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. Author manuscript; available in PMC: 2011 Feb 9.
Published in final edited form as: Cell Microbiol. 2008 Aug 26;10(12):2568–2578. doi: 10.1111/j.1462-5822.2008.01230.x

G-CSF induction early in uropathogenic Escherichia coli infection of the urinary tract modulates host immunity

Molly A Ingersoll 1,†, Kimberly A Kline 1, Hailyn V Nielsen 1, Scott J Hultgren 1,*
PMCID: PMC3036167  NIHMSID: NIHMS249173  PMID: 18754853

Summary

Uropathogenic Escherichia coli (UPEC), the causative agent of approximately 85% of urinary tract infections (UTI), is a major health concern primarily affecting women. During infection, neutrophils infiltrate the bladder, but the mechanism of recruitment is not well understood. Here, we investigated the role of UPEC-induced cytokine production in neutrophil recruitment and UTI progression. We first examined the kinetics of cytokine expression during UPEC infection of the bladder, and their contribution to neutrophil recruitment. We found that UPEC infection induces expression of several pro-inflammatory cytokines including granulocyte colony-stimulating factor (G-CSF, CSF-3), not previously known to be involved in the host response to UTI. G-CSF induces neutrophil emigration from the bone marrow; these cells are thought to be critical for bacterial clearance during infection. Upon neutralization of G-CSF during UPEC infection, we found fewer circulating neutrophils, decreased neutrophil infiltration into the bladder and, paradoxically, a decreased bacterial burden in the bladder. However, depletion of G-CSF resulted in a corresponding increase in macrophage-activating cytokines, such as monocyte chemotactic protein-1 (MCP-1, CCL-2) and Il-1β, which may be key in host response to UPEC infection, potentially resolving the paradoxical decreased bacterial burden. Thus, G-CSF acts in a previously unrecognized role to modulate the host inflammatory response during UPEC infection.

Introduction

Cystitis, resulting from infection with uropathogenic Escherichia coli (UPEC), is a severely debilitating disease characterized by frequent and urgent voiding, dysuria, nausea and fever (Bower et al., 2005) lasting ~6 days (Foxman, 2002; Hooton et al., 2004). UPEC is a clinically important Gram-negative pathogen due to the high infection rate (Griebling, 2004), frequent recurrence of infection (Ikaheimo et al., 1996; Hooton and Stamm, 1997) and dissemination of antibiotic resistance (Johnson et al., 2002; France et al., 2005). Additionally, UPEC is genetically tractable and establishes a disease in the murine bladder similar to human cystitis (Garofalo et al., 2007; Rosen et al., 2007), making it an ideal model organism with which to study the host innate immune response to a Gram-negative pathogen.

During the course of infection, Gram-negative bacteria, such as UPEC, frequently breach protective host barriers, such as the skin or mucosa (Mulvey, 2002; Bower et al., 2005; Parsot, 2005). The host must respond appropriately to prevent unchecked bacterial growth by recognition of invading pathogens, activation of tissue resident macrophages and dendritic cells, and release of pro-inflammatory cytokines. While UPEC pathogenic mechanisms have been well described (Andersson et al., 1991; Svanborg and Godaly, 1997; Mulvey et al., 1998; 2001; Mulvey, 2002; Anderson et al., 2003; 2004; Justice et al., 2004; Bergsten et al., 2007; Wright et al., 2007), a comprehensive understanding of the host response to UPEC infection is lacking. UPEC infection is characterized by acute inflammation including neutrophil infiltration (Ko et al., 1993; Agace et al., 1995; Haraoka et al., 1999; Schilling et al., 2001), which is currently thought to play a key role in bacterial containment and eradication. Treatment with an α-Gr-1 antibody, to deplete neutrophils, resulted in a higher bacterial burden in the host compared with control IgG-treated animals (Haraoka et al., 1999). Urothelium-secreted IL-8 is important for neutrophil recruitment to the bladder during cystitis (Hedges et al., 1994; Agace et al., 1995; Sampson, 2000) and is present in human urine from patients experiencing acute urinary tract infections (UTI) (Ko et al., 1993). MIP-2 [macrophage inflammatory protein-2; a murine functional orthologue of IL-8 (Hang et al., 1999)] can be detected in the urine, bladders and kidneys of infected mice (Hang et al., 1999; Mittal et al., 2004). Additional signals mediating the pro-inflammatory response to UPEC are less well characterized. Schilling et al. (2003) demonstrated diminished neutrophil recruitment during UPEC infection in chimeric mice with TLR4 signalling-competent urothelium and TLR4-defective haematopoietic cells, providing evidence that NF-κB-regulated cytokines (Dziarski and Gupta, 2000) acting downstream of TLR4 signalling are important for neutrophil recruitment. Whereas IL-8 is a chemotactic cytokine that induces mature neutrophils to leave the circulation and enter infected tissue (Bozic et al., 1995; Menten et al., 2002), another cytokine potentially involved in neutrophil recruitment, granulocyte colony-stimulating factor (G-CSF, CSF-3), acts on neutrophils at the level of the bone marrow. G-CSF induces immature progenitor cells to exit the bone marrow, enter the bloodstream and mature into neutrophils (Metcalf and Nicola, 1983; Barreda et al., 2004). Administration of recombinant G-CSF increases the number of circulating neutrophils (Marshall, 2005) indicating that G-CSF plays an important role in maintaining appropriate neutrophil levels in the blood.

