC were plated on 60-mm diameter tissue-culture dishes (Corning Glass Works) at 1.7 x 106 cells in 3 ml of 20% medium and grown to confluence. The HUVEC were washed three times with assay medium (medium 199 supplemented with 25 mM HEPES (pH 7.2) and 20% FBS that was heat inactivated at 56°C for 30 min). Next, B. burgdorferi (10 sp/EC), IFN-{gamma} (100 U/ml), B. burgdorferi and IFN-{gamma} combined, or assay medium alone was added and the cells were incubated for 8 h at 37°C. RNA was isolated with TRI Reagent (Sigma-Aldrich) according to the manufacturer’s instructions. DNA was removed from all RNA samples using the DNA-free kit (Ambion) as specified by the manufacturer. RNA samples were stored at –80°C and then analyzed by the Stony Brook University DNA Microarray Facility using the Affymetrix Human U133 Expression Array Plus 2.0.

The microarray data were analyzed using the Affy and Limma modules of the Bioconductor package for the R statistical environment (16, 17); the robust multichip averaging function was used for normalization of expression values (18). Gene ontology classifications were determined using the Gene Ontology Consortium (19) based on the current probe annotations provided by Affymetrix. A positive synergy score was determined by dividing the endothelial gene expression intensity induced by stimulation with B. burgdorferi and IFN-{gamma} combined by the sum of the gene expression intensities induced by either stimulus alone. A negative synergy score was calculated by dividing the inverse of the gene expression level induced by B. burgdorferi and IFN-{gamma} combined by the sum of the inverses of the gene expression levels attributed to the individual stimuli; this number was multiplied by –1 to give the negative synergy score a negative value for representative purposes.

All microarray data described in this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo/) with the accession no. GSE6092.

Analysis of gene-specific transcripts

HUVEC were seeded in 12-well dishes at 6 x 105 cells/well in 20% medium and cultured to confluence. Monolayers were incubated with medium alone (calibrator) or stimulated with B. burgdorferi (10 sp/EC), IFN-{gamma} (100 U/ml), or the two agents combined in 1.5 ml of 20% medium for 8 h. RNA was isolated using an RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions and stored at –80°C. Subsequently, any remaining genomic DNA was removed and cDNA was amplified from the RNA samples with a QuantiTect Reverse Transcription kit (Qiagen) in a Mastercycler gradient thermal cycler (Eppendorf). The cDNA was amplified using gene-specific primers (CXCL2, CXCL10, CXCL11, and GAPDH; QuantiTect Primer Assays) and the QuantiTect SYBR Green PCR kit provided by Qiagen. Reactions were conducted on three replicate samples in a MicroAmp Optical 96-Well Reaction Plate (Applied Biosystems) in an Applied Biosystems 7500 Real-Time PCR System. Relative quantification values were calculated via the comparative threshold cycle method using SDS software version 1.3.1 (Applied Biosystems).

Measurement of chemokines

HUVEC were plated in 48-well dishes at 1 x 105 cells/well in 0.5 ml of 20% medium. At confluence, B. burgdorferi was added at 10 sp/EC in the absence or presence of 100 U/ml IFN-{gamma} and cultures were incubated at 37°C for 24 h. Conditioned media were collected, clarified by centrifugation, and stored at –80°C before analysis. ELISAs for CXCL8, CXCL9, CXCL10, CXCL11, CCL7 (R&D Systems), and CCL8 (Ray Biotech) were performed according to the manufacturers’ instructions. To determine cell surface expression of CX3CL1, 2 x 104 HUVEC were plated in 100 µl of 20% medium in a 96-well plate and stimulated as above. A mAb to CX3CL1 was used in a whole cell ELISA as previously described (5).

To determine the effects of various cytokines and bacterial products on IFN-{gamma}-induced expression of CXCL10, HUVEC were incubated with TNF-{alpha} (1 ng/ml), IL-1beta (0.1 U/ml), or E. coli LPS (2 ng/ml) in the absence or presence of IFN-{gamma} (100 U/ml) for 24 h at 37°C. Conditioned media were collected, clarified by centrifugation, and stored at –80°C before analysis by ELISA.

Transendothelial migration assays

Human T lymphocytes were isolated from the venous blood of healthy donors, after obtaining informed consent. Collection of blood for these studies was approved by Stony Brook University’s Committee on Research Involving Human Subjects. Blood was collected in a final concentration of 0.12% EDTA as an anticoagulant, then diluted with an equal volume of PBS without Ca2+ and Mg2+ and layered over Accu-Prep density gradient medium (Accurate Chemical Scientific). Following centrifugation at 920 x g for 20 min, the PBMC were collected from the plasma/Accu-Prep interface, diluted with an equal volume of PBS without Ca2+ and Mg2+, and centrifuged at 920 x g for 6 min. The cells were next washed three times with 10 ml of PBS containing 0.5% low-LPS BSA (Serologicals) and 2 mM EDTA and centrifuged at 200 x g for 7 min to remove platelets. T lymphocytes were isolated from the remaining PBMC by negative selection via the Pan T Cell Isolation kit II (Miltenyi Biotec) as specified by the manufacturer. T lymphocytes were ≥99% pure as determined by flow cytometric detection of CD3 expression. Human neutrophils were isolated from venous blood that was anticoagulated with 0.12% EDTA. In brief, RBC were sedimented with 0.6% dextran (Pharmacia) for 45–60 min. The leukocyte-rich plasma was layered over Accu-Prep density gradient medium and subjected to centrifugation as previously described (20). Neutrophils (~98% pure) were resuspended at 2 x 106 cells/ml in assay medium.

