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Infection and Immunity, September 2006, p. 5185-5190, Vol. 74, No. 9
0019-9567/06/$08.00+0     doi:10.1128/IAI.00622-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Autocrine Role for Interleukin-8 in Bartonella henselae-Induced Angiogenesis

Amy M. McCord, Sandra I. Resto-Ruiz, and Burt E. Anderson^*

Department of Molecular Medicine, School of Basic Biomedical Science, College of Medicine, University of South Florida, Tampa, Florida 33612

Received 18 April 2006/ Returned for modification 2 May 2006/ Accepted 13 June 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
The gram-negative bacterium Bartonella henselae is capable of causing angiogenic lesions as a result of infection. Previously, it has been shown that B. henselae infection can result in production of the chemokine interleukin-8 (IL-8). In this study, we demonstrated that monocytes, endothelial cells, and hepatocytes produce IL-8 in response to B. henselae infection. We also investigated the role of IL-8 in B. henselae-induced endothelial cell proliferation and capillary tube formation. Both in vitro angiogenesis assays were IL-8 dependent. B. henselae-mediated inhibition of apoptosis, as indicated by gene expression of Bax and Bcl-2, was also shown to be IL-8 dependent in endothelial cells. Furthermore, infection of endothelial cells with B. henselae stimulated upregulation of the IL-8 chemokine receptor CXCR2. Infection of human endothelial cells by B. henselae resulting in IL-8 production likely plays a central role in the ability of this organism to cause angiogenesis during infection.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The genus Bartonella consists of an expanding group of more than 20 species of arthropod-borne, gram-negative, pleomorphic bacilli. Almost one-half of these species have been associated with human disease. Two species, B. henselae and B. quintana, are the pathogenic species described most commonly throughout the world (3). Pathogenic Bartonella species are capable of causing long-lived infections of erythrocytes in their reservoir hosts, as well as diverse diseases in other mammalian hosts that may be infected incidentally, as is the case for cat scratch disease in humans caused by the zoonotic species B. henselae. Three pathogenic Bartonella species, B. bacilliformis, B. quintana, and B. henselae, are able to cause tumor-like angiomatous lesions in humans with disseminated infections (2). B. henselae and B. quintana are causative agents of bacillary angiomatosis (BA), which is characterized by unusual neoplasia of the microvascular tissue of the skin (2).

The ability of Bartonella spp. to cause the angiogenic lesions represents a fascinating aspect of the pathogenesis of these bacteria. B. henselae is a facultative intracellular bacterium which predominantly infects endothelial cells. Other cells, however, have also been implicated as host cells for B. henselae; these cells include epithelial cells, monocytes, macrophages, and dendritic cells (6, 19, 26). While the ability of this bacterium to cause angiogenesis and cell proliferation has been well researched, the details of the underlying mechanisms have not been completely clarified. B. henselae most likely causes angiogenesis through a combination of several mechanisms, including NFB-dependent proinflammatory gene activation (16, 22), direct promotion of endothelial cell proliferation (15), inhibition of endothelial cell apoptosis (9), and upregulation of angiogenic growth factors from peripheral cells (6, 7, 19).

Angiogenesis is a multistep process during which the vessel wall disassembles, the basement membrane is degraded by matrix metalloproteinases (MMPs), endothelial cells migrate and invade the extracellular matrix, endothelial cells proliferate, and a capillary lumen is formed. Interleukin-8 (IL-8) (or CXCL8) augments angiogenesis through enhanced endothelial cell survival, proliferation, and MMP production (11, 12). The IL-8 receptors CXCR1 and CXCR2 are widely expressed on normal and tumor cells (5, 23, 24, 27) and have been observed on endothelial cells (17, 21). These receptors also play a role in proliferation of endothelial cells (10).