In the present study, we sought to determine the roles of G-CSF and additional cytokine signals in the innate host response to UPEC infection in the murine urinary tract. We found that a diverse panel of cytokines is secreted upon infection, including G-CSF. We found that G-CSF depletion resulted in a significant decrease in bladder-infiltrating neutrophils during infection. Yet, bacterial clearance in G-CSF-neutralized animals was significantly improved compared with control-treated animals. As an explanation for this paradoxical result, we observed elevated levels of several cytokines able to activate macrophages accompanying G-CSF neutralization, including interleukin-1β and MCP-1. These data suggest that, in addition to neutrophils, another cell type may be important for controlling UPEC infection. These findings indicate that the host response to UPEC infection is more complex than previously appreciated and may provide some advantage to the pathogen.

Results

Kinetics of UPEC infection in the bladder

The in vivo infection kinetics, from 1 h to 2 weeks, of a wild-type UPEC clinical isolate from a human cystitis infection, were established by infecting C57BL/6 female mice by transurethral instillation of bacteria using an inoculum of 107 colony-forming units (cfu) per bladder. Importantly, this inoculum was determined empirically to be the ID100 (infectious dose 100) or the lowest inoculum that still results in a reproducible infection in all animals. Over time, UPEC amplified nearly three orders of magnitude in the bladder, peaking at 24 h post infection (PI) (Fig. 1A). After 24 h PI, cfu decreased over time to approximately 103 cfu per bladder at 2 weeks PI (Fig. 1A) illustrating the acute nature of UPEC infection. Furthermore, the kinetic data suggest that the host rapidly and efficiently clears UPEC in the C57BL/6 murine model.

Fig. 1.

Fig. 1

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UPEC robustly colonize the bladder and initiate a pro-inflammatory cytokine cascade. A total of 107 cfu of UPEC strain UTI89 (described in Experimental procedures) were inoculated trans-urethrally into C57BL/6 female mice.

A. Bladders were homogenized at the indicated time points, serially diluted, and cfu enumerated. Each point represents one mouse, bars are the geometric mean, and the graph is a composite of six experiments, 5–10 mice each (B and C). Bladders were homogenized at the indicated time points, centrifuged to remove cellular debris, and cytokine expression was measured.

B and C. (B) TNF-α, IL-12 p40 and RANTES expression, (C) MIP-1β, IL-1β, IL-6, IL-17, G-CSF, KC and MCP-1 expression. Data are representative of four experiments, 5–10 mice each, assessed individually.

Characterization of UPEC-mediated pro-inflammatory cytokine cascade

Rapid neutrophil influx, a characteristic of UTI (Ko et al., 1993), correlates with increasing bladder bacterial load (Haraoka et al., 1999; Schilling et al., 2001; Justice et al., 2004). In an effort to determine the signals that initiate neutrophil infiltration, we investigated the bladder’s global cytokine response to UPEC infection over time.