For the transmigration assay, HUVEC were plated on acellular amniotic connective tissue attached to Teflon rings, as previously described (21). After 7–10 days, when maximal transendothelial electrical resistance is achieved (15), the HUVEC-amnion cultures were washed three times with assay medium and subsequently stimulated with B. burgdorferi (10 sp/EC), IFN-{gamma} (100 U/ml), or the two stimuli combined for 24 h. The HUVEC-amnion cultures then were washed three times and freshly isolated leukocytes (1 x 106 cells/ring) were added. The T cells or neutrophils were allowed to migrate at 37°C for 4 h or 30 min, respectively. The HUVEC-amnion cultures were fixed overnight in 10% buffered formalin at 4°C and removed from the Teflon rings. They were then stained with modified Wright stain (Sigma-Aldrich) and the leukocytes associated with each culture were counted in nine fields at x400 using a bright-field microscope. To determine the percentage of leukocytes that had migrated beneath the endothelium, the HUVEC-amnion cultures were dehydrated, embedded in glycol methacrylate, and cross-sectioned perpendicular to the plane of the endothelial monolayer. The sections were stained with toluidine blue and analyzed by light microscopy (5). A minimum of 100 leukocytes was counted to determine the proportion of cells above and below the endothelium. Any loss of leukocytes from the apical surface of the endothelium during the embedding process was corrected for as previously described (22).

Statistics

Microarray data were analyzed by linear modeling and empirical Bayes-moderated t-statistic methods using the Limma module of the Bioconductor package for the R statistical environment (16, 17). All other data were subjected to an unpaired ANOVA followed by the Tukey-Kramer multiple-comparison test using InStat statistical software (GraphPad Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Synergistic induction of endothelial gene expression by B. burgdorferi and IFN-{gamma}

We sought to determine whether B. burgdorferi and IFN-{gamma} independently or cooperatively enhance the expression of proinflammatory mediators by endothelia. To this end, HUVEC were treated with B. burgdorferi, IFN-{gamma}, a combination of the two, or medium alone for 8 h and the RNA was isolated for microarray analysis. Activation of HUVEC with B. burgdorferi resulted in the differential regulation of relatively few genes. No genes were significantly down-regulated in response to B. burgdorferi, whereas the transcription of 23 genes was augmented 2-fold or more (Table I). Eleven of these genes have been reported to be induced by B. burgdorferi in various cell types or infected hosts (3, 4, 5, 23, 24, 25). A number of the up-regulated genes encode adhesion molecules or chemokines with known roles in recruitment of leukocytes; the products of four (ICAM-1, VCAM-1, CXCL8, and CCL2) have been demonstrated to promote migration of leukocytes across B. burgdorferi-activated endothelium in vitro (4, 7, 9). In contrast to treatment with B. burgdorferi, exposure of HUVEC to IFN-{gamma} alone produced changes in the levels of transcription for a relatively large number of genes. A small subset of these genes are classified as mediators of inflammation according to the Gene Ontology Consortium (12 of 215 up-regulated genes and 1 of 62 down-regulated genes; supplemental data4) and may thus contribute to the pathogenesis of LD.


View this table:
[in this window]
[in a new window]

  		Table I. Stimulation of endothelium with B. burgdorferi enhances the transcription of 23 genes

 
Notably, several of the proinflammatory genes induced by either B. burgdorferi or IFN-{gamma} were up-regulated much more strongly by a combination of the two agents. Abundant amounts of the products of these genes would likely be present in lesions of LD. Therefore, we decided to focus our attention on this group. To search systematically for cooperation between B. burgdorferi and IFN-{gamma}, a synergy score was calculated for each gene by dividing the gene expression intensity induced by the combination of B. burgdorferi and IFN-{gamma} by the sum of the intensities attributed to the individual stimuli. Genes were considered synergistically up-regulated if they had a synergy score ≥1.25. By this criterion, concomitant stimulation of endothelium with B. burgdorferi and IFN-{gamma} resulted in synergistic increases in the expression of 34 genes (Table II). Many of these encode mediators of innate and acquired immunity and thus presumably play a role in the development of LD. For example, MHC CIITA regulates expression of a variety of genes involved in processing of Ags (26). Remarkably, 9 of the 34 genes that were synergistically induced by B. burgdorferi and IFN-{gamma} encode mediators of leukocyte recruitment. Two of the genes encode adhesion molecules, namely ICAM-1 and VCAM-1, which bind to integrins on most types of leukocytes (27). Their increased expression would therefore be expected to foster inflammation in general. The other 7 genes code for chemokines, including CCL7, CCL8, CXCL9, CXCL10, CXCL11, CX3CL1, and CXCL2, the first 6 of which are attractants for T lymphocytes (28, 29, 30, 31, 32). Their presence in vivo would thus favor development of chronic inflammatory lesions. Only CXCL2 is a chemoattractant for neutrophils (33), which typically accumulate during acute inflammation.


View this table:
[in this window]
[in a new window]

  		Table II. The expression of 34 endothelial cell genes is induced synergistically by concomitant stimulation with B. burgdorferi and IFN-{gamma}

 
To assess the validity of the microarray results, levels of transcripts for selected chemokines (CXCL2, CXCL10, and CXCL11) were also measured by real-time PCR. The results obtained by this methodology (data not shown) mirrored those of the microarray analysis and thus verified the synergistic induction of all three genes in response to B. burgdorferi and IFN-{gamma}. Moreover, the cooperative production of the six chemokines that attract T cells was confirmed at the protein level, where synergistic effects were even more marked than at the transcriptional level. For these studies, HUVEC were stimulated with B. burgdorferi, IFN-{gamma}, or the two agents combined for 24 h. Chemokines were then measured in conditioned medium or on the cell surface by ELISA (Fig. 1). Little to no production of any of these chemokines occurred under basal conditions or in response to B. burgdorferi. In contrast, IFN-{gamma} alone elicited secretion of CCL8, CXCL9, CXCL10, and CXCL11, as well as cell surface expression of CX3CL1. In corroboration of the microarray findings, B. burgdorferi in combination with IFN-{gamma} stimulated a 2- to 8-fold further increase in synthesis of these five chemokines compared with IFN-{gamma} by itself. Endothelial secretion of CCL7 was significantly augmented only by the endothelium exposed to the combination of B. burgdorferi and IFN-{gamma}.


