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Infection and Immunity, November 2001, p. 6670-6675, Vol. 69, No. 11
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.11.6670-6675.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
HLA-B27 Expression Does Not Modulate Intracellular
Chlamydia trachomatis Infection of Cell Lines
J. L.
Young,*
L.
Smith,
M. K.
Matyszak, and
J. S. H.
Gaston
Department of Medicine, University of
Cambridge Clinical School, Addenbrooke's Hospital, Cambridge CB2
2QQ, United Kingdom
Received 2 April 2001/Returned for modification 23 May
2001/Accepted 24 July 2001
 |
ABSTRACT |
Chlamydia trachomatis is an obligate intracellular
pathogen. Infection of susceptible individuals with this bacterium can trigger the development of reactive arthritis, an acute inflammation that is associated with the expression of the class I major
histocompatibility antigen, HLA-B27. Other facultative intracellular
pathogens, such as Yersinia and
Salmonella spp., are also known triggers of reactive arthritis. Previous studies report conflicting results concerning whether the presence of HLA-B27 modulates the infection of cells with
these enteric pathogens. In the present study, we have examined whether
the expression of HLA-B27 can influence the infection of cell lines
with C. trachomatis and also whether the replication of
these bacteria is altered in HLA-B27-expressing cell lines. To do this,
we have used a sensitive flow cytometric approach. We fixed and
permeabilized cells and used fluorescein isothiocyanate-conjugated monoclonal antibody specific for chlamydia lipopolysaccharide to detect
intracellular bacteria. The staining pattern obtained closely resembled
the intracellular life cycle of chlamydia, with the appearance of
brightly staining cells correlating to the microscopic detection of
mature inclusion bodies. Moreover, since the percentage of cells that
stained with the antibody was proportional to the infectious inoculum
used, we were able to use the technique to quantitate the number of
infectious organisms recoverable from infected cell lines. An important
component of our study was the use of heparin to prevent reinfection of
cells and thus enable the infection to be followed from a discrete time
point. Our results suggest that HLA-B27 influences neither the
infection nor replication of C. trachomatis serovar L2
within cell lines. Consequently, the role of HLA-B27 in the
pathogenesis of reactive arthritis may lie downstream of the invasion
and replication stages of the triggering pathogenic infection.
| |
INTRODUCTION |
The major histocompatibility complex
(MHC) class I molecule HLA-B27 is strongly associated with the
spondyloarthropathies (2, 23). However, the role
which this molecule may play in disease has yet to be elucidated. A
link between HLA-B27 and bacterial infection is indicated from animal
models (8, 16), where disease fails to occur if
disease-susceptible HLA-B27 transgenic animals are maintained in
germfree (25) or pathogen-free (16) environments. Moreover, in humans, the expression of HLA-B27 is prevalent in individuals who develop reactive arthritis, a joint inflammation triggered by infection with various pathogens, including Salmonella, Yersinia, and Chlamydia
spp. Indeed, studies examining the development of arthritis in patients
after an outbreak of salmonella food poisoning suggest that HLA-B27 may
also be associated with the severity of the disease (20).
There are various theories concerning how HLA-B27 and bacteria may
interact in disease (1). For example, a number of studies have examined whether the presence of HLA-B27 influences infection of
cells with facultative intracellular gram-negative enteric pathogens.
Clearly, if HLA-B27 influences the infection and/or the survival of
pathogenic bacteria within cells, then the way in which bacteria are
cleared and/or the manner in which the immune response is triggered may
be affected and predispose to pathogenic disease, such as reactive
arthritis. However, studies addressing this issue have reported
conflicting results. For example, an inhibitory effect of HLA-B27 on
the in vitro infection of cell lines with Yersinia and
Salmonella spp. has been found (11, 12; R. Inman, B. Chiu, and U. Payne, Arthritis Rheum. 39:S297 [abstract], 1996). It was further suggested that HLA-B27 may prevent the invasion of these bacteria by competing, either indirectly or
directly, for the bacterial-invasion-mediated protein(s)
(12). This result is in disagreement with other studies
that find no effect on invasion (22) but an adverse effect
of HLA-B27 on replication or clearance of bacteria (19,
26). It was suggested that, if the results were paralleled in
vivo, the increased survival of salmonellae within HLA-B27 cells may
contribute to an increased antigenic load leading to the development of
reactive arthritis in these susceptible individuals. However, further
studies with salmonella have failed to note any impact of the
expression of HLA-B27 on either invasion or replication of salmonella
within nontransformed primary fibroblast cultures (10).