The expression levels of 23 cytokines were simultaneously evaluated in whole mouse bladder in response to UPEC infection, from 1 h to 2 weeks PI, using a mouse pro-inflammatory cytokine bead array kit. Mice were infected with 107 cfu of UPEC and their bladders homogenized at pre-determined time points. Whole bladder homogenates contained cytokines produced by cells comprising the bladder, including urothelial cells, fibroblasts, resident macrophages and dendritic cells, as well as cells infiltrating the bladder in response to infection, such as neutrophils and blood monocytes. Cytokine responses likely include those directly initiated by the bacteria, such as upon invasion of the urothelium, and by host cell–cell communication, and cannot be differentiated by this assay. Of the 23 cytokines profiled, TNF-α (tumour necrosis factor alpha), MIP-1β (macrophage inflammatory protein-1-beta, CCL4), IL-1β, IL-6, IL-17, G-CSF, KC (keratinocyte-derived cytokine), MCP-1, IL-12 p40 and RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted, CCL5) were each expressed at levels above that of PBS mock-infected animals for at least one time point over a 2-week infection in two different experiments (data not shown). Conversely, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12 p70, IL-13, eotaxin (CCL11), GM-CSF (granulocyte monocyte colony-stimulating factor, CD116), IFN-γ (interferon gamma) and MIP-1α (macrophage inflammatory protein-1-alpha, CCL3) were not expressed at levels above uninfected or mock-infected levels in the bladders of C57Bl/6 mice in these initial experiments. Using these data, we designed a custom 12-analyte assay, described in Experimental procedures, for assessing cytokine expression levels in subsequent experiments (Fig. 1B and C). Over the course of infection, we observed that TNF-α (Fig. 1B) showed a unique expression profile as compared with the other cytokines, peaking at 1000-fold over mock-infected values at 1 h PI and rapidly decreasing to no more than 10-fold over mock-infected values for the remainder of infection. RANTES and IL-12 p40 (Fig. 1B) were increased 8- to 10-fold over mock-infected values during infection, each cytokine maintaining a consistent level of expression with little variation (as compared with Fig. 1C) from 6 h PI (RANTES) or 24 h PI (IL-12 p40) to 2 weeks PI. Finally, MIP-1β, IL-1β, IL-6, IL-17, G-CSF, KC and MCP-1 had expression levels that substantially increased over mock-infected values, peaking at 24 h PI, and then decreased over time to near mock-infected levels at 2 weeks PI (Fig. 1C).

Neutralization of G-CSF decreases neutrophil infiltration

The cytokines induced during murine UTI include those important for neutrophil activation and chemotaxis (MIP-1β, KC, G-CSF), as well as those that can be secreted from neutrophils (although importantly, neutrophils are not the exclusive source for these cytokines) (TNF-α, MIP-1β, IL-1β, KC, MCP-1). Thus, we hypothesized that neutralization of cytokines important for neutrophil recruitment or activation would adversely affect the host’s ability to control UPEC infection. Therefore, we neutralized G-CSF in an effort to reduce the pool of neutrophils available (Metcalf and Nicola, 1983; Barreda et al., 2004) to respond to UPEC infection.

To assess the impact of G-CSF neutralization, we injected 50 µg mouse−1 of either control IgG or neutralizing G-CSF antibody intraperitoneally at the time of infection. Neutrophils infiltrate tissue rapidly upon infection (Haraoka et al., 1999; Mulvey et al., 2000; Justice et al., 2004). Therefore, we focused on early time points. Mice were sacrificed at either 6 or 48 h PI and G-CSF neutralization was quantified by ELISA on whole bladder homogenates. G-CSF levels in antibody-neutralized bladder tissue were significantly reduced (P = 0.0079) to approximately 33% at 6 h PI and approximately 50% at 48 h PI of G-CSF levels of control IgG-treated animals (Fig. 2A). G-CSF was not completely neutralized in treated animals, which may reflect an inability of the neutralizing antibody to wholly penetrate bladder tissue. Immunostaining of UPEC-infected bladder revealed G-CSF, expressed only during infection, was present in the lumen of the bladder (Fig. 2B; red staining) and that it colocalized with CD45+ (pan leukocyte marker) cells (Fig. 2B; green staining). Consistent with previous studies (Hang et al., 1999; Schilling et al., 2003) and indicative of the specific host response to UPEC infection, we only saw dense CD45+ populations in focal areas of the bladder suggestive of cellular infiltration (Fig. 2B).

Fig. 2.

Fig. 2

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G-CSF neutralization in the bladder. Female C57BL/6 mice were treated with either control IgG or α-G-CSF concurrent to infection with 107 cfu of UPEC strain UTI89.

