Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.1205755 on July 20, 2006

Published online before print July 20, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1205755v1
80/4/774    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ben Nasr, A.
Right arrow Articles by Klimpel, G. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ben Nasr, A.
Right arrow Articles by Klimpel, G. R.
(Journal of Leukocyte Biology. 2006;80:774-786.)
© 2006 by Society for Leukocyte Biology

Critical role for serum opsonins and complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in phagocytosis of Francisella tularensis by human dendritic cells (DC): uptake of Francisella leads to activation of immature DC and intracellular survival of the bacteria

Abdelhakim Ben Nasr*, Judith Haithcoat*, Joseph E. Masterson*, John S. Gunn{dagger}, Tonyia Eaves-Pyles* and Gary R. Klimpel*,1

* Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas; and
{dagger} Tzagournis Medical Research Facility, The Ohio State University, Columbus, Ohio

1 Correspondence: Department of Microbiology and Immunology, Medical Research Building, University of Texas Medical Branch, Galveston, TX 77555-1070. E-mail: gklimpel{at}utmb.edu

ABSTRACT

Francisella tularensis is one of the most infectious human pathogens known. Although much has been learned about the immune response of mice using an attenuated live vaccine strain (LVS) derived from F. tularensis subspecies holarctica (Type B), little is known about the responses of human monocyte-derived immature dendritic cells (DC). Here, we show that optimal phagocytosis of LVS by DC is dependent on serum opsonization. We demonstrate that complement factor C3-derived opsonins and the major complement receptors expressed by DC, the integrins CR3 (CD11b/CD18) and CR4 (CD11c/CD18), play a critical role in this adhesion-mediated phagocytosis. LVS induced proinflammatory cytokine production and up-regulation of costimulatory surface proteins (CD40, CD86, and MHC Class II) on DC but resisted killing. Once taken up, LVS grew intracellularly, resulting in DC death. DC maturation and cytokine production were induced by direct contact/phagocytosis of LVS or interaction with soluble products of the bacteria, and enhanced activation was seen when LVS was pretreated with serum. Sonicated LVS and supernatants from LVS cultures were potent activators of DC, but LVS LPS failed to activate DC maturation or cytokine production. Serum-treated LVS rapidly induced (within 6 h) a number of cytokines including IL-10, a potent suppressor of macrophage functions and down-regulator of Th1-like responses and the Th1 response inducer IL-12. These results suggest that the simultaneous production of an activating (IL-12, IL-1ß, and TNF-{alpha}) and a suppressing (IL-10) cytokine profile could contribute to the immunopathogenesis of tularemia.

Key Words: bacterial infection • cytokines • lipopolysacchiride • LPS • innate immunity

INTRODUCTION

Francisella tularensis is a nonspore-forming, aerobic, gram-negative bacterium, which infects a host as a facultative, intracellular pathogen [1 2 3 4 ]. It has a thin capsule, which appears to be unique from those of other gram-negative bacteria [5 6 7 ]. In addition, LPS from this bacterium seems to differ significantly from that of other gram-negative bacteria [8 9 10 ]. No toxins have been found to be associated with this bacterium [7 , 11 ]. F. tularensis has been divided into four major subspecies: tularensis, holarctica, mediasiatica, and novicida [12 , 13 ]. F. tularensis subspecies tularensis, or Type A, is highly virulent to humans and animals and is the most common biovar isolated in North America [3 , 14 ]. Subspecies holarctica, or Type B, is less virulent and common in Europe and Asia and also found in North America, causing a much milder type of disease compared with Type A [3 , 14 , 15 ].

F. tularensis is the causative agent of the zoonotic disease tularemia [16 , 17 ], which occurs throughout most of the Northern hemisphere and involves a wide range of animal reservoir hosts. Francisella is one of the most infectious pathogens known with an infectious dose as little as 10 organisms. It can be aerosolized and is extremely stable and can persist in different environments for long periods of time. Because of its ease of aerosolization, dissemination, and high lethality, F. tularensis is considered a Category A bioweapon [18 ]. However, the pathogenesis of this bacterium and its virulence factors are not well characterized, and the first pathogenicity island of this microorganism has only been reported recently [19 ].

Host immune defenses to Francisella, especially in humans, are not understood completely. Murine infections with an attenuated Type B vaccine strain [live vaccine strain (LVS)] have served as a valuable model for examining the immune response to this bacterium [20 21 22 23 24 25 ]. Furthermore, it has been shown that LVS inhibits TLR-mediated activation of intracellular signaling and secretion of TNF-{alpha} and IL-1 from murine macrophages [26 ]. Clearly, cell-mediated immunity plays a crucial role in host defense and in generating immunity in the LVS mouse model [20 , 24 , 27 ]. In fact, immunity to LVS is dependent on the production of IFN-{gamma} and TNF-{alpha} [27 28 29 30 ]. It is important that mouse macrophages can kill ingested Francisella only after exposure to IFN-{gamma} [27 , 30 ]. Bosio and Dow [31 ] reported that LVS induces up-regulation of surface markers on mouse bone marrow-derived, airway, and lung dendritic cells (DC) without induction of the proinflammatory cytokines IL-6 and TNF-{alpha}, and a recent report shows that LVS does induce the proinflammatory cytokine TNF-{alpha} in bone marrow-derived mouse DC [32 ] .

Although much information has been gained about the LVS mouse model, little is known about the human immune response to LVS or virulent Francisella or how these bacteria alter the human innate and/or adaptive immune responses. However, new insights into how human monocytes and macrophages respond to LVS have been reported recently [33 34 35 ]. In contrast to the murine model, LVS uptake by human polymorphonuclear leukocytes, macrophages, and monocytes requires opsonization with human sera [33 , 34 , 36 ]. It is to be noted that although it is reported that no serum is needed for an enhanced uptake of LVS in the mouse system, the efficiency of uptake of LVS by mouse cells, measured as the percentage of uptake from imput, is more than a 100 times less than that for opsonin-mediated phagocytosis in the human system. Recently, Clemens et al. [33 ] have shown that opsonic phagocytosis of LVS by human macrophages is mediated via C3-derived fragments and the complement receptor CR3.

In contrast, there is no information about how human DC respond to Francisella. DC occupy center stage as the most efficient antigen-presenting cell in the immune system [37 ]. They determine the class of T cell response, including the development of Th1 cells, believed to be essential for host defense against Francisella in mice [23 , 38 ]. DC not only modulate the strength and quality of the adaptive immune response but also impact the innate immune response. In fact, DC are believed to be a key cell population in the transition between innate and adaptive immunity [39 ]. Thus, understanding how human DC respond to Francisella is an important step in furthering our understanding of how this bacterium interacts with the human innate and adaptive immune system. The potential role of CRs in how human DC phagocytose bacteria is largely unknown. In this regard, recognition and adhesion of serum-opsonized bacteria to human neutrophils and macrophages were reported to be mediated by binding of C3b and iC3b to specific CRs, including CD35 and the integrins CD11b/CD18 (CR3) and CD11c/CD18 (CR4) [33 , 40 41 42 43 44 45 46 ]. It is known that human DC express high levels of CD11b and especially CD11c, which is a specific marker for DC, but the relative contribution of these receptors to complement-mediated phagocytosis of bacteria by DC has not been determined. Furthermore, whether DC can kill LVS or virulent Francisella has never been reported.