It has been observed repeatedly that IL-8 production is enhanced in response to B. henselae infection (19, 22). Additionally, IL-8 production is probably mediated through an NFB-dependent pathway. In this study, we examined the role of IL-8 in B. henselae-induced angiogenesis. Here we provide evidence that IL-8 may mediate inhibition of endothelial cell apoptosis, endothelial cell proliferation, and capillary tube formation in an in vitro B. henselae infection. Furthermore, the bacterium causes upregulation of the IL-8 receptor CXCR2, probably leading to an increase in IL-8-mediated effects.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains. B. henselae strain Houston-1 (ATCC 49882) (18) was grown on chocolate agar prepared with heart infusion agar base (Difco, Detroit, MI) supplemented with 1% bovine hemoglobin (Becton Dickinson, Cockeysville, MD). Bacterial cultures were maintained at 37°C with 5% CO₂ and humidity to saturation.

Cell lines. The immortalized human microvascular endothelial cell line HMEC-1 (1) was cultured in MCDB131 cell culture medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal calf serum (HyClone Laboratories, Logan, UT), 10 ng/ml epidermal growth factor, 1.461 g/liter L-glutamine, 1 µg/ml hydrocortisone, 50 µg/ml penicillin-streptomycin, 2.5 µg/ml amphotericin B (Sigma-Aldrich, St. Louis, MO), 2 mg/ml sodium bicarbonate, and 10 mM HEPES (Mediatech, Herndon, VA). Human THP-1 monocytes (25) were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal calf serum, 5 µM 2-mercaptoethanol (Sigma-Aldrich), 10 µg/ml vancomycin (Sigma-Aldrich), and 1 µg/ml amphotericin B. THP-1 monocytes were differentiated as described previously (28) with 10^–6 M vitamin D₃ (Sigma-Aldrich) overnight, after which the medium was changed. Human umbilical vein endothelial cells (HUVEC) were obtained from Clonetics Corporation (San Diego, CA) and were cultured in EGM (Clonetics). Human hepatocarcinoma cells (HepG2 cells) were obtained from the American Type Culture Collection (Manassas, VA) and were cultured in minimal essential medium containing 10% fetal calf serum, 1.5 g/liter sodium bicarbonate, 0.1 mM nonessential amino acids, and 1.0 µM sodium pyruvate. The cells were maintained at 37°C with 5% CO₂ and humidity to saturation. HUVEC were used in experiments at passages 4 to 7.

Infection. To generate supernatants for analysis of IL-8 secretion, HUVEC, HepG2 cells, HMEC-1 cells, or THP-1 monocytes were placed into 24-well tissue culture plates (Costar, Cambridge, Mass.) at 90% confluence. THP-1 monocytes were differentiated by overnight incubation with 1 µM vitamin D₃ (Sigma-Aldrich). Nonadherent cells were removed by washing. Cells were infected with the Houston-1 strain of B. henselae as described previously using the appropriate cell culture medium with no antibiotics (19) for 30 min, followed by washes and gentamicin treatment (50 µg/ml) for 1 h. For the downstream analyses, including real-time PCR and capillary tube formation analysis, infection and incubation after infection were carried out under serum-free conditions. Cells were infected at the multiplicities of infection (MOIs) indicated below.

ELISA. To determine IL-8 levels in supernatants from B. henselae-infected cells, the DuoSet enzyme-linked immunosorbent assay (ELISA) development system (R&D Systems, Minneapolis, Minn.) for human IL-8 was used according to the manufacturer's directions. The 3,3',5,5'-tetramethylbenzidine liquid substrate system (Sigma-Aldrich) was added and left for 20 min. The horseradish peroxidase reaction was stopped with 2 N sulfuric acid. ELISA plates were analyzed using a µQuant plate reader (Bio-Tek, Winooski, VT) at 450 nm.

HUVEC proliferation assay. HUVEC were seeded in 96-well plates at a level of 1 x 10³ cells per well in medium without antibiotics and allowed to adapt overnight. The cells were infected the following day with Houston-1 at an MOI of 50 or were incubated with recombinant human IL-8 (100 ng/ml; R&D Systems). Mouse anti-human IL-8 (10 µg/ml) or an isotype control (10 µg/ml mouse immunoglobulin G1 [IgG1]) was added to the medium during infection. After 72 h, cells were fixed and examined with an inverted microscope, and digital pictures were obtained with a Kodak DC290 digital camera. The cells were examined for qualitative differences, the cells in five high-power fields per well were counted, and the results were averaged.