A. Bladders were homogenized at 6 or 48 h PI, centrifuged to remove debris and G-CSF levels assessed by ELISA.

B. A representative example of G-CSF expression (red) in the lumen (arrow) of a UPEC-infected bladder at 6 h PI. CD45 (green) delineates leukocytes in and lining the lumen of the bladder, while Hoescht (blue) stains all nuclei.

Data are representative of three experiments, 5–10 mice each.

To determine if neutralization of G-CSF resulted in a decrease in the number of circulating neutrophils available to respond to infection, we assessed whole blood samples from UPEC-infected mice treated with either control IgG or neutralizing G-CSF antibody. At 6 and 48 h PI, using flow cytometric analysis, we counted Gr-1+/CD115− cells (neutrophils) and Gr1+/CD115+ cells (monocytes) (Fig. 3A). Gr-1 is present on neutrophils and monocytes (Geissmann et al., 2003; Daley et al., 2008), and CD115 (M-CSF receptor, CSF1R) is present only on monocytes and macrophages (Byrne et al., 1981; Sunderkotter et al., 2004; Tacke et al., 2006). We found that at 6 and 48 h PI there were significantly (6 h: P = 0.0172, 48 h: P = 0.0317) fewer circulating neutrophils (Gr-1+/CD115−) in mice receiving G-CSF neutralizing antibodies as compared with mice receiving control IgG (Fig. 3B). We observed no significant differences in the number of circulating monocytes (CD115+ cells) between the two treatment groups at either time point (Fig. 3C).

Fig. 3.

Fig. 3

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G-CSF neutralization reduces neutrophil infiltration. Female C57BL/6 mice were treated with either control IgG or α-G-CSF concurrent to infection with 107 cfu of UPEC strain UTI89.

A–C. (A) Representative flow plot demonstrating the gating strategy used to identify and quantify circulating (B) neutrophils and (C) monocytes.

D–F. Additionally, bladders were dissociated for flow cytometry (D) shown here as a representative plot to illustrate quadrant definition, and infiltrating (E) neutrophil and (F) macrophage levels assessed.

PMN = neutrophils in oval gate and monos = monocytes in rectangle in (A). Black bars, IgG control-treated; white bars, α-G-CSF-treated. Graphs are composite of three experiments, 5–10 mice each, assessed individually.

We hypothesized that as neutralization of G-CSF limited the number of neutrophils in circulation, there would be a corresponding decrease in tissue-associated neutrophils responding to UPEC infection in the α-G-CSFtreated mice. To quantitatively assess cellular infiltration we used flow cytometry to evaluate the entire bladder. We disassociated whole bladders from control IgG- or neutralizing G-CSF antibody-treated UPEC-infected mice at 6 and 48 h PI and stained single-cell suspensions with α-Gr1 and α-F4/80 antibodies. Similar to CD115, F4/80 is present only on monocytes and macrophages (Austyn and Gordon, 1981; Geissmann et al., 2003). Using flow cytometric analysis, we counted Gr1+/F4/80− cells (neutrophils) and F4/80+ cells (monocytes) (Fig. 3A) and found that at 6 h PI, correlating with circulating neutrophil numbers, there were significantly fewer neutrophils (P = 0.026) in the bladders of mice treated with G-CSF-neutralizing antibodies as compared with mice treated with control IgG (Fig. 3E). However, there was no difference in the number of infiltrating neutrophils at 48 h PI between the two treatment groups. Additionally, there was no difference in the numbers of tissue-associated macrophages in control-treated or G-CSF-neutralized bladder tissue (Fig. 3F).

G-CSF neutralization results in lower bacterial load

G-CSF neutralization resulted in a decrease in both circulating and tissue-associated neutrophils during infection. Thus, we investigated the consequence of altering the neutrophil population on bacterial clearance. We assessed bacterial burden in the bladders of UPEC-infected mice treated with either control IgG or neutralizing α-G-CSF at 6 and 48 h PI. Surprisingly, bacterial burden was comparable in both treatment groups at 6 h PI and significantly decreased (P = 0.003) at 48 h PI in mice receiving neutralizing G-CSF antibody as compared with control IgG-treated mice (Fig. 4). This result was unexpected because neutrophils, thought to be the major mediator of bacterial clearance, were reduced in number in the blood and bladder of G-CSF-neutralized mice at 6 and 48 h PI and 6 h PI respectively (Fig. 3B and E).