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 1. B. burgdorferi and IFN-{gamma} cooperate to augment endothelial production of chemokines. HUVEC were incubated with medium alone (Unstim) or B. burgdorferi (Bb) in the absence or presence of IFN-{gamma} for 24 h. Cell surface expression of the endothelial-bound form of CX3CL1 was measured by whole cell ELISA (A). Levels of secreted chemokines in cell-free conditioned media were determined by ELISA (B–F). Bars represent the means ± SD of three replicate samples. Each experiment was repeated at least once, with similar results. *, p < 0.001 compared with all other groups.

 
We next determined whether the ability to cooperate with IFN-{gamma} is shared by other proinflammatory mediators. CXCL10 was used as the readout for this study, given that it was the most abundantly produced of the six chemokines examined at the protein level (Fig. 1). In addition, CXCL10 has been implicated in the pathogenesis of LD, because amounts are elevated in the cerebrospinal fluid of patients with neuroborreliosis (34, 35). To assess cooperation of IFN-{gamma} with additional mediators, HUVEC were incubated for 24 h with IL-1beta, TNF-{alpha}, or E. coli LPS, in the absence or presence of IFN-{gamma}. Each of these stimuli alone provoked little to no endothelial secretion of CXCL10, but all elicited synergistic responses in the presence of IFN-{gamma} (Fig. 2).


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		FIGURE 2. Host- and bacterial-derived proinflammatory mediators cooperate with IFN-{gamma} to synergistically augment secretion of CXCL10 by endothelium. HUVEC were incubated with IFN-{gamma} in the absence or presence of B. burgdorferi, IL-1beta, TNF-{alpha} (A), or E. coli LPS (B) for 24 h. Control samples were incubated with medium alone (Unstim). Conditioned media were collected and analyzed by ELISA. Bars represent the means ± SD of three replicate samples. Each experiment was repeated at least twice with similar results. *, p < 0.001 compared with untreated HUVEC or HUVEC treated with a single stimulus.

 
The amount of time required for B. burgdorferi and IFN-{gamma} to augment production of CXCL10 was ascertained by incubating HUVEC with IFN-{gamma} in the absence or presence of B. burgdorferi for 4, 8, 12, 18, or 24 h. Stimulation with IFN-{gamma} elicited endothelial secretion of CXCL10 as early as 4 h, which was unaltered by the presence of B. burgdorferi (Fig. 3). By 18 h, however, B. burgdorferi cooperated with IFN-{gamma} to induce significantly more secretion of CXCL10 than did IFN-{gamma} alone. B. burgdorferi thus potentiates the IFN-{gamma}-induced endothelial secretion of CXCL10 in a time-dependent manner.


Figure 3
View larger version (8K):
[in this window]
[in a new window]

  		FIGURE 3. B. burgdorferi cooperates with IFN-{gamma} to augment the endothelial secretion of CXCL10 in a time-dependent manner. HUVEC were incubated with IFN-{gamma} in the absence or presence of B. burgdorferi (Bb). Cell-free conditioned media were collected at 4, 8, 12, 18, and 24 h after the start of the incubation, clarified, and analyzed by ELISA for CXCL10. Bars represent the means ± SD of three replicate samples. The experiment was repeated once with similar results. *, p < 0.05 compared with treatment with IFN-{gamma} alone.

 
Transendothelial migration of leukocytes

Our studies revealed a substantial increase in production of chemokines that attract T lymphocytes by endothelium incubated simultaneously with B. burgdorferi and IFN-{gamma}. To ascertain whether this increase results in enhanced recruitment of T cells, we used an in vitro model of the blood vessel wall consisting of HUVEC grown to confluence on amniotic connective tissue (21). These HUVEC-amnion cultures were treated with B. burgdorferi, IFN-{gamma}, the two stimuli combined, or medium alone for 24 h. Freshly isolated human T lymphocytes were then added to the apical side of the cultures and allowed to migrate for 4 h. Four experiments using T lymphocytes from different donors were performed, one of which is shown in Fig. 4. In all experiments, stimulation of endothelium with either B. burgdorferi or IFN-{gamma} resulted in an ~2-fold increase in the number of T cells that migrated, compared with control, untreated monolayers. Migration of T cells across HUVEC incubated with both B. burgdorferi and IFN-{gamma} was significantly greater than across resting endothelium in all experiments and, in two, the combined stimuli elicited a significantly stronger response than either agent alone.


Figure 4
View larger version (11K):
[in this window]
[in a new window]

  		FIGURE 4. B. burgdorferi and IFN-{gamma} enhance the transendothelial migration of human T lymphocytes. HUVEC-amnion cultures were incubated with B. burgdorferi (Bb), IFN-{gamma}, both stimuli combined, or medium alone (Unstim) for 24 h. Freshly isolated T lymphocytes then were added to the cultures and allowed to migrate across the endothelium for 4 h. Bars represent the mean ± SD of three to six replicate samples. The total height of the bars represents the percentage of T lymphocytes associated with the HUVEC-amnion cultures. The lower part represents the percentage of cells that migrated beneath the HUVEC, whereas the upper, hatched portion (Figure 4) indicates the percentage of cells adherent to the apical surface of the endothelium. *, p < 0.01 compared with untreated HUVEC or HUVEC treated with either stimulus alone.