In this study, we show that serum-opsonized LVS is phagocytosed efficiently by human monocyte-derived, immature DC (iDC). This uptake is dependent on C3 and is mediated by CR3 and CR4, leading to enhanced, proinflammatory cytokine production and up-regulation of important costimulatory surface proteins. Once ingested by DC, LVS grows and kills these cells. In addition, we show that soluble factors, but not LPS, produced by Francisella are responsible for DC maturation and induction of cytokine production.

MATERIALS AND METHODS

Bacteria and reagents
F. tularensis LVS (ATCC29684) was obtained from Dr. Karen Elkins (Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD). IsoVitaleX was purchased from Becton Dickinson (Cockeysville, MD). Brain heart infusion (BHI) was purchased from Difco Laboratories (Detroit, MI). Bacteria were stored frozen at –85°C until use in experiments. Bacteria were grown for 2 days on BHI agar plates enriched with IsoVitaleX. Plates were kept at 4°C, and new plates were made every third week. Plate-derived bacteria were then grown in modified Muller Hinton broth (MHB; Difco Laboratories) enriched with IsoVitaleX. The mid-logarithmic phase of growth occurred at 8–10 h of culture, at which time, bacteria consistently reached 8 x 108–2 x 109 CFU/ml. We used bacteria from cultures at this growth phase in each experiment below, and the actual concentration of bacteria was verified by a Petroff-Hausser chamber and plate counts after growing aliquots on BHI plates. LPS was purified from LVS by the hot phenol-water method, as described previously [47 , 48 ]. In some experiments, bacteria were pretreated with human serum, by incubating 2 x 109 bacteria in 1.5 ml human serum for 1 h at 37°C. Bacteria were then washed three times in PBS without calcium or magnesium and diluted in appropriate medium and used in experiments. For some experiments, bacteria were heat-killed by treating at 95°C for 5 min or 56°C for 45 min. Bacterial culture supernatants (LVS-conditioned media) were generated by culturing Francisella (109 bacteria/ml) in RPMI (Cellgro, Herndon, VA) culture medium without FBS (Hyclone, Logan, UT) and without antibiotics for 24 h. Supernatants were then cleared of bacteria by centrifugation and filtered through a 0.22-µm MillexRGP filter unit (Millipore, Carrigtwohil, Ireland). Blocking antibodies to the {alpha} chains of the CRs, Clone ICRF44 (mouse IgG1{kappa}) and Clone 3.9 (mouse IgG1 {kappa}) for CR3 and CR4, respectively, were purchased from Southern Biotech (Birmingham, AL). mAb specific for CD32 was obtained from BD Biosciences (San Diego, CA).

Human serum, monocytes, and DC isolation and culture
Human serum was prepared by drawing blood from five different healthy individuals who had no previous contact with LVS in 10 ml Vacutainer tubes without additive (Becton Dickinson Labware, Franklin Lakes, NJ). Sera were handled under endotoxin-free conditions, pooled, and stored in a manner to preserve complement activity. Heat-inactivated serum was prepared by incubation of the serum at 56°C for 45 min. C3-depleted serum was purchased from Complement Technology, Inc. (Tyler, TX).

EDTA-treated blood from healthy human donors was handled under endotoxin-free conditions, diluted 1:1 with PBS, and PBMC-purified by centrifugation over a Ficoll-sodium diatrizoate solution (Ficoll-Paque, Pharmacia Fine Chemicals, Inc., Piscataway, NJ). Monocytes were purified from PBMC by negative selection, using the magnetic column separation system from StemCell Technologies Inc. (Vancouver, BC, Canada), as described previously [49 , 50 ]. Monocyte-derived DC were generated from purified CD14+ monocytes as described previously [51 ]. Briefly, monocytes were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, L-glutamine, HEPES, sodium pyruvate, antibiotics (culture medium) plus GM-CSF (100 ng/ml), and IL-4 (50 ng/ml). Monocytes were set up in 24-well tissue-culture plates at 106 cells/ml. Nonadherent iDC were obtained at 7 days of culture, and only homogeneous iDC populations, characterized by high levels of CDla (greater than 99% positive and completely negative for other cell phenotypes) and no CD83 expression, were used in experiments. Viability was determined by trypan blue exclusion, and the cells were only used when viability exceeded 95%.

Phagocytosis and detection of intracellular and extracellular bacterial growth
DC (5x105) were washed three times in culture medium without antibiotics and then placed in 500 µl culture medium without antibiotics in 12 x 75 mm polystyrene snap-cap tubes (Becton Dickinson Labware). Varying concentrations of bacteria (in 10 µl), treated with human sera or untreated, were added to the tubes. DC and bacteria were then incubated for 1 h at 37°C. Gentamicin was added to DC/bacteria tubes at a final concentration of 50 µg/ml and incubated an additional 30 min at 37°C. DC/bacteria cultures were washed three times in RPMI containing no antibiotics and reconstituted with antibiotic-free culture medium. Tubes were then assessed immediately for intracellular bacteria (Time 0) or placed again at 37°C in culture medium containing 10 µg/ml gentamicin. To asses the effect of blocking antibodies to the CR3 and CR4 on the interaction between DC and LVS, DC were preincubated for 30 min with or without the indicated antibodies at a final concentration of 20 µg/ml and then incubated with LVS as described above. A mouse mAb of the same isotype (IgG1) was used as a control. To assess intracellular bacteria at different times post-exposure, DC/bacteria cultures (described above) were washed twice, and cells were lysed with 200 µl 0.1% SDS. Cell lysates were mixed immediately with 200 µl MHB and plated at varying dilutions on BHI agar plates. In some experiments, the effect of DC on bacterial growth was assessed using a 96-well microtiter assay or transwell culture systems. For the microtiter well assay, wells containing culture medium (RPMI plus 10% heat-inactivated FCS without antibiotics) plus Francisella (1–3x103 per well) were exposed to varying numbers of DC (103–105/well) and extracellular and intracellular bacteria growth assessed at varying times. Extracellular bacterial growth was assessed by plating 50 µl each DC culture supernatant onto BHI plates. For assessing intracellular bacterial growth, DC were harvested from each well, pretreated with 50 µg/ml gentamicin for 30 min, and then washed with antibiotic-free medium and lysed as described above. In some experiments, the effect of soluble factors produced by DC on LVS growth was assessed using a transwell culture system. For these experiments, 24-well tissue-culture plates and 0.4 µm polyethylene terephthalate (PET) transwell chambers (Falcon Cell culture insert, Becton Dickinson Labware) were used. DC and/or bacteria were placed in the bottom chamber (500 µl) and/or the top chamber (400 µl), and bacterial growth was assessed at 24 h or 48 h as described above. The SEM and statistical significance between different treatments/conditions were determined by ANOVA, followed by Tukey-Kramer’s comparison of means.

Flow cytometry
DC were assessed for the enhanced expression of surface HLA-DR, CD40, and CD86 using standard, one-color flow cytometry with FITC-conjugated mAb (BD Biosciences and Caltag, Burlingame, CA). Briefly, aliquots of DC (2–5x105) were incubated with the indicated antibodies or an isotype control antibody for 30 min at 4°C. After incubation, the cells were washed three times with PBS containing 2% FBS and subsequently, fixed with paraformaldehyde (1% in PBS). Fixed samples were then analyzed with a FACScan flow cytometer (BD Biosciences).