Fig. 4.

Fig. 4

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G-CSF neutralization reduces bacterial burden. Female C57BL/6 mice were treated with either control IgG or α-G-CSF neutralizing antibody concurrent to infection with 107 cfu of UPEC strain UTI89. Bladders were homogenized at 6 or 48 h PI, serially diluted, and cfu enumerated. Each point represents one mouse; black points, IgG control-treated; white points, α-G-CSF-treated; bars are geometric mean. Graph is a composite of three experiments, 5–10 mice each.

MCP-1, IL-1β and additional cytokines are upregulated in G-CSF-neutralized mice

G-CSF neutralization during UPEC infection resulted in reduction of circulating neutrophils, neutrophil tissue infiltration and bacterial load in the bladder. We assessed the cytokine response after neutralization, hypothesizing that G-CSF neutralization may affect the expression levels of other cytokines present during cystitis. UPEC-infected mice, injected with either control IgG or neutralizing G-CSF antibodies, were sacrificed at 6 or 48 h PI and their bladders homogenized for cytokine analysis. While the magnitude of the overall fold changes was reduced as compared with Fig. 1, which is due to plate-to-plate variation in the assay, we observed that TNF-α, MIP-1β, IL-1β, IL-6, KC and MCP-1 expression levels were significantly (P = 0.0079) increased at 6 h PI in the animals treated with neutralizing G-CSF antibody as compared with control IgG-treated animals (Fig. 5, compare white bars with black bars). In this same set of cytokines, expression levels were similar between the two treatment groups at 48 h PI. IL-12 p40 was significantly (P = 0.0159) increased at 48 h PI, but not at 6 h PI. RANTES and IL-17, cytokines important in T-cell activation, rather than neutrophil activation or recruitment, showed a similar trend of higher expression in the G-CSF-neutralized bladders as compared with the control-treated mice at 6 h PI, although the differences were not significant. IL-17 expression levels were increased in control IgG-treated animals as compared with G-CSF-neutralized animals at 48 h PI but not significantly (Fig. 5B).

Fig. 5.

Fig. 5

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Cytokine expression is altered upon G-CSF neutralization. Female C57BL/6 mice were treated with either control IgG or α-G-CSF concurrent to infection with 107 cfu of UPEC strain UTI89. Bladders were homogenized at 6 or 48 h, centrifuged to remove cellular debris and cytokine expression measured.

A. TNF-α, MIP-1β, IL-1β, IL-6, KC and MCP-1 expression levels.

B. IL-12 p40, RANTES, IL-17 expression levels.

**P = 0.0079, *P = 0.0159. Data are representative of four experiments.

Discussion

In this study, we investigated cytokine mediators involved in the host defence against UPEC over the course of acute infection. Human studies have identified several cytokines secreted into the urine during the acute phase of UTI, including IL-8 (Ko et al., 1993; Oregioni et al., 2005; Real et al., 2007), IL-6, MCP-1, MIP-3α, RANTES, MIP-1α (Godaly et al., 2007), IL-1α, IL-1β (Candela et al., 1998), G-CSF and IL-10 (Jacobson et al., 1998). These studies are limited in that cytokine expression was evaluated when symptomatic patients presented at health-care facilities, where disease onset was not precisely known, or, in a majority of studies, patients presented with pyelonephritis (Ko et al., 1993; Jacobson et al., 1998; Godaly et al., 2007; Real et al., 2007) which most likely has a different cytokine profile than cystitis. Murine studies report KC (Olszyna et al., 2001; Roelofs et al., 2006), MIP-2 (Mittal et al., 2004; Roelofs et al., 2006), IL-1β, IL-6 and TNF-a (Roelofs et al., 2006) in kidneys; MIP-2 (Hang et al., 1999; Mittal et al., 2004), KC, MIP-1α, eotaxin, MCP-1 and ENA-78 (epithelial neutrophil activating peptide-78) (Hang et al., 1999) in urine; and KC in serum of infected mice (Olszyna et al., 2001). Two studies have reported cytokines in the infected mouse bladders observe only MIP-2 (Hang et al., 1999; Mittal et al., 2004), while an additional study reports increases in KC, MIP-2 and CXCL6 mRNA in the bladder upon infection (Billips et al., 2007). Here, we focused on acute cystitis in the C57Bl/6 mouse strain, examining a comprehensive set of soluble host inflammatory mediators over a time-course (Fig. 1). In addition to determining the kinetic expression of previously reported human cystitis-associated cytokines, we uncovered unknown murine cystitis inflammatory factors such as MIP-1β, IL-12 p40, G-CSF, RANTES and IL-17 (Fig. 1) providing a more complete analysis of the cystitis-induced host response. An increased understanding of the roles of individual cytokines in host response to UTI may one day aid in the identification of biomarkers indicative of UTI outcome.

