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. 2010 Nov 24;31(4):1133–1142. doi: 10.1038/jcbfm.2010.203

Unconjugated bilirubin contributes to early inflammation and edema after intracerebral hemorrhage

Matthew C Loftspring 1,2,3,*, Holly L Johnson 1,2, Rui Feng 4, Aaron J Johnson 1,2, Joseph F Clark 1,2,3,4
PMCID: PMC3070973  PMID: 21102603

Abstract

Intracerebral hemorrhage (ICH) is a stroke subtype with significant mortality and morbidity. The role of unconjugated bilirubin (UBR) in ICH brain injury is not well understood. Therefore, we studied the effects of UBR on brain injury markers and inflammation, as well as mechanisms involved therein. We induced ICH in mice by infusion of autologous whole blood with vehicle (dimethyl sulfoxide) or UBR. We found that UBR led to an increase in edema (P⩽0.05), but a decrease in nitrate/nitrite formation (7.0±0.40 nmol/mg versus 5.2±0.70 nmol/mg protein, P⩽0.05) and no change in protein carbonyls. Unconjugated bilirubin was also associated with an increase in neutrophil infiltration compared with ICH alone, as determined by both immunofluorescence and flow cytometry (36%±3.2% versus 53%±1.3% of CD45+ cells, P⩽0.05). In contrast, we observed reduced perihematomal microglia immunoreactivity in animals receiving UBR (P⩽0.05). Using in vitro techniques, we show neutrophil activation by UBR and also show that protein kinase C participates in this signaling pathway. Finally, we found that UBR was associated with an increased expression of the leukocyte adhesion molecule intercellular adhesion molecule-1. Our results suggest that UBR possesses complex immune-modulatory and antioxidant effects.

Keywords: bilirubin, inflammation, intracerebral hemorrhage, neutrophil, protein kinase C, stroke

Introduction

Intracerebral hemorrhage (ICH), a hemorrhagic stroke, is caused by bleeding into the brain parenchyma. It occurs most commonly in the basal ganglia and thalamus. It affects ∼70,000 Americans per year and has a 48-hour mortality rate of up to 25% (Elijovich et al, 2008; Santalucia, 2008). In patients who survive the initial hemorrhage, early (i.e., 1 to 3 days) neurologic deterioration becomes a significant clinical problem (Sahni and Weinberger, 2007; Elijovich et al, 2008). A number of factors contribute to brain injury after ICH. Within 24 hours, hematoma expansion and edema have an important role (Xi et al, 2006; Elijovich et al, 2008). Acute inflammation is observed within 4 hours of ICH in animal models (Wang and Dore, 2007a). Blood products that comprise other sources of injury such as thrombin, hemoglobin, iron, and unconjugated bilirubin (UBR) have all been shown to possess toxicity (Huang et al, 2002; Xi et al, 2006). In this study, we sought to better understand the role of UBR in early brain injury after ICH.

Unconjugated bilirubin is a product of heme catabolism and is detectable in the hematoma by 8 to 12 hours in a porcine ICH model (Clark et al, 2008). Conversion of heme to biliverdin by hemeoxygenase is the rate-limiting step in bilirubin production. Biliverdin is reduced to bilirubin by biliverdin reductase (Wagner et al, 2003). Chemically, UBR is best known as a weak antioxidant because of its ability to scavenge reactive oxygen species (Farrera et al, 1994). However, it also seems to possess immune-modulatory effects in systemic diseases. For example, UBR suppresses T-cell function in liver allografts and experimental bile duct obstruction (Roughneen et al, 1986; McDaid et al, 2005). Conversely, UBR leads to an elaboration of proinflammatory cytokines from astrocytes and microglia in vitro (Gordo et al, 2006; Fernandes et al, 2007). These immune-modulatory effects have not been directly investigated in ICH. In addition, there have been conflicting results regarding the protective versus deleterious roles of UBR and hemeoxygenase in the brain and spinal cord (Wang and Dore, 2007b; Lin et al, 2007; Liu et al, 2008).