Cytokine measurements
Cytokine levels present in DC culture supernatants were determined using commercially purchased ELISA assays. The different cytokines measured were as follows: IL-6 (BD Biosciences, 50–20,000 pg), IL-8 (BD Biosciences, 50–20,000 pg), IL-10 (BD Biosciences, 50–10,000 pg), IL-12p70 (BD Biosciences 50–20,000 pg), TNF-{alpha} (BD Biosciences, 50–10,000 pg), and IL-1ß (eBiosciences, San Diego, CA, 5–500 pg). The SEM and statistical significance between different treatments/conditions were determined by ANOVA, followed by Tukey-Kramer’s comparison of means.

Protease, DNase, RNase, and heat treatment of sonicated LVS lysates
Sonicated LVS lysates were obtained by sonicating 2 x 109 bacteria in 1 ml PBS. The lysate was heat-treated by boiling for 5 min or made 10 mM Tris-HCl (pH 7.5), 10 mM CaCl, 10 mM MgCl and was then treated for 30 min at 37°C with DNase I (Amersham Biosciences, Piscataway, NJ) and RNase A (Sigma Chemical Co., St. Louis, MO) at a concentration of 100 unit/ml and 20 µg/ml for DNase I and RNase A, respectively. For protease treatment, the lysate was treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated, immobilized Trypsin (Pierce, Rockford, IL) for 2 h at 37°C. The treated lysate was separated from the immobilized Trypsin by centrifugation and was used to stimulate DC.

RESULTS

Phagocytosis of LVS by DC is critically dependent on serum opsonization and is inhibited by blocking CR3 and CR4
Our initial experiments assessed the role of opsonization in phagocytic uptake of LVS by DC. As previously reported for human neutrophils [36 ] and macrophages [33 , 34 ], phagocytosis by DC was critically dependent on serum opsonization (Fig. 1 ). We observed differences in the phagocitic efficiencies of DC prepared from different human donors, but the phagocytic uptake of each donor DC was always enhanced significantly by nonimmune serum opsonization. Phagocytosis efficiency was dependent on serum concentration, the duration of serum pretreatment of LVS, and whether or not the serum was heat-inactivated, thus suggesting a role for complement. Optimal complement treatment was achieved using neat serum and preincubating bacteria for at least 1 h. The major opsonins in complement are derived from complement factor C3. To determine whether complement factor C3-derived opsonins were necessary for LVS phagocytosis by DC, we pretreated LVS with C3-depleted sera prior to incubation with DC. As shown (Fig. 2A ), phagocytosis by DC of LVS coated with C3-depleted serum was reduced to levels that are not significantly different from phagocytosis of LVS preincubated with PBS. The major C3-derived opsonins in nonimmune human sera are the C3b and iC3b fragments generated through the alternative pathway of complement. Recognition of bacteria opsonized by these fragments is mediated by binding to specific CRs on the phagocyte surface. Therefore, we used antibodies that block CRs to examine whether any of the major CRs on DC, namely CR3 and CR4, were responsible for the adhesion-mediated uptake of LVS. We found that incubation of DC with blocking antibodies to CR3 or CR4 prior to and during exposure to serum-pretreated LVS inhibited phagocytosis significantly by 30–50% for CR3 or CR4 alone (Fig. 2B) . Simultaneous treatment of DC with antibodies against CR3 and CR4 reduced phagocytosis significantly by 50–80% compared with untreated or the isotype control antibody. No significant differences were observed between CR3 and CR4 antibodies in inhibiting phagocytosis, and the simultaneous treatment with both antibodies was consistently better than any of the antibodies alone, but the difference was not always statistically significant. To rule out any contribution of Fc receptor involvement with regard to natural antibody present in the serum, we also pretreated DC with mAb specific for CD32 or with different nonspecific control mAb to block FcRs. CD32 or nonspecific mouse IgG-pretreated DC and untreated DC were similar in their uptake of serum-treated bacteria and significantly different from CR3 and/or CR4-treated DC (Fig. 2B) . Collectively, these results suggest that complement C3-derived opsonins mediate phagocitic uptake of serum-opsonized LVS by DC via their functions as ligands for the major CR3 and CR4 expressed at the surface of these cells.


Figure 1
View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. Phagocytosis of LVS by DC is serum-dependent. DC were assessed for their ability to phagocytose LVS as described in Materials and Methods. DCs were incubated with varying numbers of LVS, which had been pretreated with one of the following: PBS only (LVS), serum (LVS+Serum), or heat-inactivated serum (LVS+Heat-inactivated serum). Data are presented as CFU per 5 x 105 DC. The error bars represent standard deviations of triplicate samples, and the results presented are representative of five separate experiments. Statistically significant differences between different treatments are marked (**, P<0.01). MOI, Multiplicity of infection.

 

Figure 2
View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Enhanced phagocytosis of LVS by DC depends on complement factor C3 and CR3 and CR4. DC were incubated with LVS at a MOI of 40:1 for 1 h at 37°C followed by 30 min treatment with gentamicin to kill extracellular bacteria. (A) LVS was treated with one of the following: PBS only, serum, or C3-depleted serum. (B) DC were preincubated with PBS (LVS+Serum), 20 µg/ml anti-CR3, 20 µg/ml anti-CR4, 20 µg/ml anti-CR3 and anti-CR4, 20 µg/ml anti-CD32, or 20 µg/ml mouse IgG1 at 37°C for 30 min before incubation with LVS, which had been treated with serum. Data are presented as CFU per 105 DC. The error bars represent standard deviations of triplicate samples, and the results presented are representative of five separate experiments for A and three separate experiments for B. Two different donor DC were used in A and B. Statistically significant differences between different treatments are marked (*, P<0.05; **, P<0.01).

 
Intracellular growth of LVS in DC
LVS grew inside DC, which were first exposed to the bacteria, then treated with high doses of gentamicin for 30 min, and finally, maintained in the presence of a modest concentration (10 µg/ml) of gentamicin (Table 1 ). The intracellular bacterial number increased over time, resulting in DC death. Variation in the kinetics of intracellular LVS growth was observed between different DC donors. LVS growth was rapid (within the first 4 h) inside DC from some individuals, which was paralleled by a more rapid DC death. To further investigate the ability of LVS to grow inside DC, we set up experiments that could assess LVS growth in the absence of antibiotics. For these experiments, cultures containing no antibiotics and low numbers of LVS (2–4x103/well) were supplemented with varying numbers of DC, and bacterial growth was determined at various times from intracellular versus extracellular environments. Results from a typical experiment (one of five) are presented in Table 2 . LVS failed to grow in RPMI medium in the absence of DC. However, LVS growth was enhanced significantly when DC were added to these cultures. This was dose-dependent, and the most LVS growth occurred when high numbers of DC were present in the cultures. This appeared to be a result of intracellular growth and dropped off significantly at 48 h when few live DC could be identified. To assess the contribution of DC-secreted factors in promoting or inhibiting extracellular LVS growth, we separated LVS from DC using a transwell culture system. Using this experimental system, we could find no evidence for DC enhancement or inhibition of extracellular growth of LVS (Table 3 ). Together, these data suggest that DC do not promote extracellular LVS growth but that the bacteria grow readily inside DC, killing them and then being released extracellularly.