G-CSF has not been previously reported to be associated with murine cystitis; therefore, we focused on its contribution to the host response to UPEC. Neutralization of G-CSF concurrent with infection (Fig. 2) resulted in a decrease in both circulating neutrophils and neutrophils infiltrating the bladder (but not monocytes and macrophages) in response to infection (Fig. 3). Surprisingly, a decrease in neutrophil infiltration correlated with a decrease in bacterial burden in the bladder (Fig. 4). In a murine peritoneal infection model, neutralizing G-CSF reduced circulating neutrophils causing increased morbidity and mortality (Barsig et al., 1996). Additionally, studies have shown that administration of recombinant G-CSF in intraperitoneal infection models is host-protective during microbial challenge (Villa et al., 1998; Marshall, 2005). However, while exogenous G-CSF administration appears to be protective during gut microbial challenges, it may be harmful in lung models of infection. Studies showed that E. coli- or Klebsiella pneumoniae-infected rats treated with recombinant G-CSF demonstrated increased mortality (Held et al., 1998; Karzai et al., 1999). These results show that in certain situations, G-CSF can be detrimental to host defence and while treatment with exogenous G-CSF may be beneficial during bacterial challenge in the gut, it is detrimental to host survival during lung infection. Our findings that G-CSF neutralization results in a reduced bacterial burden suggest that the bladder is a tissue that would not benefit from exogenous G-CSF administration during UPEC infection. Consistent with this conclusion, in a randomized human trial, administration of exogenous G-CSF reduced the frequency of certain nosocomial infections, such as primary bacteraemia, in patients with acute traumatic brain injury or cerebral haemorrhage. However, recombinant G-CSF did not protect these patients from UTI (Heard et al., 1998). In fact, while not statistically significant, the study group receiving the highest dose of G-CSF had the highest incidence of nosocomial UTI (Heard et al., 1998). Our data demonstrate that neutralization of G-CSF alters the immune response in the murine bladder to one more beneficial for the host.

Neutrophils are thought to be the primary cell type involved in controlling bacterial burden during UPEC UTI. The robust neutrophil infiltration in both human (Ko et al., 1993) and murine (Schilling et al., 2001) UTI is likely a consequence of upregulation of the diverse array of cytokines and chemokines described here and elsewhere. Additionally, previous reports have shown that treatment of mice with α-Gr-1 antibody (clone RB6-8C5) to deplete neutrophils leads to higher bacterial burden (Haraoka et al., 1999). However, in the present study, α-G-CSF antibody-treated mice had fewer bladder-associated cfu than control IgG-treated mice at 48 h PI (Fig. 4) despite reduced numbers of circulating and infiltrating neutrophils (Fig. 3). To reconcile these opposing results, it is important to recognize that the two treatments have different specificities. α-G-CSF antibody treatment effectively limits only the number of available circulating neutrophils that can respond to infection (Metcalf and Nicola, 1983; Barreda et al., 2004). α-Gr-1 antibody treatment, with the RB6-8C5 clone which recognizes both the Ly6G and Ly6C molecules, will deplete both blood neutrophils and, importantly, monocytes (Daley et al., 2008). Thus, it is possible that the increased cfu observed in the host upon α-Gr-1 antibody treatment (Haraoka et al., 1999) is due to the depletion of monocytes, which serve as the precursors for macrophages and dendritic cells.