 
Our microarray data revealed that expression of a chemoattractant specific for neutrophils, namely CXCL2, was synergistically up-regulated by concurrent stimulation of endothelium with B. burgdorferi and IFN-{gamma}. However, addition of IFN-{gamma} did not alter B. burgdorferi-induced transcription of CXCL8, which was previously shown to be the predominant chemoattractant responsible for migration of neutrophils across endothelium exposed to B. burgdorferi in vitro (4). Therefore, we tested whether IFN-{gamma} modulates the recruitment of neutrophils in response to B. burgdorferi. To this end, HUVEC-amnion cultures were incubated with medium alone, IFN-{gamma}, B. burgdorferi, or a combination of the two stimuli for 24 h. Freshly isolated human neutrophils were then added and permitted to migrate for 30 min. In corroboration of previous findings (4), preincubation of the endothelium with B. burgdorferi significantly enhanced the transmigration of neutrophils (Fig. 5). Interestingly, addition of IFN-{gamma} significantly diminished the number of neutrophils that traversed the B. burgdorferi-stimulated endothelium. In four separate experiments, the decrease in neutrophil migration averaged 65% and ranged from 40 to 89%. Despite the lack of change in the transcript levels of CXCL8 observed in our microarray analysis, we decided to determine whether a decrease in secretion of CXCL8 by endothelium exposed to B. burgdorferi and IFN-{gamma} was responsible for the diminished transmigration of neutrophils. However, the presence of IFN-{gamma} did not markedly affect endothelial secretion of CXCL8 in response to B. burgdorferi (data not shown). To search for other mechanisms by which recruitment of neutrophils might be decreased, we evaluated our microarray data for genes that were cooperatively down-regulated in response to B. burgdorferi and IFN-{gamma}. A negative synergy score was calculated as described in the Materials and Methods and genes with scores ≤–1.25 were classified according to the Gene Ontology Consortium. A large number of genes (237) were synergistically down-regulated, but only four were determined to be involved in inflammation or the immune response (supplemental data). Moreover, no genes cooperatively down-regulated by B. burgdorferi and IFN-{gamma} had an obvious role in endothelial recruitment of leukocytes to sites of inflammation.


Figure 5
View larger version (7K):
[in this window]
[in a new window]

  		FIGURE 5. IFN-{gamma} diminishes migration of human neutrophils across endothelium stimulated with B. burgdorferi. HUVEC-amnion cultures were incubated with B. burgdorferi (Bb), IFN-{gamma}, a combination of the two, or medium alone (Unstim) for 24 h. Freshly isolated neutrophils were added to the HUVEC-amnion cultures and allowed to migrate for 30 min. Bars represent the mean ± SD of four to six replicate samples. As previously reported (4 ), negligible numbers of neutrophils were bound to the apical surface of the endothelium; bars therefore depict only migrated cells. *, p < 0.001 compared with treatment with B. burgdorferi alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
LD in humans is characterized by recruitment of leukocytes to the infected tissues (2), as well as an associated increase in the levels of the host cytokine IFN-{gamma} (12, 13). In this study, microarray analysis of primary human endothelial cells was used to assess the potential role of IFN-{gamma} in mediating the pathology of human LD. We focused on the endothelium due to its central role in regulating trafficking of leukocytes by means of the adhesion molecules and chemoattractants that it synthesizes. We found that B. burgdorferi and IFN-{gamma} cooperated to up-regulate the endothelial expression of a variety of genes that are known mediators of leukocyte recruitment. In particular, IFN-{gamma} directed B. burgdorferi-activated endothelium to produce high levels of several chemoattractants for T cells. This finding is consistent with the observation that T cells predominate in chronic inflammatory lesions of the joints, skin, heart, muscles, and nervous system of patients with LD (2, 11, 36). Two of the chemokines that were synergistically up-regulated in our study, CXCL10 and CXCL11, are increased in the cerebrospinal fluids of LD patients, suggesting that they facilitate the observed recruitment of T cells to this site (34, 35, 37). Moreover, T cells that secrete IFN-{gamma} are found in the synovial fluids of patients with Lyme arthritis and their numbers are positively correlated with the severity of pathology in the joint (12). Thus, T cells are recruited to the lesions of infected humans, where they have been implicated in the pathogenesis of LD.

The functional consequences of increased endothelial expression of chemokines in response to IFN-{gamma} and B. burgdorferi were explored using a well-characterized model of the blood vessel wall. It has been shown previously that results obtained with this model are consistent with observations made in vivo. Specifically, HUVEC-amnion cultures exposed to B. burgdorferi preferentially recruit CD4+ and CD8+ T cells that secrete IFN-{gamma} (9), as well as T cells that express CD45RO (6). Similarly, studies in humans revealed a selective accumulation of CD45RO+CD27+ memory T cells and IFN-{gamma}-secreting Th1 cells in the cutaneous lesions and synovial fluids, respectively, of LD patients (12, 13). Given that B. burgdorferi and IFN-{gamma} cooperatively induce increases in secretion of T cell attractants by endothelia, we expected that the combined stimuli would enhance transendothelial migration of T lymphocytes in our in vitro model. Indeed, treatment of endothelium with both B. burgdorferi and IFN-{gamma} significantly augmented the migration of human T cells as compared with that across untreated endothelium in all experiments. However, the number of T cells that transmigrated in response to the combination of B. burgdorferi and IFN-{gamma} significantly exceeded that elicited by either stimulus alone in only two of four experiments. We speculate that in the remaining two experiments, B. burgdorferi or IFN-{gamma} alone stimulated secretion of sufficient amounts of chemokines to provoke a maximal migratory response from the T cells. In support of this notion, B. burgdorferi by itself elicits endothelial production of CCL2 at levels that are capable of inducing maximal migration of T lymphocytes in vitro (9, 38). Moreover, at the time (18 h) required for B. burgdorferi to significantly augment production of CXCL10 in response to IFN-{gamma} (Fig. 3), IFN-{gamma} on its own provoked secretion of CXCL10 at concentrations that in vitro result in maximal chemotaxis of T cells (30). Although in some instances B. burgdorferi or IFN-{gamma} individually appeared to facilitate a maximal migratory response in the HUVEC-amnion model, this is probably not the case in vivo. In the body, accumulation of chemokines is limited through dilution by tissue fluids and blood, as well as removal by scavenger receptors (39). Thus, the high concentrations of chemokines induced cooperatively by B. burgdorferi and IFN-{gamma} are likely crucial for achieving maximal endothelial recruitment of T lymphocytes in vivo.