RNA extraction and reverse transcription. Cells were infected at an MOI of 100 with Houston-1 or were incubated with 100 ng/ml recombinant IL-8 (rIL-8); 10 µg/ml anti-IL-8 or an isotype control was added at the time of infection. After 24 h, total RNA was extracted from HUVEC using the TRIzol reagent (Sigma-Aldrich) according to manufacturer's protocol. Turbo DNA-free (Ambion, Austin, TX) was used to remove the remaining DNA according to the manufacturer's protocol. Two micrograms of total RNA was transcribed with avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) and used for real-time PCR or semiquantitative reverse transcription-PCR (RT-PCR).

Real-time PCR. The primers used for real-time PCR were as follows: for ß-actin, forward primer 5'-ACCAACTGGGACGACATGGAGAAA-3' and reverse primer 5'-TAGCACAGCCTGGATAGCAACGTA-3'; for Bax, forward primer 5'-TCTACTTTGCCAGCAAACTGGTGC-3' and reverse primer 5'-TGTCCAGCCCATGATGGTTCTGAT-3'; and for Bcl-2, forward primer 5'-ATTTCCTGCATCTCATGCCAAGGG-3' and reverse primer 5'-TGTGCTTTGCATTCTTGGACGAGG-3'. ß-Actin was used as the housekeeping gene control. Real-time PCR was performed with a Bio-Rad iQ iCycler detection system (Bio-Rad Laboratories, Ltd.) with iQSYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA). The reactions were performed in 25-µl (total volume) mixtures containing primers at a concentration of 400 nM. The reaction conditions consisted of 10 min at 95°C and then 45 cycles of 15 s at 95°C, 15 s at 58°C, and 30 s at 72°C. Melting curve analysis was used to determine PCR specificity. The melting curve analysis was performed by using 80 10-s cycles, with the first cycle at 55°C and the temperature increasing 0.5°C in each succeeding cycle. All reactions were carried out at least in duplicate for each sample. The standard curve method was used to determine the amount of each transcript. The relative expression of Bcl-2 or Bax was determined by dividing the amount (ng) of Bax or Bcl-2 by the amount of ß-actin in each sample. Relative induction was determined by normalizing the relative expression in the uninfected control samples to 1. All experiments included no-template controls and untranscribed (no-RT) RNA controls.

Semiquantitative RT-PCR. RT-PCR was performed with HUVEC 24 h after infection. Total RNA was extracted as described above. cDNA preparation and subsequent PCR amplification were carried out with a One-Step RT-PCR kit (QIAGEN, Inc., Valencia, CA) in the presence of gene-specific primers and 2 µg total RNA. The PCR conditions were 1 min at 95°C, 1 min at 58°C, and 1 min at 72°C for 35 cycles. The primers used for RT-PCR were as follows: for ß-actin, forward primer 5'-AGAAAATCTGGCACCACACC-3') and reverse primer 5'-CCATCTCTTGCTCGAAGTCC-3'; for CXCR2, forward primer 5'-ATTCTGGGCATCCTTCACAG-3'; and for CXCR2, reverse primer 5'-TGCACTTAGGCAGGAGGTCT-3'. PCR products were analyzed by electrophoresis on 2% agarose gels and were visualized by ethidium bromide staining. RT-PCR data were analyzed by scanning densitometry of gel bands with the Kodak 1D image analysis software and by normalization to ß-actin signals obtained for the same times. The RT-PCRs included a no-template control and a no-reverse transcriptase control to exclude DNA or RNA contamination.

In vitro capillary tube formation assay. A 96-well plate was coated with growth factor-reduced (GFR) Matrigel (BD Biosciences, Mountain View, CA). The Matrigel contained no antibodies, a control isotype, or anti-IL-8 (10 µg/ml). The Matrigel was solidified at 37°C for 1 h, after which 10⁴ uninfected or B. henselae-infected (MOI, 100) HUVEC were added to each well. rIL-8 (100 ng/ml) was added to some wells containing uninfected cells at this time. After 18 h, the plates were examined for qualitative differences in capillary tube formation, and photographs were taken with a Kodak DC290 digital camera.