Using in vitro and in vivo techniques, we show that UBR possesses inflammatory properties that are concentration dependent. Notably, UBR increased perihematomal neutrophil infiltration and edema after ICH. Furthermore, in vitro, we show that protein kinase C (PKC) signaling has a role in mediating the proinflammatory effects of UBR. Finally, UBR was associated with increased intercellular adhesion molecule-1 (ICAM-1) expression, suggesting this as a mechanism by which UBR potentiates neutrophil infiltration.

Materials and methods

Materials

All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) unless otherwise noted. A stock solution of UBR was prepared in DMSO (dimethyl sulfoxide) at a concentration of 4.5 mmol/L.

Intracerebral Hemorrhage Model

Animal procedures were approved by the IACUC (Institutional Animal Care and Use Committee). We used male C57BL/6J mice (11 to 19 weeks of age) for this study. Mice were anesthetized with 2% isoflurane in 24% oxygen and 74% nitrous oxide, administered through an anesthesia mask (Kopf, Tujunga, CA, USA). Deep sedation was monitored throughout the procedure by the absence of pain reflexes in the toes. Body temperature was maintained at 37°C±0.5°C using a feedback-controlled heating blanket.

Our experimental ICH procedure has been described previously (Loftspring et al, 2009), with some modifications. Mice were placed in a stereotaxic frame (Kopf). A skin incision was made along the midline of the dorsal surface of the head, exposing the bregma. A 0.7-mm cranial burr hole was drilled 2.2 mm lateral and 0.2 mm anterior to the bregma. Approximately 30 μL of autologous blood was collected from the ventral tail artery by making an incision near the tail base. Further bleeding was prevented by cauterization if necessary. Blood was drawn into a 50-μL Hamilton syringe using a 26-G needle. The needle was inserted 3.5 mm ventral through the cranial hole into the brain, and 10 μL of blood plus 1 μL of DMSO (vehicle) or UBR solution was infused over 5 minutes.

Brain Homogenization and Injury Markers

Mice were deeply anesthetized with isoflurane and decapitated. The brain was removed and frozen on dry ice. Perihematomal tissue was dissected and homogenized in cold phosphate-buffered saline (137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na2HPO4, 1.8 mmol/L KH2PO4) plus protease inhibitors using a ground glass homogenizer. Samples were centrifuged at 14,000 × g for 20 minutes at 4°C. The supernatant was removed and used for analysis. Total protein was determined with the bicinchoninic acid method using bovine serum albumin as the standard (Pierce, Rockford, IL, USA). Edema was assessed by measurement of brain water content by weight essentially as described previously (Loftspring et al, 2009). In brief, the brains were removed, the perihematomal tissue was dissected, and weighed on an analytical balance with precision to 10 μg. The tissue was desiccated for 24 hours at 70°C. Brain water content was calculated according to the following equation: brain water content=((wet weight−dry weight)/wet weight) × 100. Oxidative stress was studied by quantitating nitrate/nitrite (Cayman, Ann Arbor, MI, USA) and protein carbonyls (OxyELISA, Millipore, Billerica, MA, USA) using commercially available kits. The hemoglobin in tissue homogenates was quantified by conversion to cyanmethemoglobin with Drabkin's reagent. Absorbance was read at 540 nm and values were interpolated from a standard curve. Hematoma size was also assessed by sectioning the tissue through the largest part of the hematoma, scanning mounted sections at high resolution, and calculating the hematoma area using ImageJ (NIH, Bethesda, MD, USA).

Rotarod

The rotarod task has previously been used to assess behavioral deficits in ICH (Jeong et al, 2003). The rotarod was set to accelerate from 4 to 40 r.p.m., and the time mice took to either fall or rotate with the rod was recorded. Mice were trained 1 day before surgery with four trials. Baseline was determined on the day of surgery with six trials per mouse. Six more trials were performed immediately before sacrificing. Percentage change was calculated for each mouse using the average of the six times as follows: ((Post-ICH−Pre-ICH)/Pre-ICH) × 100.