Our microarray results revealed that HUVEC stimulated with B. burgdorferi and IFN-{gamma} synergistically increased the transcription of six chemokines specific for T cells but only a single neutrophil-specific attractant, CXCL2. A deficiency in the receptor for this ligand, CXCR2, results in decreased recruitment of neutrophils and less severe arthritis in the murine model of LD (10). Therefore, we examined the potential role of IFN-{gamma} in modulating endothelial recruitment of neutrophils. Interestingly, the addition of IFN-{gamma} markedly and consistently reduced migration of human neutrophils across endothelium stimulated with B. burgdorferi. However, this reduction in neutrophil recruitment by IFN-{gamma} did not result from decreased endothelial secretion of CXCL8, a chemokine previously shown to drive migration of neutrophils across B. burgdorferi-stimulated endothelium (4). Therefore, diminished recruitment of neutrophils by endothelium stimulated with B. burgdorferi and IFN-{gamma} suggests a mechanism of specific suppression of neutrophil transmigration that is not dependent on chemokines and still allows for the increased transmigration of T cells.

The presence of IFN-{gamma}, as well as its positive correlation with severity of disease, has been thoroughly documented in human LD (12, 13, 40, 41). However, mice deficient in IFN-{gamma} or its receptor develop Lyme arthritis that is no less severe than that seen in wild-type animals (42, 43). It should be noted, however, that the inflammatory infiltrates in joints of humans and mice infected with B. burgdorferi vary considerably. In human LD patients, the infiltrate consists predominately of T lymphocytes and plasma cells (2, 11, 36). Although neutrophils are found in the synovial fluids, they are rarely seen in the synovium itself (11). In sharp contrast, the joints in mice show little to no infiltration of T lymphocytes or plasma cells and contain many neutrophils (44, 45). The stark differences between the types of leukocytes that infiltrate joints in human vs murine LD may explain the apparent discrepancy between our findings, which support a role for IFN-{gamma} in mediating severity of Lyme arthritis in humans, and those that indicate no difference after genetic ablation of IFN-{gamma} in the murine model. In addition, our data indicate that IFN-{gamma} promotes a switch toward a more chronic inflammatory response to B. burgdorferi. Specifically, we show that the presence of IFN-{gamma} decreased the recruitment of neutrophils but increased the recruitment of T lymphocytes. Therefore, IFN-{gamma} may serve as the mechanism by which infiltration of neutrophils in human Lyme arthritis is suppressed and recruitment of T lymphocytes is enhanced. Furthermore, T lymphocytes are a rich source of IFN-{gamma} and those that secrete it are selectively recruited by B. burgdorferi both in vivo (12, 13) and in vitro (9). IFN-{gamma} would then act as a positive-feedback mediator that aids B. burgdorferi in the recruitment of these T cells, which in turn secrete additional IFN-{gamma}. This process would facilitate continued renewal of IFN-{gamma} and thereby contribute to the destructive inflammation and arthritis observed in human LD.

IFN-{gamma} cooperated with not only B. burgdorferi to augment endothelial secretion of CXCL10, but also TNF-{alpha}, IL-1beta, and E. coli LPS. Previous literature supports the assertion that the synergistic induction of chemokines in cooperation with IFN-{gamma} is shared by a variety of host and bacterial proinflammatory mediators. Stimulation of endothelia with TNF-{alpha} and IFN-{gamma} synergistically augments expression of CX3CL1, CXCL9, CXCL10, and CXCL11 (3, 46, 47). In addition, ligands for TLR2 and TLR4 cooperate with IFN-{gamma} to induce synergistic secretion of CXCL9, CXCL10, and CXCL11 by fibroblasts (48, 49). Although B. burgdorferi and host cytokines share the ability to induce endothelial production of chemokines, it is important to note that activation of HUVEC by B. burgdorferi is not mediated by autologous secretion of IL-1beta or TNF-{alpha} (4). However, IL-1beta and TNF-{alpha} are secreted by human monocytes in response to B. burgdorferi (50), which suggests that these cytokines may potentiate the effects of IFN-{gamma} on synthesis of chemokines in LD. In addition, other inflammatory arthritides, such as rheumatoid and septic arthritis, are characterized by marked increases in the levels of many of the chemokines that are synergistically up-regulated by B. burgdorferi and IFN-{gamma}, including CCL8, CXCL9, CXCL10, CXCL11, and CX3CL1 (48, 49, 51, 52, 53, 54). IFN-{gamma} thus cooperates with several proinflammatory stimuli to enhance secretion of chemokines and thereby likely facilitates enhanced recruitment of leukocytes and ongoing inflammation in a variety of pathological conditions.


   Acknowledgments
 
We thank John Schwedes of the Stony Brook University DNA Microarray Facility for analysis of RNA samples and the staff of the Obstetrics Service at St. Charles Hospital for collection of umbilical cords. We also thank David Thanassi, Jorge Benach, and Sean Connolly for critical review of the manuscript, as well as friends and colleagues who offered advice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI47313 and AI055621. Back

2 Address correspondence and reprint requests to Dr. Martha B. Furie, Center for Infectious Diseases, Stony Brook University, Room 240 CMM, Stony Brook, NY 11794-5120. E-mail address: Martha.Furie{at}stonybrook.edu Back