Statistics. Significance was determined by using a Student's t test with two-tailed distribution. P values less than 0.05 were considered significant. The data were expressed as means ± standard deviations.

Top Abstract Introduction Materials and Methods Results Discussion References

 
IL-8 production by a variety of cell types infected with B. henselae. It has been widely reported that IL-8 production by endothelial cells and epithelial cells is enhanced by an NFB-dependent pathway in the presence of B. henselae (4, 22). We tested a variety of cell types for production of IL-8 in response to B. henselae at an MOI of 100. Two types of endothelial cells, HMEC-1 cells and HUVEC (see Materials and Methods), exhibited upregulated IL-8 production in response to B. henselae infection at 24 h after infection (Fig. 1A). Other cells which may be important for B. henselae pathogenesis (hepatocytes and monocyte-derived macrophages) were also examined for the ability to upregulate IL-8 production in response to B. henselae. These cells also exhibited markedly upregulated IL-8 production during infection (Fig. 1B and C). We have previously reported (19) that B. henselae-infected THP-1 monocytes do not exhibit enhanced IL-8 production at an MOI of 500, which conflicts with the results presented here. However, in this study we used vitamin D₃ for THP-1 cell differentiation (see Materials and Methods) instead of phorbol myristate acetate, which lowers background IL-8 production; thus, the differences between uninfected and infected THP-1 cells were more clearly distinguishable.

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FIG. 1. B. henselae-induced IL-8 production, as determined by ELISA. (A) B. henselae-induced IL-8 production by HUVEC and HMEC-1 cells. (B) B. henselae-induced IL-8 production by hepatocytes (HepG2 cells). (C) B. henselae-induced IL-8 production by THP-1 monocyte-derived macrophages. Un, uninfected; Bh, B. henselae infected.

Furthermore, we examined HUVEC for expression of the IL-8 receptors CXCR1 and CXCR2 in the presence of B. henselae. While CXCR1 RNA levels were not significantly different for uninfected and infected HUVEC (data not shown), CXCR2 levels were around four times higher in B. henselae-infected cells than in uninfected cells (Fig. 2).

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FIG. 2. CXCR2 expression in HUVEC. (A) RNA was extracted from uninfected and B. henselae-infected HUVEC and subjected to semiquantitative RT-PCR. (B) Scanning densitometry was used to determine the relative intensities of CXCR2 expression when expression was normalized to ß-actin housekeeping gene expression. Relative CXCR2 induction was measured by determining the ratio of CXCR2 to ß-actin when the ratio in uninfected cells was normalized to 1. UN, uninfected HUVEC; BH, B. henselae-infected HUVEC.

Effect of blocking IL-8 on B. henselae-induced endothelial cell proliferation. B. henselae causes more endothelial cell proliferation at MOIs that are less than 50 than at higher MOIs (22). This is most likely due to a cytotoxic effect from a factor encoded by the virB operon at higher MOIs. However, other aspects of B. henselae-induced angiogenesis, such as inhibition of apoptosis, capillary tube formation, and NFB-dependent proinflammatory activation, correlate positively with the number of bacteria (8, 9, 20). Since it has been reported that IL-8 directly mediates endothelial cell survival and proliferation (10), we examined the role of IL-8 in B. henselae-induced HUVEC proliferation (Fig. 3). Cells were incubated with B. henselae (MOI, 50) or rIL-8 (100 ng/ml). Cells were also treated with anti-human IL-8 or control IgG1 (10 µg/ml). After 3 days, pictures of the wells were taken (Fig. 3A), and cells were counted (Fig. 3B). Both B. henselae and rIL-8 induced proliferation compared to the results obtained with unstimulated cells (P < 0.008). The presence of an IL-8 antibody quenched the proliferative effect of B. henselae and rIL-8, while the presence of an isotype control did not. These data indicate that IL-8 has a putative role in B. henselae-induced proliferation.

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FIG. 3. Effect of IL-8 on HUVEC proliferation in response to B. henselae. HUVEC were infected with B. henselae or incubated with rIL-8 either in the presence or in the absence of a neutralizing anti-IL-8 antibody or an isotype control for 3 days. (A) Digital images of typical phase-contrast microscopic fields. (B) Cell proliferation expressed as a graph of average cell number per high-power field. UN, uninfected; BH, B. henselae infected.