Immunofluorescence and Quantitation

Mice were sacrificed as before, and the brains were immediately removed, frozen on dry ice, and stored at −80°C until sectioning. The brains were cut into 10-μm coronal sections using a cryostat. Sections were fixed with 4% paraformaldehyde for 10 minutes and blocked with 5% donkey serum in phosphate-buffered saline for 30 minutes at room temperature. Slides were incubated in primary and secondary antibodies for 1 hour each and then coverslipped with Vectashield plus DAPI (4′,6-diamidino-2-phenylindole) (Vector Laboratories, Burlingame, CA, USA). Secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Antibodies were used at the following dilutions: rabbit anti-PKC phospho-T497 (p-PKC), 1:500 (Abcam, Cambridge, MA, USA); rat anti-Ly-6G, 1:200 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rat anti-F4/80 (Abcam), 1:250; and rat anti-ICAM-1 (Abcam), 1:50.

The tissue was imaged with a Zeiss Axioplan 2 upright microscope using AxioVision software, version 4.5 (Carl Zeiss Micro Imaging, Oberkochen, Germany). Perihematomal cells positive for p-PKC, Ly-6G, or F4/80 were counted in three × 20 fields per section. Cell counts were summed and averaged with other sections of the same brain. The ICAM-1+ blood vessels were counted in six fields per section because they were fewer in number than the cells. ‘Perihematomal' was defined as being within one × 20 field of the hematoma, which corresponds to 450 μm. Samples were coded and/or counted by a blinded observer.

Confocal Microscopy

For colocalization of p-PKC to neutrophils, a Zeiss LSM 510 confocal microscope with Zeiss LSM 510 Image Browser version 4.0.0.241 was used (Carl Zeiss Micro Imaging). Sections were stained as described above using fluorescein isothiocyanate-conjugated (excitation/emission 488/518 nm) and DyLight 549 (ex/em 543/568 nm)-conjugated secondary antibodies for p-PKC and Ly-6G, respectively.

Human Neutrophil Isolation

This procedure was conducted using healthy volunteers and was in accordance with our Institutional Review Board-approved protocol. Approximately 10 mL of peripheral blood was collected into sterile ethylenediaminetetraacetic acid-coated vacuum tubes (BD, Franklin Lakes, NJ, USA) through antecubital venipuncture. Granulocytes were purified from 10 mL of whole blood using a commercially available density gradient medium (Axis-Shield, Norton, MA, USA) according to the manufacturer's instructions. The purity of neutrophils was consistently 90% to 95% as determined by cytospin and a modified Wright-Giemsa stain (Diff-Quik, Siemens, New York, NY, USA).

Superoxide Production Assay

Neutrophils were isolated as described above. Superoxide was measured using a commercially available luminol-based kit (LumiMax, Agilent Technologies, Santa Clara, CA, USA). Luminol is detectable by luminescence when it is oxidized by superoxide (Kobayashi et al, 2001). Cells were cultured at 37°C for 2 hours in a luminometer and the signal was recorded every 1 minute. The maximum luminescence (see Figure 3A) was used for analysis.

Figure 3.

Figure 3

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Unconjugated bilirubin (UBR) exerts concentration-dependent effects on neutrophils in vitro. Neutrophils were stimulated with 1.3 μg/mL phorbol 12-myristate 13-acetate (PMA) and different concentrations of UBR. (A) Kinetics and (B) analysis of superoxide production. (C, D) UBR (4.5 μmol/L) induces neutrophil degranulation, which is blocked by inhibitors of protein kinase C. (E) UBR at 45 μmol/L fails to induce degranulation. (F) Background degranulation is not blocked by ST. Bars are mean±s.e.m., *P⩽0.05 versus control, n=4 to 5 per group, representative of at least two independent experiments. ST, staurosporine.

Neutrophil Degranulation Assay

Neutrophil degranulation was assessed by measuring extracellular elastase activity. Neutrophils were isolated as described above and cultured at 37°C with 5% CO2 in 96-well V-bottom plates for 100 minutes in RPMI-1640 without phenol red (±other components as described in the ‘Results' section). Elastase activity was assessed using N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma-Aldrich), a synthetic peptide that absorbs strongly at 400 nm when cleaved by elastase (Castillo et al, 1979). After incubation, cells were pelleted and the absorbance of the supernatant was read at 400 nm. The absorbance of UBR at 400 nm was negligible at all but the highest concentration, i.e., at 45 μmol/L. For these incubations, we accounted for the baseline contribution of UBR.