View this table:
[in this window]
[in a new window]
  		Table 1. Kinetics of Francisella Growth in DC

 

View this table:
[in this window]
[in a new window]
  		Table 2. DC Enhances the Growth of Francisella

 

View this table:
[in this window]
[in a new window]
  		Table 3. DC Do Not Release Inhibitory or Growth Factors for LVS Growth

 
Cytokine production by LVS-stimulated DC
In these initial experiments, LVS was not pretreated with human sera but added directly to DC, which then produced significant levels of proinflammatory cytokines at 24 h post-exposure to varying numbers of LVS. The results were dose-dependent, and optimal levels of cytokines were induced at the higher MOIs (data not shown). As in vivo, it is likely that LVS would be opsonized with complement or other blood proteins, and we investigated cytokine production at 6 and 24 h following DC exposure to serum-treated versus -untreated LVS. At 24 h, serum-treated LVS always induced higher cytokine levels when compared with untreated LVS (Fig. 3 ). Cytokine levels induced by serum-treated LVS were also always higher at 24 h when compared with 6 h. When compared with untreated-LVS, serum-treated LVS also induced significantly higher levels of IL-1ß, IL-6, and TNF-{alpha} at 6 h post-stimulation. It should be stressed that we found variability between different DC donors with regards to the levels of different cytokines produced following LVS exposure. The data presented show that the proinflammatory and/or DC maturation-inducing cytokines IL-6, IL-8, TNF-{alpha}, and IL-1ß are up-regulated early after exposure to serum-treated LVS. In addition, IL-10, a potent suppressor of macrophage functions and down-regulator of Th1-like responses, and the Th1 response inducer IL-12 are also up-regulated significantly.


Figure 3
View larger version (32K):
[in this window]
[in a new window]
  		Figure 3. LVS-induced cytokine production by DC is enhanced by serum opsonization. DC were assessed for cytokine production following exposure to one of the following: medium only, LVS at a MOI of 20:1, or LVS at a MOI of 20:1, which had been pretreated with human serum. Cytokine production was assessed in supernatants obtained at 6 h and 24 h. Data are presented for IL-1ß (A), IL-6 (B), IL-8 (C), IL-10 (D), IL-12p70 (E), and TNF-{alpha} (F). The error bars represent standard deviations of triplicate samples, and the results presented are representative of three separate experiments. Statistically significant differences between different treatments are marked (*, P<0.05; **, P<0.01). ND, Not determined.

 
LVS-LPS does not induce cytokine production
We next investigated what bacterial component might be involved in LVS-induced cytokine production by DC. LPS purified from LVS failed to induce cytokine production, even when used at high concentrations (500 ng/ml; Table 4 ). In contrast, LPS from E. coli induced high levels of cytokine production. However, conditioned medium from 24 h cultures of LVS grown in RPMI induced significant levels of cytokine production, as did sonicated bacteria (Tables 4 and 5 and Fig. 4 ). However, sonicated bacterial preparations, which were treated with protease, but not RNAase and DNAase, lost their ability to induce cytokine production, suggesting that a protein might be the mediator of this activation. Further, LVS-conditioned medium and sonicated LVS-induced cytokine production were not a result of LVS-LPS, as polymyxin B failed to inhibit production of these cytokines, and LVS-LPS failed to induce cytokines (Tables 4 and 5 and Fig. 4 ). In contrast, heat-killed LVS failed to induce the production of any significant levels of the cytokines IL-1ß, TNF-{alpha}, and IL-12p70, although low levels of IL-6 and IL-8 could be detected (Table 6 ). These data show that the cytokine-inducing capacity of LVS is not a result of LPS and that soluble factors produced by this organism could contribute toward DC activation.


View this table:
[in this window]
[in a new window]
  		Table 4. LVS-Conditioned Medium but Not LVS-LPS Activates DC

 

View this table:
[in this window]
[in a new window]
  		Table 5. Sonicated LVS Activates DC

 

Figure 4
View larger version (10K):
[in this window]
[in a new window]
  		Figure 4. LVS culture supernatants induce IL-12p70 production by DC. LVS culture supernatants (1:4 final dilutions) were assessed for their ability to induce cytokine production in DC using the 96-well microtiter culture system. DC were exposed to one of the following: medium alone, E. coli LPS (20 ng/ml), or E. coli LPS + polymyxin B (2 µg/ml), LVS-conditioned medium, or LVS-conditioned medium + polymyxin B (2 µg/ml). Data presented are from one experiment, which was representative of three separate experiments. Statistically significant differences between different treatments are marked (**, P<0.01).

 

View this table:
[in this window]
[in a new window]
  		Table 6. Cytokine Production by Heat-Killed LVS

 
LVS-induced up-regulation of costimulatory molecules
Monocyte-derived iDC express low levels of MHC Class II molecules and costimulatory molecules CD40, CD80, and CD86. After exposure to bacteria, LPS, or inflammatory mediators, DC undergo a maturation process characterized by cytokine production and increased expression of MHC and costimulatory molecules. To further explore LVS activation of DC, we assessed DC for increased expression of HLA-DR, CD40, and CD86 following 24 h of exposure to LVS versus E. coli LPS (100 ng/ml). As seen in Figure 5 , LVS induced an up-regulation in the expression of all of these surface proteins. This enhancement was comparable with that induced by E. coli LPS. As observed with cytokine production, LPS purified from LVS failed to induce up-regulation of any of the costimulatory molecules (Fig. 6 ). However, LVS culture supernatant (data not shown) or sonicated LVS induced up-regulation of these surface proteins, and this activation was not inhibited by polymyxin B (Fig. 7 ).


Figure 5
View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. LVS induces enhanced expression of costimulatory molecules on DC, which were exposed to one of the following: medium only, LVS at a MOI of 40:1, E. coli LPS (100 ng/ml). After 24 h, DC were collected, washed, and stained with antibodies specific for HLA-DR, CD40, and CD86. The histogram presented is from one experiment, which was representative of five separate experiments.

 

Figure 6
View larger version (13K):
[in this window]
[in a new window]
  		Figure 6. LPS from LVS fails to activate DC, which were exposed to one of the following: medium only, E. coli LPS (100 ng/ml), LVS-LPS (100 ng/ml), LVS-LPS (500 ng/ml), or LVS (MOI: 40:1). After 24 h of incubation, DC were collected, washed, and stained for HLA-DR, CD40, and CD86. Data presented are from one experiment, which was representative of six different experiments.

 

Figure 7
View larger version (18K):
[in this window]
[in a new window]
  		Figure 7. Sonicated LVS activates DC and is not inhibited by polymyxin. DC were incubated with one of the following: medium only, E. coli LPS (20 ng/ml), E. coli LPS (20 ng/ml) + polymyxin (2 µg/ml), sonicated LVS (equivalent to MOI 40:1), sonicated LVS + polymyxin, or LVS (MOI 40:1). After 24 h of culture, DC were collected, washed, and stained for HLA-DR, CD40, and CD86. Data presented are from one experiment, which was representative of three different experiments.

 
DISCUSSION

In this study, we have shown that LVS is taken up by human DC and that optimal phagocytosis is dependent on serum opsonization and is mediated by CRs; LVS grows within DC, resulting in DC death; LVS induces proinflammatory cytokine production by DC, which is enhanced when LVS is opsonized by serum; LVS activates DC to have increased expression of costimulatory molecules; LPS purified from LVS is not capable of activating DC; and bacterial components released by LVS or associated with LVS can activate DC. Thus, our studies have shown that although LVS induces DC maturation, LVS is still able to grow intracellularly, which leads to DC death.