TNF-α, MIP-1β, IL-1β, IL-6, G-CSF, MCP-1 and IL-12 p40 can be produced by monocytes or macrophages and MIP-1β and MCP-1 are chemotactic for macrophages, suggesting that macrophages may play an important role in the innate response to UPEC infection. G-CSF has previously been shown to have immunomodulatory effects on macrophages by suppressing TNF-α (Gorgen et al., 1992; Nishiki et al., 2004; Kim et al., 2006) and IL-1β (Boneberg and Hartung, 2002) production in response to infection or LPS treatment. In this study, we did not find differences between the control IgG and the G-CSF neutralizing antibody treatment groups in the number of either circulating or tissue-associated monocyte populations at 6 or 48 h PI (Fig. 3C and F). However, while the percentages did not change significantly, the increase in cytokine production (Fig. 4) suggests that macrophages may be more activated or less suppressed when G-CSF is neutralized.

The data presented here suggest the following model: UPEC are sensed within minutes upon ascension into the bladder by cells such as resident macrophages that, in turn, secrete TNF-α in response to infection (Fig. 1B). Resident cells must be the source of TNF-α because neutrophils have not infiltrated the bladder at this very early time point. Upon UPEC invasion, the urothelium secretes KC (Fig. 1C) and MIP-2 (Mittal et al., 2004), which in combination with TNF-α, serve to recruit neutrophils (Fig. 3) and blood monocytes to the bladder. By 6 h PI, an immune infiltrate including neutrophils and likely macrophages secretes MIP-1β, IL-1β, IL-6, G-CSF, MCP-1 and IL-12 p40 (Fig. 1B and C). G-CSF released from the immune infiltrate, paradoxically, leads to less efficient bacterial clearance from the bladder (Fig. 4, IgG-treated), by suppressing pro-inflammatory cytokine secretion and macrophage activation (Gorgen et al., 1992; Boneberg and Hartung, 2002; Nishiki et al., 2004; Kim et al., 2006). Upon G-CSF neutralization, additional cytokines are secreted (Fig. 5) and the inflammatory response is better able to clear UPEC from the urinary bladder (Fig. 4, 48 h PI, G-CSF-treated). Finally, cytokines such as IL-17, IL-12 p40 and RANTES recruit and activate T cells to initiate adaptive immunity. Thus, macrophages, other phagocytic cells, and T cells may play a critical role in bladder innate defences in addition to neutrophils. Further study of these cell types will provide insight in the overall mechanisms of host defence against UTI.

Experimental procedures

Bacterial strains

For all experiments, wild-type UPEC strain UTI89, a human cystitis isolate (Mulvey et al., 2001), was grown statically at 37°C for 16 h overnight in Luria–Bertani broth.

Reagents

The mouse group I cytokine 23-plex Bio-Plex kit and cytokine reagent kit were from Bio-Rad Laboratories (Hercules, CA). A custom 12-analyte kit was developed, based on experimental results, containing antibodies to mouse IL-6, KC, IL-12 p40, IL-12 p70, TNF-α, IL-1β, G-CSF, IL-17, eotaxin, MCP-1, MIP-1β and RANTES. Importantly, to minimize plate-to-plate variation in fold-change magnitude, all samples from any given experiment were always analysed on the same plate. G-CSF ELISA DuoSet, neutralizing antibodies to G-CSF (clone 67604.111) and isotope control IgG (clone 4341.11) were from R&D Systems (Minneapolis, MN).

Mouse infections

The murine cystitis model has been described previously (Mulvey et al., 1998). Briefly bacteria, grown as described above, were re-suspended in sterile phosphate-buffered saline (PBS) to 2 × 108 cfu ml−1 for a final inoculum of 1 × 107 cfu in 50 µl. Six- to eight-week-old wild-type C57BL/6 female mice from The Jackson Laboratory (Bar Harbor, ME) were anaesthetized by inhalation of 4% isoflurane and infected via transurethral catheterization. For neutralization experiments, mice were injected intraperitoneally with 50 µg of α-G-CSF neutralizing antibody or isotype control IgG (R&D Systems, Minneapolis, MN), re-suspended in sterile PBS at the time of infection. At indicated times post infection, mice were sacrificed and bladders aseptically removed and processed as follows: for cfu enumeration, bladders were each individually homogenized in 1 ml of PBS for serial dilution in PBS and plated on Luria–Bertani broth agar. For cytokine analysis, individual bladder homogenates were microcentrifuged at 14 000 r.p.m. for 5 min and supernatants frozen at −20°C until the time of the assay. Bio-Plex assays and ELISA were carried out according to manufacturers’ protocols. All animal studies were performed in accordance with the Committee for Animal Studies at Washington University School of Medicine.