Flow Cytometry

Mice were deeply anesthetized with isoflurane and transcardially perfused with 5 mL of cold RPMI-1640. The brains were removed and placed in cold RPMI. Immune cell isolation from the brains was performed as described previously (Loftspring et al, 2009). In brief, the excised mouse brains were pushed through a nylon cell strainer (BD Biosciences, San Jose, CA, USA) with a 100-μm pore width and incubated at 37°C for 45 minutes in 250 μg/mL collagenase type 4 (Worthington Biochemical Corp., Lakewood NJ, USA). Myelin was removed by centrifugation at 12,000 × g for 30 minutes in 45% Percoll.

Cells isolated from the central nervous system were stained with anti-CD3 allophycocyanin, anti-CD45 phycoerythrin-Cy7, anti-Ly-6G biotin, and anti-CD11b fluorescein isothiocyanate (BD catalog numbers 553066, 552848, 127603, and 557396, respectively). Samples were analyzed on a BD LSRII instrument (BD Biosciences). Raw data were displayed with side scatter along the Y axis and with forward scatter along the X axis. A gate was placed according to the profile of inflammatory cells as determined previously (Johnson et al, 1999, 2001). Cell populations were defined as follows, according to previously published data (Rynkowski et al, 2009; Olson, 2010): neutrophils (CD45hi, CD11b+, Ly-6G+), microglia (CD45int, CD11b+, Ly-6G−), macrophages (CD45hi, CD11b+, Ly-6G−), and T cells (CD45hi, CD3+, CD11b−) (Figure 4A). Cell populations were analyzed using FACSDiva (BD Biosciences) and are expressed as a percentage of CD45+ cells.

Figure 4.

Figure 4

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Unconjugated bilirubin (UBR) is associated with increased neutrophil infiltration 1 day after intracerebral hemorrhage (ICH). (A) Representative flow cytometry contour plots showing different populations of parenchymal inflammatory cells. (B) Quantitation of inflammatory cell populations in the brain after ICH. P1, total CD45+ cells; P2, neutrophils (CD45hi CD11b+Ly-6G+); P3, microglia (CD45int CD11b+ Ly-6G−); P4, macrophages (CD45hi CD11b+ Ly-6G−). Bars are mean±s.e.m., *P⩽0.05 versus ICH+vehicle, #P⩽0.05 versus ICH+4.5 μmol/L UBR, n=4 to 6 mice per group.

Statistics

Data were analyzed using Student's t-test (two groups) or one-way ANOVA (analysis of variance) (three or more groups) using Fisher's post hoc test. Analysis was performed using KaleidaGraph (Synergy Software, Reading, PA, USA). The level of significance was set at P⩽0.05.

Results

Unconjugated Bilirubin is Associated with Increased Edema and Decreased Nitrate/Nitrite 1 Day after Intracerebral Hemorrhage

An example of our mouse ICH model is shown in Figure 1A. We observed a well-defined hematoma centered in the striatum. We did not find a difference in hemoglobin content or hematoma area between the two groups, suggesting that equal blood volumes were delivered to the brain (Figure 1B). Unconjugated bilirubin (450 μmol/L) was associated with increased edema, as measured by brain water content (77.0%±0.18% in ICH versus 77.5%±0.15% in ICH+UBR, P⩽0.05, Figure 1C). Although there was a trend towards poorer rotarod performance in the ICH+UBR group, we did not find a statistically significant difference (P=0.18, data not shown). This may be attributed to the fact that rotarod is not sensitive enough to detect subtle differences in laterality. Finally, we assessed oxidative stress by measuring nitrate/nitrite and protein carbonyls. We found less nitrate/nitrite in mice receiving UBR (7.0±0.40 nmol/mg versus 5.2±0.70 nmol/mg protein, P⩽0.05, Figure 1D) but no change in protein carbonyls (data not shown).