In the present study, we examined whether HLA-B27 influences the
infection of cell lines with the obligate intracellular pathogen Chlamydia trachomatis serovar L2. We also examined
whether replication of the bacteria is altered within
HLA-B27-expressing cells. To do this, we have used a flow
cytometric approach to visualize and quantitate intracellular
chlamydiae infecting a panel of cell lines. The results of this
study indicate that the presence of an HLA-B27 transgene does not
specifically influence infection of cell lines or the replication of
chlamydiae within the infected cell lines studied. Moreover, using a
panel of B lymphoblastoid cell lines (BLCL) which express or lack
native HLA-B27, no effects of HLA-B27 on Chlamydia infection
could be identified. Our results suggest that HLA-B27 does not have a
major influence on the infection or replication of C. trachomatis serovar L2 within transformed cell lines.
| |
MATERIALS AND METHODS |
Cell lines.
The C1R parent cell line and transfectants
expressing HLA-B2705 or HLA-A201 (6, 30) were a generous
gift from K. Granfors, National Public Health Institute, Turku,
Finland. BLCL were derived by transforming peripheral blood mononuclear
cells (PBMC) with Epstein-Barr virus (EBV) obtained from the
supernatant of the cell line B95.8, as previously described
(29). PBMC were obtained from four healthy donors (one of
whom was HLA-B27+) or from
HLA-B27+ spondyloarthropathy patients. HLA-B27
expression was determined serologically or by PCR (results not shown).
HeLa cells were used for propagating chlamydiae, and the elementary
bodies (EB) were purified as described previously (3). The
cell lines were maintained in RPMI 1640 (Gibco-BRL, Paisley, Scotland)
supplemented with 2 mM glutamine (Sigma-Aldrich Company, Ltd., Poole,
England), 20 mM HEPES (Sigma), and 5% heat-inactivated fetal bovine
serum (First Link UK, Ltd., Brierley Hill, England). Transfected cell lines were maintained in 1 mg of G418 (Gibco-BRL)/ml. All cell lines
tested negative for mycoplasma by using PCR and/or enzyme-linked immunosorbent assay techniques (Roche Diagostics, Ltd., Lewes, England).
Infection protocol.
The infectious EB titer of the purified
C. trachomatis L2 was determined using immunofluorescent
microscopy by counting inclusion bodies formed on infected monolayer
cultures of the HeLa cell line, stained 24 to 48 h after infection
with an anti-chlamydia lipopolysaccharide (LPS)-fluorescein
isothiocyanate (FITC) monoclonal antibody (Dako, Ltd., Ely, England).
These EB were used to infect the C1R cell lines and BLCL. Briefly, the
cell lines were added to 24-well plates (Gibco-BRL) in 2-ml volumes at
0.5 × 106 to 1 × 106/ml and C. trachomatis L2 was added
at the indicated multiplicity of infection. Control wells lacking
chlamydiae (mock) were also included. The plates were spun at
1,400 × g for 30 to 60 min. Most of the medium was
removed, and fresh medium supplemented as described above was added.
Unless otherwise indicated, 200 U of heparin (Monoparin; CP
Pharmaceuticals Ltd., Wrexham, England)/ml was also added to the cells
after the centrifugation step. The cells were then incubated for 18 to
26 h or for the indicated periods of time.
Quantification of intracellular chlamydiae.