Optimal phagocytosis of LVS by human monocytes, macrophages, and neutrophils is dependent on serum opsonization and appears to differ from that observed using mouse macrophages [27 , 33 , 34 , 36 ]. We now show that optimal phagocytosis of LVS by human DC also requires serum opsonization and is mediated by CR3 and CR4. These data are in agreement with those reported by Clemens et al. [33 ], who also demonstrated a role for CR3 in uptake of LVS by human monocyte-derived macrophages but are in disagreement with the data published by Bolger et al. [35 ], who concluded that serum opsonization is not necessary for enhanced phagocytosis. However, their conclusion is based on an experimental approach in which bacteria are pelleted by centrifugation onto human macrophages. Although such an experimental approach will synchronize the phagocytic process, it also could exclude detection of any initial opsonin-dependent adhesions between the bacteria and the phagocyte. Indeed, we have confirmed (data not shown) that when bacteria and DC or monocytes are copelleted by centrifugation following similar procedures as Bolger et al. [35 ], the phagocytosis is enhanced without need for serum opsonization.

The requirement for serum opsonization and the fact that C3-deficient serum does not promote phagocytosis suggest the involvement of the complement factor C3-derived opsonins C3b and/or iC3b. The results of this study show that CR3 (CD11b/CD18) and CR4 (CD11c/CD18) are the major receptors responsible for phagocytosis of serum-opsonized LVS. CR3 has been reported to mediate phagocytosis of different complement opsonized bacteria by human neutrophils and macrophages, but CR4 has been directly shown to mediate phagocytosis by human macrophages only in the case of Mycobacterium leprae and Mycobacterium tuberculosis [41 , 44 , 46 ]. To our knowledge, neither CR3 nor CR4 has been shown to be involved directly in adhesion-mediated phagocytosis by DC. Human iDC have been shown to phagocytose a number of gram-negative and -positive bacteria, as well as different fungal and yeast organisms [52 53 54 55 56 57 58 59 60 ]. In most of these studies, no opsonization was necessary, and phagocytosed organisms were killed or growth-inhibited when intracellular growth was investigated. However, opsonization is required for optimal uptake of Cryptococcus neoformans [57 ] and Listeria monocytogenes [59 ] by human iDC. In addition, Trypanosoma cruzi survives and multiplies within human iDC [61 ], and L. monocytogenes induces cell death in a small percentage of human iDC. Similarly, one study reported that M. tuberculosis replicates inside human iDC [58 ].

Although we found little difference in the uptake of LVS by monocytes (data not shown) versus iDC, Nagl et al. [60 ] reported that iDC were much less efficient at taking up and destroying Staphylococcus aureus or E. coli when compared with monocytes or monocyte-derived macrophages. The plasticity of DC was recently demonstrated in a study that showed different pathogens induce different changes in gene-expression patterns in monocyte-derived DC [62 ]. Thus, our results demonstrate a unique aspect of F. tularensis entry into DC, which could play an important role in the pathogenesis and immune responses.

LPS from F. tularensis is not biologically active when compared with LPS derived from other gram-negative bacteria [8 9 10 ]. The nonendotoxic nature of this LPS appears to be attributed to the unusual structure of the lipid A molecule. We found that LPS from LVS contained little, if any, ability to activate human DC. This was observed using a wide range of concentrations. Thus, the ability of LVS to activate human DC appears to be mediated via other bacterial components. In fact, we found culture supernatant from overnight cultures of LVS or sonicated LVS could activate DC, and this was not inhibited by polymyxin B. The fact that protease treatment of sonicated LVS resulted in a significant inhibition of DC activation suggests that a protein is possibly involved in this stimulation. Similar results were also observed with culture supernatants (data not presented). The identification and characterization of these factors are currently in progress.

Recently, it was reported that Francisella induced IL-1ß production by human monocyte-required internalization and phagosome escape of the bacteria [63 ]. In this study, heat-killed Francisella failed to induce IL-1ß but could induce IL-8 production. In our study, we found that DC activation was inhibited significantly when LVS was killed via treatment at 56°C or 95°C. We consistently found that killed LVS failed to induce the production of any significant levels of the cytokines IL-1ß, TNF-{alpha}, and IL-12p70, although low levels of IL-6 and IL-8 could be detected. In our studies, DC were significantly unresponsive to heat-killed bacteria. This was observed when assessing cytokine production as well as up-regulation of surface costimulatory proteins (data not shown).

DC exposure to LVS resulted in enhanced expression of costimulatory proteins (CD40, CD86, and MHC II) as well as the production of IL-6, IL-8, IL-10, TNF-{alpha}, IL–1ß, and IL-12p70. The proinflammatory cytokines IL-6, TNF-{alpha}, IL-1ß, IL-8, and IL-12 are crucial in the innate and adaptive immune response to infections. These cytokines are important in enhancing the bactericidal capacity of phagocytes, recruiting additional innate cell populations such as neutrophils to sites of infection, inducing DC maturation, and directing the specific immune response to invading microbes [64 65 66 ]. However, IL-10 can mediate potent immunosuppression: It has been shown to inhibit macrophage activation and production of reactive oxygen species, prevent the maturation of DC [67 68 69 ], induce T cell tolerance and development of T regulatory cells [70 , 71 ], and suppress TNF-{alpha} production [72 ]. Our finding that LVS induces high levels of IL-10 and IL-12 could play a role in altering the host protective immune response.

Recently, F. tularensis was shown to induce proinflammatory changes in human endothelial cells and macrophages [35 , 73 ]. This was not dependent on living bacteria, as killed bacteria could also activate endothelial cells with regard to chemokine production, up-regulation of E-selectin, VCAM-1, ICAM-1, and transendothelial migration of neutrophils. In our study, we also found that activation of DC was not dependent on live bacteria or phagocytic uptake, as naked LVS, sonicated LVS, and culture supernatants from LVS could induce cytokine production and up-regulation of costimulatory proteins. However, serum-treated LVS induced higher levels of cytokines by DC, and the kinetics of cytokine production was significantly faster when serum-treated LVS was used to stimulate DC. This was also observed in the up-regulation of costimulatory proteins (data not presented). It is unclear whether this was mediated via enhanced avidity of LVS to these cells, with LVS components stimulating via some receptor(s) or via some receptor-activation pathway independent of LVS (CR activation).

In the mouse model, cell-mediated immunity is essential for host defense and protection against LVS infection [20 21 22 23 24 25 , 27 ]. Bosio and Dow [31 ] have shown that LVS induces only up-regulation of CD86 and MHC II expression but failed to induce any proinflammatory cytokine production. Although these authors do not discuss serum dependency for uptake of LVS by mouse DC, their data show an efficiency of uptake of only 0.01% compared with an efficiency of uptake of 1–4%, which we see with serum opsonization. In contrast to the report of Bosio and Dow [31 ], a study recently published has shown that mouse bone marrow-derived DC were activated via TLR2 by LVS to produce TNF-{alpha} and to have up-regulation of costimulatory molecules MHC Class II, CD86, and CD80 [32 ]. Phagocytic uptake of LVS by mouse DC was not investigated in this study. Thus, how mouse DC (bone marrow-derived) compare with human monocyte-derived DC is unclear. However, the crucial role of DC in cell-mediated immunity and innate immunity makes it important to determine how human DC respond to LVS. We now show that human DC respond to LVS with cytokine production and up-regulation of costimulatory proteins, which appears to be similar to mouse bone marrow-derived DC, as reported by Katz et al. [32 ]. In the pathogenesis of LVS in mouse studies, it was shown that a 30-kb pathogenicity island of Francisella is necessary for intramacrophage growth [19 ]. The Francisella pathogenicity island protein IglC is necessary for intracellular growth and induction of apoptosis in murine macrophages by Francisella [74 ]. In addition, Mgla, a transcriptional activator, which regulates the transcription of the pathogenicity island components iglC, pdpD, and pdpA, as well as the pdpA gene itself, was shown to be necessary for the induction of macrophage cell death [19 , 75 ]. It is unclear whether these will also play important roles in the pathogenesis of LVS in human studies and LVS-DC interactions, but the homologous IglC in F. tularensis subspecies novicida has recently been shown to be essential for modulating phagosome biogenesis and survival of the bacteria in human macrophages [76 , 77 ]. It is more important that it is unknown how human DC will respond to virulent Francisella (F. tularensis strain schu 4). In summary, we have shown that LVS induces maturation of human iDC. This activation/maturation was characterized by proinflammatory and suppressive cytokine production and up-regulation of costimulatory proteins and was independent of phagocytosis. The fact that LVS and bacterial components, other than LPS, induce DC maturation/activation could be important for vaccine development and furthering our understanding of the pathogenesis of this bacterium. Finally, we show in this report that human DC take up LVS via CRs and that LVS grows intracellularly and results in DC death.