Figure 1.

Figure 1

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Unconjugated bilirubin (UBR) is associated with increased edema and decreased nitrate/nitrite 1 day after intracerebral hemorrhage (ICH). (A) Example of experimental ICH in the mouse. (B) Hemorrhage size, (C) edema, and (D) nitrate/nitrite between ICH+vehicle and ICH+450 μmol/L UBR. Bars are mean±s.e.m., *P⩽0.05 versus ICH+vehicle, n=4 to 7 mice per group; NS, not significant (P=0.38).

Unconjugated Bilirubin Modulates Inflammation at 1 Day after Intracerebral Hemorrhage

Next, we examined markers of the inflammatory process in the presence of ICH+UBR or ICH plus vehicle (DMSO, Figure 2). Unconjugated bilirubin (450 μmol/L) was associated with an increase in perihematomal neutrophils (P⩽0.05), as determined by immunofluorescence for Ly-6G, which is a component of the Gr-1 complex and is selectively expressed by neutrophils, with limited expression by other cell types (Daley et al, 2008). An opposite effect was observed for microglia/macrophage F4/80 immunoreactivity (P⩽0.05). The F4/80 marker is expressed by microglia and macrophages and is upregulated on activation (Austyn and Gordon, 1981).

Figure 2.

Figure 2

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Unconjugated bilirubin (UBR) modulates inflammation by 1 day after intracerebral hemorrhage (ICH). (A, B) Representative neutrophil Ly-6G immunofluorescence in ICH+vehicle (panel A) and in ICH+450 μmol/L UBR (panel B). (D, E) Representative microglia/macrophage F4/80 immunofluorescence in ICH+vehicle (panel D) and in ICH+450 μmol/L UBR (panel E). Cell counts of (C) neutrophils and (F) microglia/macrophages. Bars are mean±s.e.m., *P⩽0.05 versus ICH+vehicle, n=4 to 5 mice per group.

Unconjugated Bilirubin Exerts Concentration-Dependent Effects on Neutrophils In Vitro

As neutrophils accumulate as soon as 4 hours after ICH (Wang and Dore, 2007a), we studied whether UBR activates freshly isolated human neutrophils (Figure 3). We used two functional markers of activation: superoxide production and elastase activity (i.e., degranulation). For superoxide anion production, neutrophils were stimulated with 1.3 μg/mL phorbol 12-myristate 13-acetate and supplemented with varying amounts of UBR, which (45 μmol/L) inhibited superoxide anion production, whereas UBR (0.45 μmol/L) potentiated superoxide anion production (Figures 3A and 3B, P⩽0.05). Unstimulated neutrophils did not have detectable superoxide anion production (data not shown). For elastase activity, 4.5 μmol/L UBR promoted degranulation (P⩽0.05), whereas 45 μmol/L UBR had no effect (Figures 3C–3E). Addition of staurosporine (500 nmol/L) or Gö 6983 (1 μmol/L, Tocris, Ellisville, MO, USA) prevented UBR-induced degranulation (Figures 3C and 3D). Both compounds inhibit most PKC isoforms found in neutrophils (Peterman et al, 2004).

Unconjugated Bilirubin is Associated with Increased Neutrophil Infiltration at 1 Day

In view of the concentration-dependent effects of UBR observed in vitro, we induced ICH with two different concentrations of UBR and performed flow cytometry (Figure 4). These experiments serve as a more sensitive assay to confirm our immunofluorescence findings and to better define inflammatory cell populations, because microglia and macrophages are distinguishable from each other by flow cytometry (Olson, 2010). No discernable population of T cells was found (Figure 4A). Unconjugated bilirubin (450 μmol/L) was associated with an increase in CD45hi CD11b+ Ly-6G+ cells (neutrophils) compared with ICH plus vehicle (36%±3.2% versus 53%±1.3% of CD45+ cells, P⩽0.05, Figure 4B).