3 Abbreviations used in this paper: LD, Lyme disease; sp/EC, spirochetes per endothelial cell. Back

4 The online version of this article contains supplemental material. Back

Received for publication August 3, 2006. Accepted for publication October 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Benach, J. L., E. M. Bosler, J. P. Hanrahan, J. L. Coleman, G. S. Habicht, T. F. Bast, D. J. Cameron, J. L. Ziegler, A. G. Barbour, W. Burgdorfer, et al 1983. Spirochetes isolated from the blood of two patients with Lyme disease. N. Engl. J. Med. 308: 740-742. [Abstract]
  2. Duray, P. H.. 1987. The surgical pathology of human Lyme disease: an enlarging picture. Am. J. Surg. Pathol. 11: (Suppl. 1):47-60. [Medline]
  3. Ebnet, K., M. M. Simon, S. Shaw. 1996. Regulation of chemokine gene expression in human endothelial cells by proinflammatory cytokines and Borrelia burgdorferi. Ann. NY Acad. Sci. 797: 107-117. [Medline]
  4. Burns, M. J., T. J. Sellati, E. I. Teng, M. B. Furie. 1997. Production of interleukin-8 (IL-8) by cultured endothelial cells in response to Borrelia burgdorferi occurs independently of secreted IL-1 and tumor necrosis factor {alpha} and is required for subsequent transendothelial migration of neutrophils. Infect. Immun. 65: 1217-1222. [Abstract]
  5. Sellati, T. J., M. J. Burns, M. A. Ficazzola, M. B. Furie. 1995. Borrelia burgdorferi upregulates expression of adhesion molecules on endothelial cells and promotes transendothelial migration of neutrophils in vitro. Infect. Immun. 63: 4439-4447. [Abstract]
  6. Gergel, E. I., M. B. Furie. 2001. Activation of endothelium by Borrelia burgdorferi in vitro enhances transmigration of specific subsets of T lymphocytes. Infect. Immun. 69: 2190-2197. [Abstract/Free Full Text]
  7. Burns, M. J., M. B. Furie. 1998. Borrelia burgdorferi and interleukin-1 promote the transendothelial migration of monocytes in vitro by different mechanisms. Infect. Immun. 66: 4875-4883. [Abstract/Free Full Text]
  8. Sellati, T. J., L. D. Abrescia, J. D. Radolf, M. B. Furie. 1996. Outer surface lipoproteins of Borrelia burgdorferi activate vascular endothelium in vitro. Infect. Immun. 64: 3180-3187. [Abstract]
  9. Gergel, E. I., M. B. Furie. 2004. Populations of human T lymphocytes that traverse the vascular endothelium stimulated by Borrelia burgdorferi are enriched with cells that secrete {gamma} interferon. Infect. Immun. 72: 1530-1536. [Abstract/Free Full Text]
  10. Brown, C. R., V. A. Blaho, C. M. Loiacono. 2003. Susceptibility to experimental Lyme arthritis correlates with KC and monocyte chemoattractant protein-1 production in joints and requires neutrophil recruitment via CXCR2. J. Immunol. 171: 893-901. [Abstract/Free Full Text]
  11. Duray, P. H., A. C. Steere. 1988. Clinical pathologic correlations of Lyme disease by stage. Ann. NY Acad. Sci. 539: 65-79. [Medline]
  12. Gross, D. M., A. C. Steere, B. T. Huber. 1998. T helper 1 response is dominant and localized to the synovial fluid in patients with Lyme arthritis. J. Immunol. 160: 1022-1028. [Abstract/Free Full Text]
  13. Salazar, J. C., C. D. Pope, T. J. Sellati, H. M. Feder, Jr, T. G. Kiely, K. R. Dardick, R. L. Buckman, M. W. Moore, M. J. Caimano, J. G. Pope, et al 2003. Coevolution of markers of innate and adaptive immunity in skin and peripheral blood of patients with erythema migrans. J. Immunol. 171: 2660-2670. [Abstract/Free Full Text]
  14. Colvin, R. A., G. S. Campanella, J. Sun, A. D. Luster. 2004. Intracellular domains of CXCR3 that mediate CXCL9, CXCL10, and CXCL11 function. J. Biol. Chem. 279: 30219-30227. [Abstract/Free Full Text]
  15. Huang, A. J., M. B. Furie, S. C. Nicholson, J. Fischbarg, L. S. Liebovitch, S. C. Silverstein. 1988. Effects of human neutrophil chemotaxis across human endothelial cell monolayers on the permeability of these monolayers to ions and macromolecules. J. Cell. Physiol. 135: 355-366. [Medline]
  16. Gentleman, R. C., V. J. Carey, D. M. Bates, B. Bolstad, M. Dettling, S. Dudoit, B. Ellis, L. Gautier, Y. Ge, J. Gentry, et al 2004. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5: R80[Medline]
  17. Gautier, L., L. Cope, B. M. Bolstad, R. A. Irizarry. 2004. affy—analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20: 307-315. [Abstract/Free Full Text]
  18. Irizarry, R. A., B. M. Bolstad, F. Collin, L. M. Cope, B. Hobbs, T. P. Speed. 2003. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 31: e15[Abstract/Free Full Text]
  19. Ashburner, M., C. A. Ball, J. A. Blake, D. Botstein, H. Butler, J. M. Cherry, A. P. Davis, K. Dolinski, S. S. Dwight, J. T. Eppig, et al 2000. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25: 25-29. [Medline]
  20. Anderson, D. C., M. L. Mace, B. R. Brinkley, R. R. Martin, C. W. Smith. 1981. Recurrent infection in glycogenosis type Ib: abnormal neutrophil motility related to impaired redistribution of adhesion sites. J. Infect. Dis. 143: 447-459. [Medline]