Infected cells
were washed to remove exogenous heparin. The cells were then lysed by
using sonication, and the lysate was centrifuged at 380 × g for 5 min to pellet the cell debris. The bacterium-rich
supernatant was transferred to Eppendorf tubes and spun at 16,000 × g for 1 h at 4°C to pellet the chlamydiae. For
immediate use, the chlamydia-rich pellet was resuspended in phosphate-buffered saline, and serial dilutions were used to infect aliquots of C1R cells, as described above. Alternatively, as in the
experiment depicted in Fig. 5, the pellet was resuspended in
sucrose-phosphate-glutamate buffer and frozen at 
70°C until required. To check that copurifying cell debris was not toxic, a
control preparation from mock-infected cells was also prepared. Intracellular staining followed by flow cytometric analysis (see below)
was performed on the cells 18 to 24 h after the infection.
Staining protocol.
For intracellular staining, cells were
harvested and fixed and permeabilized by using the commercial Fix and
Permeabilisation Reagent (BD Pharmingen, Oxford, England) according to
the manufacturer's instructions. The cells were stained with a
predetermined optimal amount of FITC-conjugated monoclonal antibody
specific for chlamydia LPS (Dako, Ltd.) or an appropriate isotype
control. No staining with the isotype control was detected (results not
shown). To confirm that the staining was due to intracellular staining,
infected cells that were fixed only with 4% formaldehyde were also
incubated with the antibodies; no or very little staining was apparent
with the anti-chlamydia monoclonal antibody (results not shown). A Becton Dickinson Facsort machine was used to collect the cells, with
10,000 events usually being acquired (except for Fig. 2, wherein 5,000 events were collected), and the data were subsequently analyzed by
using WinMDI software (Joseph Trotter, http://facs.scripps.edu/). For
quantitating the infectious inoculum of bacteria, the percentage of
cells giving fluorescence above that of the negative control was
calculated and plotted against the dilution of bacterial preparation used to infect the cells.
| |
RESULTS |
Heparin inhibits reinfection of cell lines with
Chlamydia.
We used intracellular staining, followed
by fluorescence-activated cell sorting (FACS) analysis to visualize
infection of cell lines with C. trachomatis L2. As shown in
Fig. 1, no staining was evident with
mock-infected cells while a proportion of the cells (at 20 h, 66%
in the absence of heparin and 68% in the presence of heparin) that had
been exposed to C. trachomatis stained with the anti-LPS
antibody. Infected cells did not stain when they were fixed only,
indicating that the C. trachomatis was intracellular (results not shown). Both bright and intermediately staining
populations of infected cells could be seen, with the former likely to
represent mature inclusion bodies. Because we wished to monitor the
course of an infection in cell lines that differed for the expression of HLA-B27, we tested whether the addition of heparin would inhibit ongoing infection within the cell cultures. As is apparent, after 48 h, in the absence of heparin, all of the cells became infected with C. trachomatis. However, a substantial number of
uninfected cells remained (22%) in cultures to which heparin had been
added, while there was a loss of brightly staining cells. This suggests that, in the absence of heparin, cells not infected at early time points could later be infected by C. trachomatis elementary
bodies released into the culture from the early infected cells. Heparin inhibits this secondary infection. Accordingly, to facilitate a
synchronous infection, heparin was added in all of the following experiments.
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FIG. 1.
Heparin inhibits the reinfection of cell lines in
culture. Dot plots of forward scatter against fluorescence are shown
for mock-infected C1R cells or C1R cells infected with C.
trachomatis at a multiplicity of five EB to one cell and
stained after 20 or 48 h with anti-EB-FITC. This result is
representative of four similar experiments.
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|
HLA-B27 does not affect infection of BLCL with
Chlamydia.