Received December 21, 2005; revised January 27, 2006; accepted May 18, 2006.

REFERENCES

  1. Tarnvik, A., Ericsson, M., Golovliov, I., Sandstrom, G., Sjostedt, A. (1996) Orchestration of the protective immune response to intracellular bacteria: Francisella tularensis as a model organism FEMS Immunol. Med. Microbiol. 13,221-225[CrossRef][Medline]
  2. Sandstrom, G., Sjostedt, A., Tarnvik, A. (1996) The first international conference on tularemia: Umea, Sweden, 23–25 August 1995—introduction FEMS Immunol. Med. Microbiol. 13,179[Medline]
  3. Jacobs, R. F. (1996) Tularemia Adv. Pediatr. Infect. Dis. 12,55-69[Medline]
  4. Titball, R. W., Sjostedt, A. (2003) Francisella tularensis: an overview ASM News 69,558-563
  5. Jantzen, E., Berdal, B. P., Omland, T. (1979) Cellular fatty acid composition of Francisella tularensis J. Clin. Microbiol. 10,928-930[Abstract/Free Full Text]
  6. Nichols, P. D., Mayberry, W. R., Antworth, C. P., White, D. C. (1985) Determination of monounsaturated double-bond position and geometry in the cellular fatty acids of the pathogenic bacterium Francisella tularensis J. Clin. Microbiol. 21,738-740[Abstract/Free Full Text]
  7. Hood, A. M. (1977) Virulence factors of Francisella tularensis J. Hyg. (Lond.) 79,47-60[Medline]
  8. Ancuta, P., Pedron, T., Girard, R., Sandstrom, G., Chaby, R. (1996) Inability of the Francisella tularensis lipopolysaccharide to mimic or to antagonize the induction of cell activation by endotoxins Infect. Immun. 64,2041-2046[Abstract]
  9. Dreisbach, V. C., Cowley, S., Elkins, K. L. (2000) Purified lipopolysaccharide from Francisella tularensis live vaccine strain (LVS) induces protective immunity against LVS infection that requires B cells and {gamma} interferon Infect. Immun. 68,1988-1996[Abstract/Free Full Text]
  10. Sandstrom, G., Sjostedt, A., Johansson, T., Kuoppa, K., Williams, J. C. (1992) Immunogenicity and toxicity of lipopolysaccharide from Francisella tularensis LVS FEMS Microbiol. Immunol. 5,201-210[Medline]
  11. Skrodzki, E., Roszkiewicz, A. (1968) Investigations on the pathogenesis of tularemia. 8. Histopatological changes in the organs of white mice due to the effect of F. tularensis endotoxin Biul. Inst. Med. Morsk. Gdansk. 19,77-84[Medline]
  12. Forsman, M., Sandstrom, G., Sjostedt, A. (1994) Analysis of 16S ribosomal DNA sequences of Francisella strains and utilization for determination of the phylogeny of the genus and for identification of strains by PCR Int. J. Syst. Bacteriol. 44,38-46[Abstract/Free Full Text]
  13. Svensson, K., Larsson, P., Johansson, D., Bystrom, M., Forsman, M., Johansson, A. (2005) Evolution of subspecies of Francisella tularensis J. Bacteriol. 187,3903-3908[Abstract/Free Full Text]
  14. Tarnvik, A. (1989) Nature of protective immunity to Francisella tularensis Rev. Infect. Dis. 11,440-451[Medline]
  15. Sjostedt, A., Tarnvik, A., Sandstrom, G. (1996) Francisella tularensis: host-parasite interaction FEMS Immunol. Med. Microbiol. 13,181-184[Medline]
  16. Larsson, P., Oyston, P. C., Chain, P., Chu, M. C., Duffield, M., Fuxelius, H. H., Garcia, E., Halltorp, G., Johansson, D., Isherwood, K. E., et al (2005) The complete genome sequence of Francisella tularensis, the causative agent of tularemia Nat. Genet. 37,153-159[CrossRef][Medline]
  17. Oyston, P. C., Sjostedt, A., Titball, R. W. (2004) Tularaemia: bioterrorism defence renews interest in Francisella tularensis Nat. Rev. Microbiol. 2,967-978[CrossRef][Medline]
  18. Dennis, D. T., Inglesby, T. V., Henderson, D. A., Bartlett, J. G., Ascher, M. S., Eitzen, E., Fine, A. D., Friedlander, A. M., Hauer, J., Layton, M., et al (2001) Tularemia as a biological weapon: medical and public health management JAMA 285,2763-2773[Abstract/Free Full Text]
  19. Nano, F. E., Zhang, N., Cowley, S. C., Klose, K. E., Cheung, K. K., Roberts, M. J., Ludu, J. S., Letendre, G. W., Meierovics, A. I., Stephens, G., Elkins, K. L. (2004) A Francisella tularensis pathogenicity island required for intramacrophage growth J. Bacteriol. 186,6430-6436[Abstract/Free Full Text]
  20. Conlan, J. W., Sjostedt, A., North, R. J. (1994) CD4+ and CD8+ T-cell-dependent and -independent host defense mechanisms can operate to control and resolve primary and secondary Francisella tularensis LVS infection in mice Infect. Immun. 62,5603-5607[Abstract/Free Full Text]
  21. Stenmark, S., Sunnemark, D., Bucht, A., Sjostedt, A. (1999) Rapid local expression of interleukin-12, tumor necrosis factor {alpha}, and {gamma} interferon after cutaneous Francisella tularensis infection in tularemia-immune mice Infect. Immun. 67,1789-1797[Abstract/Free Full Text]
  22. Sjostedt, A., Conlan, J. W., North, R. J. (1994) Neutrophils are critical for host defense against primary infection with the facultative intracellular bacterium Francisella tularensis in mice and participate in defense against reinfection Infect. Immun. 62,2779-2783[Abstract/Free Full Text]
  23. Cowley, S. C., Elkins, K. L. (2003) Multiple T cell subsets control Francisella tularensis LVS intracellular growth without stimulation through macrophage interferon {gamma} receptors J. Exp. Med. 198,379-389[Abstract/Free Full Text]
  24. Elkins, K. L., Cowley, S. C., Bosio, C. M. (2003) Innate and adaptive immune responses to an intracellular bacterium, Francisella tularensis live vaccine strain Microbes Infect. 5,135-142[CrossRef][Medline]
  25. Fulop, M., Mastroeni, P., Green, M., Titball, R. W. (2001) Role of antibody to lipopolysaccharide in protection against low- and high-virulence strains of Francisella tularensis Vaccine 19,4465-4472[CrossRef][Medline]
  26. Telepnev, M., Golovliov, I., Grundstrom, T., Tarnvik, A., Sjostedt, A. (2003) Francisella tularensis inhibits Toll-like receptor-mediated activation of intracellular signaling and secretion of TNF-{alpha} and IL-1 from murine macrophages Cell. Microbiol. 5,41-51[CrossRef][Medline]
  27. Fortier, A. H., Polsinelli, T., Green, S. J., Nacy, C. A. (1992) Activation of macrophages for destruction of Francisella tularensis: identification of cytokines, effector cells, and effector molecules Infect. Immun. 60,817-825[Abstract/Free Full Text]