  21. Furie, M. B., E. B. Cramer, B. L. Naprstek, S. C. Silverstein. 1984. Cultured endothelial cell monolayers that restrict the transendothelial passage of macromolecules and electrical current. J. Cell Biol. 98: 1033-1041. [Abstract/Free Full Text]
  22. Randolph, G. J., M. B. Furie. 1995. A soluble gradient of endogenous monocyte chemoattractant protein-1 promotes the transendothelial migration of monocytes in vitro. J. Immunol. 155: 3610-3618. [Abstract]
  23. Behera, A. K., E. Hildebrand, J. Scagliotti, A. C. Steere, L. T. Hu. 2005. Induction of host matrix metalloproteinases by Borrelia burgdorferi differs in human and murine Lyme arthritis. Infect. Immun. 73: 126-134. [Abstract/Free Full Text]
  24. Habicht, G. S., L. I. Katona, J. L. Benach. 1991. Cytokines and the pathogenesis of neuroborreliosis: Borrelia burgdorferi induces glioma cells to secrete interleukin-6. J. Infect. Dis. 164: 568-574. [Medline]
  25. Grygorczuk, S., S. Pancewicz, J. Zajkowska, M. Kondrusik, R. Rwierzbinska, T. Hermanowska-Szpakowicz. 2004. Concentrations of macrophage inflammatory proteins MIP-1{alpha} and MIP-1beta and interleukin 8 (IL-8) in Lyme borreliosis. Infection 32: 350-355. [Medline]
  26. Reith, W., S. LeibundGut-Landmann, J. M. Waldburger. 2005. Regulation of MHC class II gene expression by the class II transactivator. Nat. Rev. Immunol. 5: 793-806. [Medline]
  27. Albelda, S. M., C. W. Smith, P. A. Ward. 1994. Adhesion molecules and inflammatory injury. FASEB J. 8: 504-512. [Abstract]
  28. Loetscher, P., M. Seitz, I. Clark-Lewis, M. Baggiolini, B. Moser. 1994. Monocyte chemotactic proteins MCP-1, MCP-2, and MCP-3 are major attractants for human CD4+ and CD8+ T lymphocytes. FASEB J. 8: 1055-1060. [Abstract]
  29. Liao, F., R. L. Rabin, J. R. Yannelli, L. G. Koniaris, P. Vanguri, J. M. Farber. 1995. Human Mig chemokine: biochemical and functional characterization. J. Exp. Med. 182: 1301-1314. [Abstract/Free Full Text]
  30. Taub, D. D., A. R. Lloyd, K. Conlon, J. M. Wang, J. R. Ortaldo, A. Harada, K. Matsushima, D. J. Kelvin, J. J. Oppenheim. 1993. Recombinant human interferon-inducible protein 10 is a chemoattractant for human monocytes and T lymphocytes and promotes T cell adhesion to endothelial cells. J. Exp. Med. 177: 1809-1814. [Abstract/Free Full Text]
  31. Cole, K. E., C. A. Strick, T. J. Paradis, K. T. Ogborne, M. Loetscher, R. P. Gladue, W. Lin, J. G. Boyd, B. Moser, D. E. Wood, et al 1998. Interferon-inducible T cell {alpha} chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3. J. Exp. Med. 187: 2009-2021. [Abstract/Free Full Text]
  32. Bazan, J. F., K. B. Bacon, G. Hardiman, W. Wang, K. Soo, D. Rossi, D. R. Greaves, A. Zlotnik, T. J. Schall. 1997. A new class of membrane-bound chemokine with a CX3C motif. Nature 385: 640-644. [Medline]
  33. Schroder, J. M., N. L. Persoon, E. Christophers. 1990. Lipopolysaccharide-stimulated human monocytes secrete, apart from neutrophil-activating peptide 1/interleukin 8, a second neutrophil-activating protein: NH2 terminal amino acid sequence identity with melanoma growth stimulatory activity. J. Exp. Med. 171: 1091-1100. [Abstract/Free Full Text]
  34. Lepej, S. Z., O. D. Rode, T. Jeren, A. Vince, A. Remenar, B. Barsic. 2005. Increased expression of CXCR3 and CCR5 on memory CD4+ T-cells migrating into the cerebrospinal fluid of patients with neuroborreliosis: the role of CXCL10 and CXCL11. J. Neuroimmunol. 163: 128-134. [Medline]
  35. Giunti, D., G. Borsellino, R. Benelli, M. Marchese, E. Capello, M. T. Valle, E. Pedemonte, D. Noonan, A. Albini, G. Bernardi, et al 2003. Phenotypic and functional analysis of T cells homing into the CSF of subjects with inflammatory diseases of the CNS. J. Leukocyte Biol. 73: 584-590. [Abstract/Free Full Text]
  36. Duray, P. H.. 1989. Histopathology of clinical phases of human Lyme disease. Rheum. Dis. Clin. North Am. 15: 691-710. [Medline]
  37. Rupprecht, T. A., U. Koedel, B. Muhlberger, B. Wilske, A. Fontana, H. W. Pfister. 2005. CXCL11 is involved in leucocyte recruitment to the central nervous system in neuroborreliosis. J. Neurol. 252: 820-823. [Medline]
  38. Taub, D. D., P. Proost, W. J. Murphy, M. Anver, D. L. Longo, J. Van Damme, J. J. Oppenheim. 1995. Monocyte chemotactic protein-1 (MCP-1), -2, and -3 are chemotactic for human T lymphocytes. J. Clin. Invest. 95: 1370-1376. [Medline]
  39. Locati, M., Y. M. Torre, E. Galliera, R. Bonecchi, H. Bodduluri, G. Vago, A. Vecchi, A. Mantovani. 2005. Silent chemoattractant receptors: D6 as a decoy and scavenger receptor for inflammatory CC chemokines. Cytokine Growth Factor Rev. 16: 679-686. [Medline]
  40. Widhe, M., S. Jarefors, C. Ekerfelt, M. Vrethem, S. Bergstrom, P. Forsberg, J. Ernerudh. 2004. Borrelia-specific interferon-{gamma} and interleukin-4 secretion in cerebrospinal fluid and blood during Lyme borreliosis in humans: association with clinical outcome. J. Infect. Dis. 189: 1881-1891. [Medline]