Unconjugated Bilirubin is Associated with an Increase in p-PKC+ Neutrophils 1 Day after Intracerebral Hemorrhage

As we found that PKC was involved in UBR-induced neutrophil activation in vitro, we investigated whether the same was true after ICH (Figure 5). We stained for an activation loop epitope shared by all PKC isoforms. Phosphorylated threonine (T497 in PKC-α) (referred to as p-PKC) in the activation loop is essential for full activation in most PKC isoforms with the notable exception of PKC-δ (Steinberg, 2008). We observed an increase in cells immunoreactive for p-PKC in the perihematomal region after ICH+UBR (P⩽0.05, Figures 5A–5C). To determine which cell type(s) contained active p-PKC, we performed double immunofluorescence. We found that active p-PKC does not colocalize with NeuN (neurons) or F4/80 (microglia/macrophages, data not shown), but does colocalize with Ly-6G, a marker of neutrophils (Figures 5D–5F). Finally, we asked whether an increase in ICAM-1 might be a mechanism by which UBR led to an increase in parenchymal neutrophils. We observed an increase in ICAM-1+ blood vessels in mice with ICH+UBR (Figures 5G–5I, P⩽0.05).

Figure 5.

Figure 5

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Unconjugated bilirubin (UBR) is associated with an increase in perihematomal p-PKC+ neutrophils 1 day after intracerebral hemorrhage (ICH). (A–C) Representative p-PKC immunofluorescence in (panel A) ICH+vehicle, in (panel B) ICH+450 μmol/L UBR, and (panel C) counts of p-PKC+ cells. (D–F) Confocal micrographs showing colocalization of active p-PKC with neutrophil marker Ly-6G. (G–I) Immunofluorescence and quantitation of ICAM-1 in (panel G) ICH+vehicle and (panel H) ICH+450 μmol/L UBR. Bars are mean±s.e.m., *P⩽0.05 versus ICH+vehicle. PKC, protein kinase C.

Discussion

In this report, we show that UBR increases inflammatory cell infiltration and edema after ICH. Furthermore, our in vitro experiments provide potential mechanisms by which the in vivo effects occur. Intracerebral hemorrhage causes edema in humans and in mouse models (Garrett et al, 2009; Xi et al, 2006). The exact magnitude of edema observed in animal ICH models depends on the method by which it is measured, the brain region, and the specifics of the ICH model. We used similar edema measurement methods as used by Garrett et al (2009). Although their data were obtained at 72 hours, these authors found that the brain water content of sham and ICH groups was ∼76% and 77%, respectively. The ICH group in their study corresponds to that observed in this study.

With regard to neutrophil function, it seems that low UBR has proinflammatory properties, whereas high UBR suppresses certain functions. For example, in vitro high UBR (45 μmol/L) suppressed superoxide production (Figures 3A and 3B). In vivo, UBR was associated with decreased nitrate/nitrite. This could occur either by direct scavenging of reactive oxygen species or by altering signaling pathways (Datla et al, 2007; Farrera et al, 1994). Datla et al (2007) observed a concentration-dependent inhibition of superoxide production by UBR in HL-60 cells. Therefore, our in vitro and in vivo results are in agreement with previous reports showing the antioxidant effects of UBR. Our novel finding that low UBR (0.45 μmol/L) potentiates superoxide production may be attributed to the fact that we used a higher phorbol 12-myristate 13-acetate concentration than did Datla et al or may be related to differences between primary neutrophils and the HL-60 cell line.

Neutrophils are a significant source of reactive oxygen species, which are an important injury mechanism in ICH (Tang et al, 2005; Pun et al, 2009). Similarly, UBR may induce degranulation of neutrophil collagenases in vivo leading to increased brain edema (Borregaard et al, 1995; Joice et al, 2009). This is plausible because higher UBR was required for degranulation than for potentiation of superoxide production in vitro. This possibility is in accordance with our finding of increased PKC+ neutrophils in the UBR group. Unconjugated bilirubin has previously been shown to cause edema in experimental ICH (Huang et al, 2002).