Role of IL-8 in B. henselae-induced endothelial cell survival. We examined HUVEC infected with B. henselae at an MOI of 100 for expression of the Bcl-2 family members Bcl-2 (antiapoptotic) and Bax (apoptotic). IL-8 induces increased Bcl-2 expression and decreased Bax expression (12). While it has been reported that B. henselae inhibits apoptosis of HUVEC though inhibition of caspases (9), the Bcl-2 and Bax levels in uninfected and B. henselae-infected HUVEC have not been compared previously. We examined Bax and Bcl-2 levels in HUVEC by real-time PCR. We found that in B. henselae-infected HUVEC there was almost undetectable Bax expression and about fourfold-enhanced Bcl-2 expression (Fig. 4A) compared with uninfected controls, as normalized to a ß-actin housekeeping gene. The increased Bcl-2/Bax ratio probably biases the cell into an antiapoptotic state. We examined the role of IL-8 in B. henselae-enhanced HUVEC survival. When anti-IL-8 was added to HUVEC in the presence of B. henselae, the cells responded with increased Bax levels and decreased Bcl-2 levels (Fig. 4B and C). Bax levels were increased about fivefold in the presence of anti-IL-8 but not in the presence of control IgG1 (Fig. 4B). Conversely, the Bcl-2 levels induced by B. henselae infection decreased sixfold in the presence of an IL-8 neutralizing antibody (Fig. 4C). These results revealed a possible autocrine role for IL-8 in B. henselae-stimulated endothelial cell survival.

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FIG. 4. Effect of anti-IL-8 on inhibition of HUVEC apoptosis induced by B. henselae. (A) B. henselae caused enhanced Bcl-2 expression and decreased Bax expression in HUVEC. The results are expressed in relative expression units (ratio of the amount of Bcl-2 or Bax transcripts to the amount of ß-actin transcripts). (B) Bax expression that was reduced by B. henselae increased in the presence of a neutralizing antibody to IL-8. (C) Bcl-2 expression that was increased by B. henselae decreased in the presence of anti-IL-8. UN, uninfected; BH, B. henselae infected.

Role of IL-8 in B. henselae-induced capillary tube formation. Previous in vitro angiogenesis assays revealed a proangiogenic response of HUVEC to B. henselae infection (8). HUVEC infected with B. henselae seeded on a GFR Matrigel exhibited advanced capillary tube formation compared to the formation with uninfected HUVEC (Fig. 5). HUVEC incubated with rIL-8 also exhibited enhanced capillary tube formation. Furthermore, when anti-IL-8 was present in the Matrigel, the capillary tube formation was visibly diminished (Fig. 5). The presence of an isotype control, however, had no such effect on tube formation, indicating that IL-8 has another autocrine role during B. henselae infection.

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FIG. 5. Effect of anti-IL-8 on B. henselae-induced capillary formation in a GFR Matrigel. UN, uninfected HUVEC; BH, B. henselae-infected HUVEC; rIL-8, HUVEC treated with 100 ng/ml rIL-8. Preparations were treated with no antibody, with an isotype control (10 µg/ml IgG1), or anti-IL-8 (10 µg/ml).

Top Abstract Introduction Materials and Methods Results Discussion References

 
The human pathogens B. henselae, B. quintana, and B. bacilliformis stimulate vasoproliferation. B. henselae and B. quintana are causative agents of BA, which manifests as lesions similar to those seen during Kaposi's sarcoma, particularly in immunocompromised individuals (2). Bartonella spp. have a unique ability to induce angiogenesis. Angiogenesis is a complex process involving several key steps. These steps include (i) inhibition of endothelial cell apoptosis, (ii) endothelial cell proliferation, (iii) breakdown of the extracellular matrix by MMPs, and (iv) capillary tube formation. IL-8 can promote each of these steps. Since B. henselae upregulates IL-8 production by endothelial cells (3, 19), we investigated a putative autocrine role for IL-8 in B. henselae-induced angiogenesis.