  28. Elkins, K. L., Rhinehart-Jones, T., Nacy, C. A., Winegar, R. K., Fortier, A. H. (1993) T-cell-independent resistance to infection and generation of immunity to Francisella tularensis Infect. Immun. 61,823-829[Abstract/Free Full Text]
  29. Elkins, K. L., Leiby, D. A., Winegar, R. K., Nacy, C. A., Fortier, A. H. (1992) Rapid generation of specific protective immunity to Francisella tularensis Infect. Immun. 60,4571-4577[Abstract/Free Full Text]
  30. Anthony, L. S., Ghadirian, E., Nestel, F. P., Kongshavn, P. A. (1989) The requirement for {gamma} interferon in resistance of mice to experimental tularemia Microb. Pathog. 7,421-428[CrossRef][Medline]
  31. Bosio, C. M., Dow, S. W. (2005) Francisella tularensis induces aberrant activation of pulmonary dendritic cells J. Immunol. 175,6792-6801[Abstract/Free Full Text]
  32. Katz, J., Zhang, P., Martin, M., Vogel, S. N., Michalek, S. M. (2006) Toll-like receptor 2 is required for inflammatory responses to Francisella tularensis LVS Infect. Immun. 74,2809-2816[Abstract/Free Full Text]
  33. Clemens, D. L., Lee, B. Y., Horwitz, M. A. (2005) Francisella tularensis enters macrophages via a novel process involving pseudopod loops Infect. Immun. 73,5892-5902[Abstract/Free Full Text]
  34. Clemens, D. L., Lee, B. Y., Horwitz, M. A. (2004) Virulent and avirulent strains of Francisella tularensis prevent acidification and maturation of their phagosomes and escape into the cytoplasm in human macrophages Infect. Immun. 72,3204-3217[Abstract/Free Full Text]
  35. Bolger, C. E., Forestal, C. A., Italo, J. K., Benach, J. L., Furie, M. B. (2005) The live vaccine strain of Francisella tularensis replicates in human and murine macrophages but induces only the human cells to secrete proinflammatory cytokines J. Leukoc. Biol. 77,893-897[Abstract/Free Full Text]
  36. Lofgren, S., Tarnvik, A., Bloom, G. D., Sjoberg, W. (1983) Phagocytosis and killing of Francisella tularensis by human polymorphonuclear leukocytes Infect. Immun. 39,715-720[Abstract/Free Full Text]
  37. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J., Pulendran, B., Palucka, K. (2000) Immunobiology of dendritic cells Annu. Rev. Immunol. 18,767-811[CrossRef][Medline]
  38. Cowley, S. C., Hamilton, E., Frelinger, J. A., Su, J., Forman, J., Elkins, K. L. (2005) CD4–CD8– T cells control intracellular bacterial infections both in vitro and in vivo J. Exp. Med. 202,309-319[Abstract/Free Full Text]
  39. Ardavin, C., Amigorena, S., Reis e Sousa, C. (2004) Dendritic cells: immunobiology and cancer immunotherapy Immunity 20,17-23[CrossRef][Medline]
  40. Weineisen, M., Sjoring, U., Fallman, M., Andersson, T. (2004) Streptococcal M5 protein prevents neutrophil phagocytosis by interfering with CD11b/CD18 receptor-mediated association and signaling J. Immunol. 172,3798-3807[Abstract/Free Full Text]
  41. Schlesinger, L. S., Horwitz, M. A. (1991) Phagocytosis of Mycobacterium leprae by human monocyte-derived macrophages is mediated by complement receptors CR1 (CD35), CR3 (CD11b/CD18), and CR4 (CD11c/CD18) and IFN-{gamma} activation inhibits complement receptor function and phagocytosis of this bacterium J. Immunol. 147,1983-1994[Abstract]
  42. Schlesinger, L. S., Horwitz, M. A. (1990) Phagocytosis of leprosy bacilli is mediated by complement receptors CR1 and CR3 on human monocytes and complement component C3 in serum J. Clin. Invest. 85,1304-1314[Medline]
  43. Schlesinger, L. S., Bellinger-Kawahara, C. G., Payne, N. R., Horwitz, M. A. (1990) Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3 J. Immunol. 144,2771-2780[Abstract]
  44. Zimmerli, S., Edwards, S., Ernst, J. D. (1996) Selective receptor blockade during phagocytosis does not alter the survival and growth of Mycobacterium tuberculosis in human macrophages Am. J. Respir. Cell Mol. Biol. 15,760-770[Abstract]
  45. Ernst, J. D. (2000) Bacterial inhibition of phagocytosis Cell. Microbiol. 2,379-386[CrossRef][Medline]
  46. Schlesinger, L. S. (1993) Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors J. Immunol. 150,2920-2930[Abstract]
  47. Kieffer, T. L., Cowley, S., Nano, F. E., Elkins, K. L. (2003) Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS, and contributes to F. novicida murine pathogenesis Microbes Infect. 5,397-403[CrossRef][Medline]
  48. Cowley, S. C., Myltseva, S. V., Nano, F. E. (1996) Phase variation in Francisella tularensis affecting intracellular growth, lipopolysaccharide antigenicity and nitric oxide production Mol. Microbiol. 20,867-874[CrossRef][Medline]
  49. Tseng, C. T., Klimpel, G. R. (2002) Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions J. Exp. Med. 195,43-49
  50. Tseng, C. T., Miskovsky, E., Klimpel, G. R. (2001) Crosslinking CD81 results in activation of TCR{gamma}{delta} T cells Cell. Immunol. 207,19-27[CrossRef][Medline]
  51. Rissoan, M. C., Soumelis, V., Kadowaki, N., Grouard, G., Briere, F., de Waal Malefyt, R., Liu, Y. J. (1999) Reciprocal control of T helper cell and dendritic cell differentiation Science 283,1183-1186[Abstract/Free Full Text]
  52. Kolb-Maurer, A., Gentschev, I., Fries, H. W., Fiedler, F., Brocker, E. B., Kampgen, E., Goebel, W. (2000) Listeria monocytogenes-infected human dendritic cells: uptake and host cell response Infect. Immun. 68,3680-3688[Abstract/Free Full Text]
  53. Giacomini, E., Iona, E., Ferroni, L., Miettinen, M., Fattorini, L., Orefici, G., Julkunen, I., Coccia, E. M. (2001) Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response J. Immunol. 166,7033-7041[Abstract/Free Full Text]
  54. Dixon, G. L., Newton, P. J., Chain, B. M., Katz, D., Andersen, S. R., Wong, S., van der Ley, P., Klein, N., Callard, R. E. (2001) Dendritic cell activation and cytokine production induced by group B Neisseria meningitidis: interleukin-12 production depends on lipopolysaccharide expression in intact bacteria Infect. Immun. 69,4351-4357[Abstract/Free Full Text]
  55. Subauste, C. S., Wessendarp, M. (2000) Human dendritic cells discriminate between viable and killed Toxoplasma gondii tachyzoites: dendritic cell activation after infection with viable parasites results in CD28 and CD40 ligand signaling that controls IL-12-dependent and -independent T cell production of IFN-{gamma} J. Immunol. 165,1498-1505[Abstract/Free Full Text]