Gordo et al (2006) have shown that UBR activates and then damages the microglia in vitro, causing them to become necrotic. Our observation of decreased perihematomal F4/80 immunofluorescence is in agreement with this finding. Unconjugated bilirubin activates specific signaling pathways in the microglia as opposed to only causing generalized toxicity. Silva et al (2010) have recently found in vitro that UBR is associated with mitogen-activated protein kinase and nuclear factor-κB activation within 2 hours of UBR exposure. From 2 to 24 hours, the authors observed propidium iodide-positive microglia and apoptotic microglia, as shown by activation of caspases 3, 8, and 9. Therefore, UBR in ICH may be associated with hyperacute microglia activation followed by later cell death.

The highest in vivo UBR concentration (450 μmol/L) in this study was chosen on the basis of that observed in porcine hematoma by 8 to 12 hours after ICH (Clark et al, 2008). Theoretically, it is possible for the blood we infused to produce >450 μmol/L UBR (Huang et al, 2002). However, we did not observe levels this high in our porcine model or in the spinal fluid of patients with subarachnoid hemorrhage (Clark et al, 2008; Pyne-Geithman et al, 2005). Therefore, we used concentrations similar to what we have observed. As UBR is derived from the hematoma, we mixed UBR with the infused blood. Our goal was to model the effects of relevant UBR levels after ICH. The approach of mixing an agent of study with the infused ICH blood has been reported previously (Wagner et al, 2000). An alternative would have been to infuse UBR alone or to measure UBR after ICH and correlate different levels seen to injury markers. We opted not to try these because our intent was to study the effects of UBR in the hemorrhagic milieu.

Conjugated (direct) bilirubin is formed in the liver and is of limited relevance to ICH. Unconjugated (indirect) bilirubin is hydrophobic, is produced after hemolysis, and is primarily bound to albumin, which is abundant in the hemorrhagic milieu. However, albumin-bound UBR is in equilibrium with nanomolar concentrations of unbound UBR and it is this form that can passively cross the plasma membrane and exert intracellular effects (Zucker et al, 1999; Ostrow et al, 1994). Therefore, the concentrations we studied in vitro contain higher free UBR concentrations than those studied in vivo because our cell culture system did not contain significant amounts of protein. Furthermore, because of complicated binding curves to albumin, it is difficult to predict free UBR concentrations in vivo (Ostrow et al, 1994). For this reason, we caution against direct comparison of in vitro with in vivo UBR concentrations because the ‘available' UBR is higher in vitro for a given concentration of total UBR. A further complication, beyond the scope of this paper, is that UBR has four isomers (namely EE, EZ, ZE, and ZZ). We did not monitor for isomerization changes during the study. Further studies to assess isomer subtypes may be warranted.

The mechanism leading to increased neutrophil infiltration in UBR-treated mice remains to be clarified. In vitro, UBR leads to production of interleulin-1β and tumor necrosis factor-α by astrocytes and microglia (Fernandes et al, 2007; Gordo et al, 2006). These mediators of inflammation may then lead to increased activation of the endothelium and greater extravasation of leukocytes. Although this possibility was not directly examined in the current study, we performed immunofluorescent staining for ICAM-1, which along with related proteins are major adhesion molecules necessary for firm adhesion of marginating leukocytes. Furthermore, the soluble form of ICAM-1 is elevated in the cerebrospinal fluid after human ICH (Kraus et al, 2007), suggesting the importance of this molecule in ICH. We found an increase in ICAM-1 immunoreactive blood vessels in mice with ICH+UBR. This finding leads us to suggest that an increase in ICAM-1 is a potential mechanism, whereby UBR leads to greater neutrophil infiltration into the brain.

One limitation of our study is that we can only speculate that the effects we observe in vitro are the mechanisms leading to the observed in vivo phenomena. Nonetheless, we show a number of novel immune-modulatory effects of UBR and one putative signaling pathway. These results are significant because they suggest that the concentration of UBR and cell type(s) on which it acts are important determinants of whether it is proinflammatory or antiinflammatory. Finally, we suggest that future experiments use similar methods to elucidate additional mechanisms of inflammation in ICH.

The authors declare no conflict of interest.

Footnotes

This study was supported by NIH grants R01NS050569 and U54EB007954 to JFC and R01NS060881 to AJJ. MCL is supported by the UC University Research Council Fellowship.

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