There are conflicting reports concerning whether endothelial cells actively proliferate or whether they simply exhibit enhanced survival in the presence of B. henselae (9, 22). Endothelial cell proliferation in BA most likely results from a combination of inhibition of apoptosis and mitogenic stimulation. In addition, endothelial cell proliferation and angiogenesis probably result from the effects of the bacterium on both the endothelial cells and peripheral cells, such as epithelial cells and macrophages (7, 19). While in this study we focused on the autocrine role of IL-8, a paracrine role should not be overlooked as many types of cells produce IL-8 after infection with B. henselae (Fig. 1A and B). Furthermore, this bacterium causes upregulation of expression of one of the IL-8 receptors, CXCR2 (Fig. 2). This may represent a mechanism by which the effects of IL-8 on the endothelial cells are enhanced because elevated levels of the receptor are present. The fact that IL-8 production is upregulated from endothelial and other cells, combined with the finding that CXCR2 expression is also enhanced during endothelial cell infection, indicates that the level of IL-8 signaling is extremely elevated in the endothelial cells during B. henselae infection.

The balance between Bax and Bcl-2 is important for endothelial cell survival or apoptosis. IL-8 induces an increase in Bcl-2 expression and a decrease in Bax expression, which most likely favors survival over apoptosis in endothelial cells (11). It has been shown that B. quintana can modulate the cell-programmed death of endothelial cells by increasing Bcl-2 expression (14). In this study, we examined expression of two Bcl-2 family members, Bcl-2 (antiapoptotic) and Bax (apoptotic), in HUVEC. In the presence of B. henselae, the expression of Bax is decreased and the expression of Bcl-2 is increased. The increases and decreases are quite dramatic alone; however, when the ratio of Bcl-2 to Bax is considered, the comparison is even more dramatic. This is the first report that B. henselae mediates Bax and Bcl-2 expression in endothelial cells. Liberto et al. (13) showed that Bcl-2 protein levels increased in endothelial cells and that mitotic activity was also induced by B. quintana. In our study, we found increased levels of Bcl-2 and decreased levels of Bax in B. henselae-infected primary HUVEC. In addition, the data revealed a possible role for IL-8 in the prevention of apoptosis.

The data also indicate that IL-8 may have a role in endothelial cell proliferation and capillary tube formation. Both of these aspects of angiogenesis were decreased in the presence of an IL-8-neutralizing antibody. However, other mechanisms are probably also involved in proliferation, including the activity of growth factors such as vascular endothelial growth factor from other cells. It has been shown that while B. henselae causes endothelial cells to proliferate, the proliferation is inhibited at higher MOIs as a result of a cytotoxic effect from the virB-encoded type four secretion system (22). Our proliferation results agreed with this phenomenon; at MOIs greater than 50, endothelial cell proliferation decreased. However, the other aspects of angiogenesis (capillary tube formation, enhanced endothelial cell survival, and IL-8 production) were greater at an MOI of 100 than at an MOI of 50. These results suggest that the cytotoxic effect of virB does not have an effect on expression of Bcl-2 family members or capillary tube formation. Thus, the proangiogenic effect of B. henselae may consist of a complicated fusion of many host cell and bacterial factors. Nevertheless, IL-8 seems to play an autocrine role and possibly a paracrine role in B. henselae-induced angiogenesis, representing a mechanism by which the bacterium causes upregulation of IL-8, thereby increasing its survival by expanding its host cell reservoir. Assessment of the contribution of each of these in vitro components to the overall angiogenesis mediated by B. henselae is not complete and requires development of a practical animal model.

 
This research was supported by Public Health Service grant R01-AI38178 from the National Institutes of Health.

We thank Thomas Lawley of Emory University and The Biological Products Branch, Centers for Disease Control and Prevention, for providing the HMEC-1 cell line used in this study.

 
* Corresponding author. Mailing address: Department of Molecular Medicine, School of Basic Biomedical Science, College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd., MDC10, Tampa, FL 33612. Phone: (813) 974-2608. Fax: (813) 974-4151. E-mail: banderso{at}hsc.usf.edu.

Editor: J. T. Barbieri

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, September 2006, p. 5185-5190, Vol. 74, No. 9
0019-9567/06/$08.00+0     doi:10.1128/IAI.00622-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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