Flow cytometry

For flow cytometric analysis of bladder tissue, bladders from 5 to 10 mice per experimental time point were bisected and incubated individually at 37°C for 1 h in 1 mg ml−1 collagenase D (Sigma, St Louis, MO) and 100 µg ml−1 DNase (Sigma, St Louis, MO) and then manually passed through a 40 µm cell strainer to obtain a single-cell suspension. Bladder cell suspensions were washed once in PBS, fixed in 0.5% paraformaldehyde overnight and stored at 4°C until all samples were obtained, 2 days time. Samples were subsequently stained with an α-F4/80-APC-conjugated antibody (clone BM8, eBiosciences, San Diego, CA) and an α-Gr1-FITC-conjugated antibody (clone 5B6-8C5, BD Pharmingen, San Jose, CA). For flow cytometric analysis of blood, animals were sacrificed by cervical dislocation and blood samples taken by cardiac puncture. Samples were mixed with BD Pharmlyse (BD Pharmingen, San Jose, CA) to lyse red blood cells and washed once with HBSS– (no calcium, no magnesium) (Sigma, St Louis, MO). Leukocytes were re-suspended in HBSS– and stained with an α-Gr-1-APC-conjugated antibody (clone RB6-8C5, BD Pharmingen, San Jose, CA) and an α-CD115-PE-conjugated antibody (clone AFS98, eBiosciences, San Diego, CA). Data were acquired on a modified five-colour FACScan maintained by the High Speed Cell Sorter Core at Washington University. Data were analysed using FlowJo v8.6.3 (Tree Star, Ashland, OR); blood samples were gated on live cells and then gated on either Gr-1hi, CD115lo/− for neutrophil percentages or CD115hi, Gr-1hi or Gr-1lo for monocytes. Bladder samples were marked with quadrants to define Gr-1+/F4/80− (neutrophils) or F4/80+ (macrophages) cells. Graphs were generated from per cent of parent analysis from FlowJo in GraphPad Prism 4 (San Diego, CA). To determine bladder-infiltrating cells, the percentage of resident cells (neutrophils > 1%, macrophages ~12% in PBS mock-infected bladders) was determined fist and subtracted from the per cent measured by flow cytometry during experimentation.

Immunohistochemical analysis

Bladders were bisected, immediately embedded in Tissue Tek OCT compound (Sakura Finetek) and frozen for sectioning. Sections (10 µM) were fixed in ice-cold acetone, blocked in PBS containing 3% bovine serum albumin, 0.3% Triton X-100 and CD16/CD32 Mouse Fc block (BD Biosciences, San Jose, CA), and stained for G-CSF using rabbit α-mouse G-CSF clone sc-13102 (Santa Cruz Biotechnology, Santa Cruz, CA) followed by secondary donkey-α-rabbit-Alexa594 (Molecular Probes, Eugene, OR) and leukocytes using a pan-leukocyte marker CD45-FITC (α-mouse CD45-FITC, clone 30-F11, eBioscience, San Diego, CA). Tissue was counterstained with Hoescht dye to reveal nuclear morphology.

Statistical analysis

Experiments described in this report were performed a minimum of three times (experimental replicates are reported in all figure legends) with 5–10 mice per cfu determination, Bio-plex, ELISA or flow cytometric analysis time point. Error bars are standard error of the mean (SEM), horizontal lines in dot graphs are geometric means. All statistics were performed using the non-parametric, Mann–Whitney U-test (InStat®; GraphPad Prism 4 software, San Diego, CA).

Acknowledgements

The authors thank Mark Miller and Andrew Pekosz for many helpful discussions and Swaine Chen, Kelly Wright and Karen Dodson for critical manuscript review and discussion. The authors also acknowledge the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St Louis, MO, for the use of the High Speed Cell Sorter Core, which provided support for the Bio-Rad Bio-Plex System and the flow analysis, as well as William Eades, who provided expert advice and help with flow analysis. The Siteman Cancer Center is supported in part by an NCI Cancer Center Support Grant #P30 CA91842. This work was supported by NIH Grants R01DK51406 and ORWH SCOR P50DK64540 (with the FDA) (to S.J.H.) and K.A.K. received support from an American Heart Association Postdoctoral Fellowship Award No. 0625736Z.

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