Various BLCL were exposed to
Chlamydia, and the percentage of infected cells was
determined by flow cytometry after 24 h (Fig. 2). Although the cell lines showed minor
variations in their intracellular staining patterns, there was no
difference associated with either the presence or absence of HLA-B27
expression. Thus, HLA-B27 does not appear to influence the ability to
infect a BLCL with C. trachomatis.
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FIG. 2.
HLA-B27 expression does not affect infection of BLCL
with C. trachomatis. Dot plots of forward scatter
against fluorescence are shown for various BLCL infected with C.
trachomatis at a multiplicity of 10:1 and stained after 24 h with anti-EB-FITC. The presence or absence of HLA-B27 expression and
the donor origin of the cell lines are indicated. No staining was
apparent with mock-infected cells (not shown). The results shown are
representative of five similar experiments.
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|
HLA-B27 does not affect the course of an infection with
Chlamydia or the rate of replication of bacteria within
infected cell lines.
Aliquots of cells were infected with various
numbers of C. trachomatis, incubated, and stained after
24 h. As shown in Fig. 3, the
percentage of cells giving positive staining was directly proportional
to the number of bacteria used to infect the cells. Thus, we reasoned
that intracellular staining and FACS analysis can provide a useful
technique for quantitating the numbers of bacteria used to initiate an
infection. It was therefore possible to compare the numbers of bacteria
recovered from infected cell lines differing for expression of HLA-B27.
Thus, in addition to assessing whether HLA-B27 modulates cell infection
by C. trachomatis, we could also assay whether HLA-B27
influenced replication of C. trachomatis.
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FIG. 3.
The percentage of infected cells is proportional to the
infectious inoculum used. The percentage of C1R cells giving positive
staining with anti-EB-FITC 24 h after their infection with the
indicated amounts of C. trachomatis was determined from
analysis of flow cytometry data. Fewer than 1% of mock-infected cells
stained with the anti-EB-FITC (result not shown). The results shown
are representative of three similar experiments.
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|
We analyzed aliquots of cell lines infected with
C. trachomatis over a 30-h time period (Fig.
4). The parental cell line C1R
was
compared with cell lines transfected with HLA-A201 or HLA-B2705.
In all
cases, infection could be detected as early as 2 h after
the
addition of the
C. trachomatis. At this time point,
inclusion
bodies could not be detected microscopically, suggesting that
the flow cytometry technique is more sensitive than conventional
microscopy. A very bright staining subpopulation, which correlated
with
the microscopic detection of mature inclusion bodies, was
not apparent
until the 24-h time point. A higher proportion of
the parental cell
line was infected compared to the transfectants.
However, cells
transfected with either HLA-B2705 or HLA-A201 exhibited
similar
infection, indicating that HLA-B2705 did not have a specific
inhibitory
effect. The numbers of infectious bacteria that could
be recovered from
the infected cell lines 24 h after infection
were compared (Fig.
5). In agreement with the higher
percentage
of parental cell line infection, more infectious bacteria
were
recovered from the infected C1R cells. Similar numbers of
infectious
bacteria were recovered from transfected cell lines
expressing
either HLA-B2705 or HLA-A201. Thus, we conclude that
replication
of
C. trachomatis is similar in these
transfected cell lines and
not influenced by the expression of
HLA-B2705.
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FIG. 4.
Time course of C. trachomatis infection
in C1R, C1R-A201, and C1R-B2705 cell lines. Dot plots of forward
scatter against fluorescence are shown for the indicated cell lines,
stained with anti-EB-FITC at the indicated times after addition of
C. trachomatis at a multiplicity of 5:1. Examples of the
negative staining obtained with mock-infected cells are also shown.
Similar results to those shown were observed in a repeat experiment
(not shown).
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FIG. 5.