  41. Forsberg, P., J. Ernerudh, C. Ekerfelt, M. Roberg, M. Vrethem, S. Bergstrom. 1995. The outer surface proteins of Lyme disease Borrelia spirochetes stimulate T cells to secrete interferon-{gamma} (IFN-{gamma}): diagnostic and pathogenic implications. Clin. Exp. Immunol. 101: 453-460. [Medline]
  42. Brown, C. R., S. L. Reiner. 1999. Experimental Lyme arthritis in the absence of interleukin-4 or {gamma} interferon. Infect. Immun. 67: 3329-3333. [Abstract/Free Full Text]
  43. Glickstein, L., M. Edelstein, J. Z. Dong. 2001. {gamma} Interferon is not required for arthritis resistance in the murine Lyme disease model. Infect. Immun. 69: 3737-3743. [Abstract/Free Full Text]
  44. Barthold, S. W., D. H. Persing, A. L. Armstrong, R. A. Peeples. 1991. Kinetics of Borrelia burgdorferi dissemination and evolution of disease after intradermal inoculation of mice. Am. J. Pathol. 139: 263-273. [Abstract]
  45. Barthold, S. W., D. S. Beck, G. M. Hansen, G. A. Terwilliger, K. D. Moody. 1990. Lyme borreliosis in selected strains and ages of laboratory mice. J. Infect. Dis. 162: 133-138. [Medline]
  46. Mohan, K., Z. Ding, J. Hanly, T. B. Issekutz. 2002. IFN-{gamma}-inducible T cell {alpha} chemoattractant is a potent stimulator of normal human blood T lymphocyte transendothelial migration: differential regulation by IFN-{gamma} and TNF-{alpha}. J. Immunol. 168: 6420-6428. [Abstract/Free Full Text]
  47. Fraticelli, P., M. Sironi, G. Bianchi, D. D’Ambrosio, C. Albanesi, A. Stoppacciaro, M. Chieppa, P. Allavena, L. Ruco, G. Girolomoni, et al 2001. Fractalkine (CX3CL1) as an amplification circuit of polarized Th1 responses. J. Clin. Invest. 107: 1173-1181. [Medline]
  48. Proost, P., A. K. Vynckier, F. Mahieu, W. Put, B. Grillet, S. Struyf, A. Wuyts, G. Opdenakker, J. V. Damme. 2003. Microbial Toll-like receptor ligands differentially regulate CXCL10/IP-10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN-{gamma} and provide a mechanism for enhanced synovial chemokine levels in septic arthritis. Eur. J. Immunol. 33: 3146-3153. [Medline]
  49. Proost, P., S. Verpoest, B. K. Van de, E. Schutyser, S. Struyf, W. Put, I. Ronsse, B. Grillet, G. Opdenakker, J. Van Damme. 2004. Synergistic induction of CXCL9 and CXCL11 by Toll-like receptor ligands and interferon-{gamma} in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids. J. Leukocyte Biol. 75: 777-784. [Abstract/Free Full Text]
  50. Radolf, J. D., L. L. Arndt, D. R. Akins, L. L. Curetty, M. E. Levi, Y. Shen, L. S. Davis, M. V. Norgard. 1995. Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytes/macrophages. J. Immunol. 154: 2866-2877. [Abstract]
  51. Haringman, J. J., T. J. Smeets, P. Reinders-Blankert, P. P. Tak. 2006. Chemokine and chemokine receptor expression in paired peripheral blood mononuclear cells and synovial tissue of patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis. Ann. Rheum. Dis. 65: 294-300. [Abstract/Free Full Text]
  52. Ruth, J. H., M. V. Volin, G. K. Haines, III, D. C. Woodruff, K. J. Katschke, Jr, J. M. Woods, C. C. Park, J. C. Morel, A. E. Koch. 2001. Fractalkine, a novel chemokine in rheumatoid arthritis and in rat adjuvant-induced arthritis. Arthritis Rheum. 44: 1568-1581. [Medline]
  53. Ueno, A., M. Yamamura, M. Iwahashi, A. Okamoto, T. Aita, N. Ogawa, H. Makino. 2005. The production of CXCR3-agonistic chemokines by synovial fibroblasts from patients with rheumatoid arthritis. Rheumatol. Int. 25: 361-367. [Medline]
  54. Nanki, T., T. Imai, K. Nagasaka, Y. Urasaki, Y. Nonomura, K. Taniguchi, K. Hayashida, J. Hasegawa, O. Yoshie, N. Miyasaka. 2002. Migration of CX3CR1-positive T cells producing type 1 cytokines and cytotoxic molecules into the synovium of patients with rheumatoid arthritis. Arthritis Rheum. 46: 2878-2883. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. M. Petzke, A. Brooks, M. A. Krupna, D. Mordue, and I. Schwartz
Recognition of Borrelia burgdorferi, the Lyme Disease Spirochete, by TLR7 and TLR9 Induces a Type I IFN Response by Human Immune Cells
J. Immunol., October 15, 2009; 183(8): 5279 - 5292.
[Abstract] [Full Text] [PDF]

Home page
 haematolHome page
R. J. Bende, F. van Maldegem, and C. J.M. van Noesel
Chronic inflammatory disease, lymphoid tissue neogenesis and extranodal marginal zone B-cell lymphomas
Haematologica, August 1, 2009; 94(8): 1109 - 1123.
[Abstract] [Full Text] [PDF]

Home page
 CVIHome page
D. T. Nardelli, S. M. Callister, and R. F. Schell
Lyme Arthritis: Current Concepts and a Change in Paradigm
Clin. Vaccine Immunol., January 1, 2008; 15(1): 21 - 34.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dame, T. M.
Right arrow Articles by Furie, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dame, T. M.
Right arrow Articles by Furie, M. B.
Right arrowPubmed/NCBI databases
*GEO DataSet
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS