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Rheumatology 2001; 40: 889-895
© 2001 British Society for Rheumatology

Original Papers

Limited endothelial E- and P-selectin expression in MRL/lpr lupus-prone mice

O. A. Harari, D. Marshall, J. F. McHale, S. Ahmed and D. O. Haskard

BHF Cardiovascular Medicine Unit, National Heart & Lung Institute, Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, UK


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. Inflammation in MRL/lpr mice may involve dysfunctional leucocyte–endothelial cell (EC) interactions. Previously, we have shown that intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) increase with age in a tumour necrosis factor {alpha} (TNF{alpha})- and interleukin-1 (IL-1)-dependent manner. The object of this study was to determine the expression of E- and P-selectin.

Methods. Selectin expression was quantified in MRL/lpr mice and BALB/c controls by intravenous injection of differentially radio-labelled antibodies.

Results. E-selectin, but not P-selectin, was up-regulated in the kidneys of older mice. Neither was up-regulated elsewhere. There was no defect in selectin inducibility, as a further inflammatory stimulus (intraperitoneal lipopolysaccharide) resulted in up-regulation. Serum from older MRL/lpr did not induce selectin expression by EC in vitro.

Conclusion. The increase in E-selectin in the kidney may contribute to the development of glomerulonephritis. However, the lack of systemic E- and P-selectin expression may represent a protective mechanism which limits the interaction between leucocytes and the endothelium in the chronic inflammatory context.

KEY WORDS: Inflammation, Endothelium, Adhesion, E-selectin, P-selectin, MRL/lpr, Lupus, Glomerulonephritis.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The MRL/Mp-lpr/lpr (MRL/lpr) mouse strain has a defect in Fas, leading to an accumulation of CD4-CD8-{alpha}ß T cells in lymphoid and other tissues [1, 2]. It spontaneously develops a chronic, systemic inflammatory disease with similarities to systemic lupus erythematosus (SLE), including the appearance of a lymphocytic vascular and perivascular infiltration [3]. As early as 8 weeks after birth there is mononuclear cell infiltration of tissues, including the lung, kidney, heart and brain, and this increases steadily with age [4–7]. Renal failure is the principal cause of death, which occurs between 7 and 9 months of age [3].

Endothelial cell (EC) adhesion molecules might be expected to play a role in the recruitment of chronic inflammatory cells from the circulation in MRL/lpr mice. Using a recently developed technique for the relative quantification of EC surface molecules in mice, we found that endothelial expression of the integrin ligands intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) increased with advancing age in the heart, kidney and brain, with VCAM-1 also increasing in the lung [8]. These increases were shown to be dependent in part on the increasing elaboration of tumour necrosis factor {alpha} (TNF{alpha}) and interleukin-1 (IL-1).

E- and P-selectin mediate the rolling of leucocytes on EC, prior to integrin-mediated arrest and subsequent transmigration into tissues [9, 10]. They are expressed on the surface of EC in response to inflammatory stimuli such as TNF{alpha} and IL-1 [11]. Whilst expression of E- and P-selectins in vitro tends to be transient, it is still unclear whether this is also true in vivo, or whether E- and P-selectin expression becomes persistent in chronic inflammatory disease.

In view of the lack of published data on selectin expression in murine models of SLE, we performed a study designed to investigate the expression of E- and P-selectins during the course of disease progression in MRL/lpr mice and to relate the results to our previous work with ICAM-1 and VCAM-1.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Antibodies and cell lines
MES1 is an IgG2a monoclonal antibody (mAb) against mouse E-selectin, kindly supplied by Dr Derek Brown (Celltech Chiroscience, Slough, UK). RB40.3414 (RB40) is a rat IgG1 mAb against mouse P-selectin. The hybridoma cell line was obtained from Professor Dietmar Vestweber (University of Munster, Germany). Hybridoma cell lines producing rat IgG1 and IgG2a mAbs against dinitrophenol (DNP) were obtained from Dr David Gray (Imperial College School of Medicine, London, UK). mAb were analysed by size exclusion chromatography using a Superose 12 column on a fast performance liquid chromatography system (Pharmacia, Uppsala, Sweden). All preparations were found to be greater than 90% monomeric. The murine endothelioma cell line sEnd.1 was obtained from Dr Martyn Robinson (Celltech) [12]. It was grown in Dulbecco's Modified Eagle's Medium supplemented with 1 mM sodium pyruvate (Sigma, Poole, UK) and 10% heat-inactivated fetal calf serum (FCS), 50 U/ml penicillin and 50 µg/ml streptomycin (Gibco, Paisley, UK).

Animals
The mice (all females) were housed in the Biological Services Unit at Imperial College School of Medicine, Hammersmith Campus. They were obtained from Harlan Olac (Oxon, UK) and housed in filter cages supplied with irradiated bedding and food and acidified water. They were housed in proximity to sentinels used for regular pathogen screening. Lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4 (Sigma) in Hanks buffered saline solution (HBSS) or HBSS alone was administered by intraperitoneal (i.p.) injection. Four hours later, E- and P-selectin expression in the heart, lung, liver, kidney, or brain was determined by radio-labelled mAb targeting.

Anti-selectin mAb localization in vivo
The method used was the same as that previously described [13]. Briefly, anti-E-selectin, anti-P-selectin and anti-DNP mAb were differentially radio-labelled with 99mTc, 111In and 125I. mAb were mixed together and injected via the tail vein. After 5 min the mice were placed under terminal anaesthesia, exsanguinated and the vasculature perfused with heparinized saline. Organs were harvested, weighed, placed in vials and counted simultaneously for 99mTc, 111In and 125I in a gamma counter. Counts per minute (c.p.m.) were corrected for background, spill between channels and 99mTc decay over the counting period, and for organ weight in grams. After correction, c.p.m./g were divided by the total c.p.m. injected into that animal (the injected dose, ID). This proportion was then made into a percentage (referred to as the %ID/g). Alternatively, when experiments involved mice of different sizes, it was normalized for plasma volume by multiplying by the weight of the animal in grams (referred to as the normalized %ID/g). Specific uptake of anti-selectin mAb was calculated by subtracting the %ID/g for the control mAb. Mice with little or no target molecule expression sometimes showed negative values for specific %ID/g, due to minor differences in non-specific uptake of control and targeting mAb. As this technique offers a relative, rather than an absolute, measure of selectin expression, data from separate experiments were never pooled. Each experiment included appropriate internal controls, both positive and negative, for statistical comparison with test groups.

Flow cytometry
Serum was obtained from BALB/c or MRL/lpr mice under clean conditions, pooled and stored at -70°C prior to use. To assess the capacity of sera to induce E- and P-selectin expression, sEnd.1 cells were plated on gelatin-coated 24-well plates at 105 cells per well, cultured overnight and then incubated for 4 h with sera diluted 1: 2 with cell culture medium. The cells were then washed twice in HBSS without calcium and magnesium and detached with 0.125% trypsin-ethylene diamine tetraacetic acid (EDTA) (ICN, Oxon, UK). They were then stained with FITC-conjugated mAb MES1, RB40 or isotype-matched anti-DNP controls. After staining they were washed twice, fixed in 1% paraformaldehyde and analysed on a Coulter Epics XL cell sorter (Coulter, Miami, FL, USA). Control cells were incubated with varying concentrations of recombinant murine TNF{alpha} (a gift from Dr Tony Meager) in HBSS for 4 h.

Statistics
The correlation between two variables was determined without the assumption of a linear relationship, using Spearman's two-tailed test. Linear and non-linear regression analyses were performed using the least squares method. Multiple comparisons were made using the one-tailed analysis of variance (ANOVA) followed by Bonferroni's multiple comparison of means test. Differences between groups were considered to be significant where P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Acute systemic inflammation induced by LPS
The ability to quantify EC selectin expression was demonstrated in BALB/c mice by showing a relationship between the size of an acute inflammatory stimulus (i.p. LPS) and the degree of up-regulation of endothelial selectins. Mice were administered LPS at varying doses or HBSS alone. After 4 h, selectin expression was quantified in the heart, lung, liver, brain and kidney. Figure 1A shows specific anti-E-selectin mAb uptake (%ID/g) in the heart. The %ID/g progressively increased with each increment in LPS dose. LPS significantly increased E-selectin expression with a correlation between LPS dose and anti-E-selectin %ID/g (r = 0.91, P < 0.0001, Spearman). Similar findings were in evidence for E-selectin in other organs, with r and P values shown in Fig. 1B–E. Figure 1F–J shows correspondingly significant increases in anti-P-selectin %ID/g in the heart, lung, liver and brain. Unexpectedly, we found no evidence for LPS-induced P-selectin expression in the kidney (Fig. 1J). To confirm these findings, some mice were intravenously (i.v.) administered 50 µg of either unlabelled anti-E-, -P-selectin, -CD31 or control mAb, and the tissues snap frozen, sectioned and immunostained with an anti-rat Ig antibody. Lung tissue from BALB/c mice not challenged with LPS exhibited constitutive CD31 expression on all vessels, but no E- or P-selectin was seen. LPS-treated mice showed E- and P-selectin expression on medium and large vessels, but not on pulmonary capillaries.



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  		FIG. 1. Effect of varying i.p. LPS dose on the localization of anti-E-selectin mAb MES1 and anti-P-selectin mAb RB40. BALB/c mice were treated i.p. with varying doses of LPS or with the vehicle (HBSS) alone. Four hours later they were injected i.v. with 2 µg of 99mTc-labelled MES1, 125I-labelled RB40 and 111In-labelled control IgG1 mAb. After 5 min the blood pool was perfused out and the following organs harvested: heart (A, F), lung (B, G), liver (C, H), brain (D, I) and kidney (E, J). Localization (as %ID/g) of anti-E-selectin mAb MES1 (A–E) and anti-P-selectin mAb RB40 (F–J) was ascertained (see Materials and methods). Treatment with LPS significantly increased localization of mAb MES1 and mAb RB40 (comparing each dose of LPS with the HBSS group using the one-tailed ANOVA with Bonferroni correction, *P<0.05; **P<0.001), with the exception of mAb RB40 localization in the kidney (J). Furthermore, increased doses of LPS correlated with increased mAb MES1 and mAb RB40 localization (Spearman correlation coefficient r and associated P value are shown). There were five mice in each group except for the 0.25 mg/kg dose, in which there were four. Values shown are means±standard deviation.

 

Vascular selectin expression in MRL/lpr mice
Because MRL/lpr are considerably larger than BALB/c, and thus have a greater intravascular volume, injection of the same amount of mAb into the two strains could therefore be expected to result in lower circulating mAb levels and a lower %ID/g in MRL/lpr than BALB/c. Therefore it was necessary initially to determine how the strains could be compared. The relationship between the circulating concentration of control mAb at 5 min after injection and mouse weight was determined in both strains at different ages. As shown in Fig. 2Go, increasing body weight was associated with decreasing whole blood mAb level. A non-linear regression analysis was performed on the data assuming an inverse relationship (curve shown in Fig. 2Go). The coefficient of determination (R2) was high (0.76), suggesting that the relationship between these two variables was indeed one of inverse proportionality. On the basis of this result, comparisons between mice of different weights were achieved by multiplying the proportion of ID localizing to 1 g tissue by the weight of the animal in grams, resulting in a ‘normalized %ID/g’.



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  		FIG. 2. Relationship between circulating concentration of control mAb at 5 min after injection and mouse weight. BALB/c mice at 20 weeks of age and MRL/lpr mice at 6 and 20 weeks of age (n=5 per group), were injected i.v. with 10 µg of 125I-labelled anti-DNP IgG1 mAb. They were placed under terminal anaesthesia and, after 5 min, were exsanguinated. Whole blood 125I c.p.m. were ascertained and, after correction for background, expressed as %ID/ml and plotted against animal weight. The equation of the interpolation line came from a non-linear regression analysis, performed using the least squares method, assuming an inverse relationship. The coefficient of determination, R2, was 0.76.

 
Endothelial expression of E- and P-selectin was then determined in the heart, lung, kidney and brain of MRL/lpr mice at different ages and in BALB/c controls. In the heart, lung and brain, there was no significant difference in E-selectin expression (Fig. 3A–C). However, the kidneys of both 16- and 22-week-old MRL/lpr mice (Fig. 3D) appeared to show a low level of E-selectin up-regulation that was significant when compared with 7-week-old mice and with age-matched BALB/c controls (P < 0.001). There was no detectable up-regulation of P-selectin (Fig. 3E–H) in any of the MRL/lpr organs studied, at any age. Immunohistochemistry confirmed the absence of E- and P-selectin expression in the heart, lung, liver and brain, and did not show E- or P-selectin staining in kidney sections.



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  		FIG. 3. E- and P-selectin expression in MRL/lpr and BALB/c mice. E-selectin (A–D) and P-selectin (E–H) expression was determined, as previously described, in the heart (A, E), lung (B, F), brain (C, G) and kidney (D, H) of BALB/c (white bars) and MRL/lpr (filled bars) mice at 7, 16 and 22 weeks (n=6 per group). Bars represent mean+standard deviation. There was no general up-regulation of E-selectin or P-selectin expression in the organs of MRL/lpr mice. However, a low level of persistent E-selectin expression was seen in the renal vasculature of 16- and 22-week-old mice (D). The data were analysed using a one-tailed ANOVA with Bonferroni correction. **P<0.001 comparing MRL/lpr at 16 or 22 weeks with age-matched BALB/c, and with MRL/lpr at 7 weeks of age. The results from this experiment are representative of three separate experiments.

 

LPS inducibility of selectin expression in MRL/lpr mice
In view of the failure to detect increasing endothelial E- and P-selectin expression with developing disease in MRL/lpr mice, we checked whether MRL/lpr mice had the capacity to express E- and P-selectins following stimulation with LPS. As shown in Fig. 4Go, E-selectin expression increased significantly with increasing dose of LPS in the heart, lung, brain and kidney (Fig. 4A–D). Although it appeared that the E-selectin response to LPS in the pulmonary and renal vasculature was greater in MRL/lpr than in BALB/c mice, this observation was not reproduced in subsequent experiments. P-selectin expression was significantly LPS inducible in the heart, lung and brain (Fig. 4E–G), with no difference between MRL/lpr and BALB/c. As previously observed for BALB/c mice, P-selectin was not induced by LPS in the renal vasculature of MRL/lpr mice (Fig. 4H).



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  		FIG. 4. LPS inducibility of E- and P-selectin expression in MRL/lpr and BALB/c mice. E-selectin (A–D) and P-selectin (E–H) expression was determined in the heart (A, E), lung (B, F), brain (C, G) and kidney (D, H) of BALB/c (open square) and MRL/lpr (solid square) mice at 20 weeks of age, 4 h after i.p. injection of varying doses of LPS (n=6 per group). Values shown are mean±standard deviation. With the exception of P-selectin expression in the kidney, both selectins were strongly LPS inducible in the organs of MRL/lpr as well as BALB/c mice. The data were analysed using the one-tailed ANOVA with Bonferroni correction. For each strain, mice administered 0.025 and 1 mg/kg LPS were compared with those given 0.0025 mg/kg: *P<0.05, **P<0.001. The results from this experiment are representative of two separate experiments.

 

Effect of MRL/lpr serum on EC selectin expression in culture
In our previous study, we found, in accordance with other reports [14, 15], that the serum of 20-week-old MRL/lpr mice contained raised levels of immunoreactive TNF{alpha}, in the order of 200 pg/ml, and IL-1{alpha} and IL-1ß at 50 and 100 pg/ml, respectively. Moreover, incubation of sEnd.1 cells in vitro with this serum led to up-regulation of ICAM-1 and VCAM-1, and this was inhibited by a cocktail of anti-TNF{alpha} and anti-IL-1 antibodies [8]. When sEnd.1 cells were incubated with varying concentrations of TNF{alpha}, we found that concentrations similar to those found in the serum of 20-week-old MRL/lpr mice detectably induced both E- and P-selectin (Fig. 5A). However, incubation with serum from 20-week-old MRL/lpr mice failed to stimulate E- and P-selectin expression (Fig. 5B, C).



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  		FIG. 5. Effect of TNF{alpha} and serum from 20-week-old MRL/lpr mice on selectin expression by mouse EC. (A) Expression of E-selectin (circles, dashed line) and P-selectin (triangles, solid line) by sEnd.1 cells was ascertained by flow cytometry. Relative fluorescence intensity (RFI) was obtained by dividing the mean FI of anti-selectin mAb-incubated cells by that of control mAb-incubated cells. Selectin up-regulation from the basal level is expressed as a percentage maximum response. sEnd.1 cells expressed both selectins in response to TNF{alpha}. Expression of E-selectin (B) and P-selectin (C) by sEnd.1 cells was then determined after 4 h incubation with HBSS, TNF{alpha} (10 ng/ml), or serum from 20-week-old BALB/c or MRL/lpr mice. Neither MRL/lpr nor BALB/c serum up-regulated E- or P-selectin expression on sEnd.1 cells. The data shown are from one experiment, representative of three.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The measurement of EC surface determinants using i.v. injected radio-labelled mAb is a relatively new technique that allows a detailed analysis of changes in the EC surface over the course of inflammatory responses. The correlation between this measure and the amount of LPS administered suggested that it was indeed a quantitative measure of expression of E- and P-selectins.