Quantification of infectious bacteria recovered from
C1R, C1R-B2705, or C1R-A201 cell lines at 24 h. Serial dilutions
of C. trachomatis prepared from C1R (squares), C1R-A201
(triangles), or C1R-B2705 (circles) cells 24 h after their
infection with C. trachomatis were used to infect C1R
cells. After 24 h, the percentage of positively staining cells was
determined after flow cytometry analysis of the infected C1R cells and
is plotted against the dilution of C. trachomatis used
for the infection. The percentage of staining cells treated with a
control preparation prepared from mock-infected cells (diamond) and the
percentage of mock-infected C1R cells (inverted triangle) staining with
anti-EB-FITC are also indicated. The results shown are representative
of three such experiments.
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| |
DISCUSSION |
In the present study we used a flow cytometric approach to analyze
infections of cell lines with the obligate intracellular pathogen,
C. trachomatis. C. trachomatis is more commonly
examined using direct microscopy and counting the inclusion bodies
which are formed by the replicating bacteria (7, 18). This
latter technique has severe limitations in the number of cells that can be examined and can lead to large errors when one is quantitating infectious bacteria. Moreover, if replication of C. trachomatis within a cell is not optimal, then it may not be
possible to visualize inclusion bodies. The use of flow cytometry
provides a way to analyze accurately a large number of cells for
evidence of chlamydia infection. Moreover, since staining is apparent
as early as 2 h postincubation with C. trachomatis,
abnormal and suboptimal infections can still be detected using this
technique. Although the use of intracellular staining and flow
cytometry has been previously reported for analyzing the infection of
cell lines with Chlamydia, the technique has not been
extensively used since its initial description 14 years ago
(27). The advent of commercially available reagents for
fixation and permeabilization has greatly simplified this method, and
we would advocate its use as a standard technique in studying
chlamydial infections. The staining pattern obtained by using
intracellular staining and flow cytometry (see Fig. 4) closely
resembles the infectious cycle of C. trachomatis (21). Thus, dim staining was seen in the first few hours
after addition of the C. trachomatis with the appearance of
bright staining populations correlating to the microscopic detection of
mature inclusion bodies. Similarly, the reduction in the numbers of
bright staining cells at a late time in the infectious cycle is
consistent with the lysis of these cells and release of the mature
elementary bodies. As expected by the correlation of staining with the
infectious cycle, the total percentage of cells staining positive for
evidence of Chlamydia infection was found to be dependent
upon the infectious inoculum used. Thus, flow cytometry also provided a
method for quantifying bacteria present within a preparation. We
believe that this technique provides an ideal tool to address cellular issues relating to C. trachomatis infection.
In the present study, we examined whether HLA-B27 expression exerts an
influence on the infection of cell lines with C. trachomatis and/or the replication of C. trachomatis within the cell. We
infected various cell lines, which differed for the expression of
HLA-B27, with C. trachomatis and monitored the infection by
intracellular staining and flow cytometry. An important technical
consideration in this study was the inclusion of heparin after the
initial infection. Heparin has been shown to inhibit uptake of C. trachomatis by various cell lines (31) and its
inclusion in the present study inhibited secondary infection of cells
with C. trachomatis serovar L2. It should be noted that
heparin may not be as successful in inhibiting infection of cell lines
with other serovars of C. trachomatis. For example, although
trachoma and LGV serovars have both been shown to use a
glucoaminoglycan-dependent infection pathway (5), trachoma
serovars are less dependent on the host cell heparin sulfate than is L2
for their infectivity (4, 24). However, our use of serovar
L2 in the present study enabled us to examine discrete time points
after the inception of a C. trachomatis infection. Using
BLCL, variations in infectivity pattern were noted, but there was no
correlation between any particular C. trachomatis staining
profile and the expression of HLA-B27 or with the disease status of the
donor of the blood from which the cell lines were established. When we
used the C1R cell line (6, 30), an EBV-positive mutant of