  56. Newman, S. L., Holly, A. (2001) Candida albicans is phagocytosed, killed, and processed for antigen presentation by human dendritic cells Infect. Immun. 69,6813-6822[Abstract/Free Full Text]
  57. Kelly, R. M., Chen, J., Yauch, L. E., Levitz, S. M. (2005) Opsonic requirements for dendritic cell-mediated responses to Cryptococcus neoformans Infect. Immun. 73,592-598[Abstract/Free Full Text]
  58. Fortsch, D., Rollinghoff, M., Stenger, S. (2000) IL-10 converts human dendritic cells into macrophage-like cells with increased antibacterial activity against virulent Mycobacterium tuberculosis J. Immunol. 165,978-987[Abstract/Free Full Text]
  59. Kolb-Maurer, A., Kurzai, O., Goebel, W., Frosch, M. (2003) The role of human dendritic cells in meningococcal and listerial meningitis Int. J. Med. Microbiol. 293,241-249[CrossRef][Medline]
  60. Nagl, M., Kacani, L., Mullauer, B., Lemberger, E. M., Stoiber, H., Sprinzl, G. M., Schennach, H., Dierich, M. P. (2002) Phagocytosis and killing of bacteria by professional phagocytes and dendritic cells Clin. Diagn. Lab. Immunol. 9,1165-1168
  61. Van Overtvelt, L., Vanderheyde, N., Verhasselt, V., Ismaili, J., De Vos, L., Goldman, M., Willems, F., Vray, B. (1999) Trypanosoma cruzi infects human dendritic cells and prevents their maturation: inhibition of cytokines, HLA-DR, and costimulatory molecules Infect. Immun. 67,4033-4040[Abstract/Free Full Text]
  62. Huang, Q., Liu, D., Majewski, P., Schulte, L. C., Korn, J. M., Young, R. A., Lander, E. S., Hacohen, N. (2001) The plasticity of dendritic cell responses to pathogens and their components Science 294,870-875[Abstract/Free Full Text]
  63. Gavrilin, M. A., Bouakl, I. J., Knatz, N. L., Duncan, M. D., Hall, M. W., Gunn, J. S., Wewers, M. D. (2006) Internalization and phagosome escape required for Francisella to induce human monocyte IL-1ß processing and release Proc. Natl. Acad. Sci. USA 103,141-146[Abstract/Free Full Text]
  64. Hornef, M. W., Wick, M. J., Rhen, M., Normark, S. (2002) Bacterial strategies for overcoming host innate and adaptive immune responses Nat. Immunol. 3,1033-1040[CrossRef][Medline]
  65. Sundquist, M., Wick, M. J. (2005) TNF-{alpha}-dependent and -independent maturation of dendritic cells and recruited CD11(int)CD11b(+) cells during oral Salmonella infection J. Immunol. 175,3287-3298[Abstract/Free Full Text]
  66. Sundquist, M., Rydstrom, A., Wick, M. J. (2004) Immunity to Salmonella from a dendritic point of view Cell. Microbiol. 6,1-11[CrossRef][Medline]
  67. Buelens, C., De Groote, D., Goldman, M., Willems, F. (1996) Differential effects of interleukin-10 on the production of interleukin-12 and interleukin-8 by human dendritic cells generated from peripheral blood Transplant. Proc. 28,3255-3256[Medline]
  68. Buelens, C., Verhasselt, V., De Groote, D., Thielemans, K., Goldman, M., Willems, F. (1997) Human dendritic cell responses to lipopolysaccharide and CD40 ligation are differentially regulated by interleukin-10 Eur. J. Immunol. 27,1848-1852[Medline]
  69. Buelens, C., Verhasselt, V., De Groote, D., Thielemans, K., Goldman, M., Willems, F. (1997) Interleukin-10 prevents the generation of dendritic cells from human peripheral blood mononuclear cells cultured with interleukin-4 and granulocyte/macrophage-colony-stimulating factor Eur. J. Immunol. 27,756-762[Medline]
  70. McGuirk, P., McCann, C., Mills, K. H. (2002) Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis J. Exp. Med. 195,221-231[Abstract/Free Full Text]
  71. Akbari, O., DeKruyff, R. H., Umetsu, D. T. (2001) Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen Nat. Immunol. 2,725-731[CrossRef][Medline]
  72. Sing, A., Roggenkamp, A., Geiger, A. M., Heesemann, J. (2002) Yersinia enterocolitica evasion of the host innate immune response by V antigen-induced IL-10 production of macrophages is abrogated in IL-10-deficient mice J. Immunol. 168,1315-1321[Abstract/Free Full Text]
  73. Forestal, C. A., Benach, J. L., Carbonara, C., Italo, J. K., Lisinski, T. J., Furie, M. B. (2003) Francisella tularensis selectively induces proinflammatory changes in endothelial cells J. Immunol. 171,2563-2570[Abstract/Free Full Text]
  74. Lai, X. H., Golovliov, I., Sjostedt, A. (2004) Expression of IglC is necessary for intracellular growth and induction of apoptosis in murine macrophages by Francisella tularensis Microb. Pathog. 37,225-230[Medline]
  75. Mariathasan, S., Weiss, D. S., Dixit, V. M., Monack, D. M. (2005) Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis J. Exp. Med. 202,1043-1049[Abstract/Free Full Text]
  76. Santic, M., Molmeret, M., Abu Kwaik, Y. (2005) Modulation of biogenesis of the Francisella tularensis subsp. novicida-containing phagosome in quiescent human macrophages and its maturation into a phagolysosome upon activation by IFN-{gamma} Cell. Microbiol. 7,957-967[CrossRef][Medline]
  77. Santic, M., Molmeret, M., Klose, K. E., Jones, S., Kwaik, Y. A. (2005) The Francisella tularensis pathogenicity island protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm Cell. Microbiol. 7,969-979[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
C. D. Clay, S. Soni, J. S. Gunn, and L. S. Schlesinger
Evasion of Complement-Mediated Lysis and Complement C3 Deposition Are Regulated by Francisella tularensis Lipopolysaccharide O Antigen
J. Immunol., October 15, 2008; 181(8): 5568 - 5578.
[Abstract] [Full Text] [PDF]

Home page
 J Med MicrobiolHome page
P. C. F. Oyston
Francisella tularensis: unravelling the secrets of an intracellular pathogen
J. Med. Microbiol., August 1, 2008; 57(8): 921 - 930.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
A. Ben Nasr and G. R. Klimpel
Subversion of complement activation at the bacterial surface promotes serum resistance and opsonophagocytosis of Francisella tularensis
J. Leukoc. Biol., July 1, 2008; 84(1): 77 - 85.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
B. Tamilselvam and S. Daefler
Francisella Targets Cholesterol-Rich Host Cell Membrane Domains for Entry into Macrophages
J. Immunol., June 15, 2008; 180(12): 8262 - 8271.
[Abstract] [Full Text] [PDF]