Our studies in MRL/lpr mice showed, first, that vascular endothelium did not systemically up-regulate E- and P-selectins during the development of disease, with the possible exception of a persistent low level of E-selectin expression in the kidneys of older mice. However, immunostaining failed to confirm this observation, presumably because the low level of expression lay below the sensitivity of the technique. Second, the lack of selectin up-regulation did not reflect an underlying defect in the inducibility of these molecules. When challenged with i.p. LPS, MRL/lpr mice expressed the selectins in several organs at least to the same degree as BALB/c controls. Third, serum collected from older MRL/lpr animals did not induce selectin expression on mouse endothelioma cells in culture. These results were surprising, as TNF{alpha} and IL-1 are known to be up-regulated in MRL/lpr mice and the induction of E- and P-selectin in response to these cytokines in mice is well described [16].

The most obvious explanation for these observations is that the static or slowly changing local concentrations of cytokines that characterize chronic inflammation result in progressive down-regulation of E- and P-selectin expression by EC. The mechanism of this down-regulation might involve the re-synthesis of the intracellular NF{kappa}B inhibitor, I{kappa}B [17, 18]. However, if this was the sole mechanism, MRL/lpr serum should have induced selectin expression on cytokine-naive sEnd.1 cells in culture. It is therefore possible that MRL/lpr serum contains a factor (or factors) which down-regulates the selectin response to EC activation (but not the ICAM-1 and VCAM-1 response [8]). While the identity of this putative factor(s) is a matter of speculation, the cytokines interferon {gamma} (IFN{gamma}), IL-4 and transforming growth factor ß (TGFß) are candidate molecules, as each has been shown to have such an action in vitro [19–22]. Furthermore, their over-production has been reported in the MRL/lpr model [14, 23, 24]. Further studies that address this issue are planned. While the precise mechanism of E- and P-selectin down-regulation remains to be defined, one can speculate that it might be protective, by limiting leucocyte–EC interactions and thereby reducing vascular injury and tissue damage.

The observation that P-selectin is not inducible in the mouse kidney has been reported elsewhere [25], and noted by us in a model of nephritis induced by nephritogenic rabbit serum (data not shown). These studies are at variance with earlier reports of functional P-selectin expression in murine nephritis [26, 27], and argue against P-selectin having an important role in leucocyte recruitment to the kidney. The mechanisms underlying the heterogeneity of selectin gene expression in different vascular beds remain unknown.

In spite of the lack of selectin expression, on-going mononuclear cell accumulation in and around blood vessels clearly occurs in MRL/lpr mice [3]. It is likely that the tethering and rolling of leucocytes on EC in this context is mediated by other molecules, including VCAM-1 [8, 28, 29] and/or L-selectin ligands [30]. Furthermore, at some sites of pathology, rolling on EC is not an essential prerequisite to leucocyte extravasation, as, due to the narrow luminal diameter, leucocytes remain in contact with EC throughout their passage through the capillary bed. There is evidence that rolling is not necessary in the pulmonary vasculature [31] and hepatic sinusoidal system [32], and it may be supposed that similar considerations might apply to the glomerular tuft.

Little is known about EC activation in lupus. This study suggests that, unlike the integrin ligands ICAM-1 and VCAM-1 [8], the role of endothelial selectins in on-going basal leucocyte–EC interactions may be limited. However, as endothelium remains able to express E- and P-selectin in response to LPS, one could postulate that a rapid shift in the inflammatory milieu (such as that brought about by infection) might lead to the brief expression of selectins and a wave of leucocyte recruitment. Such a potentiation of leucocyte flux in ‘acute-on-chronic’ inflammation could explain the observation that mortality after LPS administration is higher in MRL/lpr mice than controls [33] and that low-dose LPS administration exacerbates glomerulonephritis in this strain [34]. An analogous observation in human SLE is that infective episodes stimulate lupus disease activity [35].

In summary, we have found that E- and P-selectins are not expressed by endothelium in multiple organs during the development of disease in MRL/lpr mice. However, the two selectins remained inducible by LPS. These observations lead to a model of acute-on-chronic endothelial activation in which activation of E- and P-selectin expression in response to intercurrent infection might exacerbate established dysfunctional leucocyte–EC interactions. In patients with SLE, such a mechanism might provide an important link between infections and disease activity.


   Acknowledgments
 
O.A.H. was funded by the Wellcome Trust and D.M. and J.F.McH were funded by the Medical Research Council. D.O.H. is funded by the British Heart Foundation. We thank Dr Michael Robson for his assistance.


   Notes
 
Correspondence to: D. O. Haskard Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 1 November 2000; Accepted 7 March 2001


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