the human plasma cell leukemia-derived line Hmy2, which expresses very
low levels of MHC class I, we found that this cell line was more
susceptible to infection with C. trachomatis than other
conventional BLCL. Precisely why this cell line is more readily
infected than BLCL is unknown, but it may be due to the loss of
regulatory genes from the mutated C1R cell line and/or related to the
differing status of EBV infectivity. The expression of either the
HLA-B2705 or HLA-A201 transgene by C1R cells reduced their infectivity
with C. trachomatis. However, no significant differences
were found in the kinetics of the infection or the percentage of
infected cells between C1R-B2705 and C1R-A201 cells. Similarly, in
agreement with the larger percentage of parental cells exhibiting
evidence of intracellular C. trachomatis, a greater number
of C. trachomatis organisms were harvested from these cells after 24 h in comparison to the two transfectants which yielded similar numbers of C. trachomatis. Thus, the expression of
HLA class I, and not specifically HLA-B27, may exert an inhibitory effect on C. trachomatis infection. However, it is also
noteworthy, as is apparent from the greater numbers of the parental C1R
cells with a large forward-scatter signal (see Fig. 4), that more of the parental C1R cells were in the exponential phase of growth compared
to the MHC class I transfectants. This, rather than the lack of MHC
class I, may have led to the enhanced infection with C. trachomatis. Nonetheless, the results with both the BLCL and the
transfectants show that no effect of HLA-B27 on the infection or
replication of C. trachomatis could be discerned. This
result is in disagreement with a study that described a decreased
replication of C. trachomatis within C1R cells that
correlated with the expression of HLA-B2705 intracellular domain
(17). However, the methods used for quantitating the
C. trachomatis in that study were subject to large errors,
and no heparin was added to the cultures to prevent continuing
infection. Indeed, in a previous report the same authors using the same
methods found no effect of HLA-B27 expression on C. trachomatis infection (J. G. Kuipers et al., Abstr. 62nd Annu. Sci. Meet. Am. Coll. Rheumatol., Arthritis Rheum. 41[9S], abstr. 699, 1998).
The apparent lack of a modulating role of HLA-B27 expression on
C. trachomatis infection seen in the present study does not necessarily exclude that such a mechanism functions in reactive arthritis disease. It may be that our use of transformed cell lines
and/or the use of the invasive L2 serovar of C. trachomatis prevented the detection of this proposed inhibitory mechanism of
HLA-B27. We used the L2 serovar because of its relative ease of culture
and susceptibility to inhibition of infection by heparin. This highly
invasive serovar is not generally associated with reactive arthritis,
although there have been reports of joint symptoms and erythema
nodosum, a rash associated with reactive arthritis, after
LGV infection (13). While we cannot detect an
effect of HLA-B2705 on C. trachomatis replication, we have a
robust technique to permit the reciprocal study of the effects of
bacterial infection on HLA-B27 expression. Indeed, alterations in the
expression of HLA-B27 (28) after the infection of cells with Yersinia and Salmonella spp. have been
reported. Moreover, infection of cells with C. trachomatis
also leads to a downregulation of MHC class I (32) which
allows the recognition of infected cells by NK cells (C. E. Hook
and J. S. H. Gaston, Abstr. 4th Meet. Eur. Soc. Chlamydia
Res., p. 184, 2000). It is also possible that C. trachomatis
infection may increase the expression of aberrant forms of
HLA-B27, such as homodimers or free heavy chains, which have been
suggested as pathologic targets of immune responses (14,
15). Indeed, an alteration in the splicing of B27 mRNA leading
to the production of soluble HLA-B27 has been detected after infection
of cell lines with Yersinia and Salmonella spp. (9).
| |
ACKNOWLEDGMENT |
This work was supported by the Medical Research Council (United Kingdom).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: University of
Cambridge Clinical School, Department of Medicine, Level 5, Box 157, Addenbrooke's Hospital, Hills Rd., Cambridge CB2 2QQ, United Kingdom. Phone: 1223-330157. Fax: 1223-330160. E-mail:
jly21{at}medschl.cam.ac.uk.
Editor:
B. B. Finlay
| |
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Infection and Immunity, November 2001, p. 6670-6675, Vol. 69, No. 11
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.11.6670-6675.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
