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The Journal of Immunology, 1999, 163: 3993-4000.
Copyright © 1999 by The American Association of Immunologists

TNF-{alpha} and IL-1 Sequentially Induce Endothelial ICAM-1 and VCAM-1 Expression in MRL/lpr Lupus-Prone Mice1

Julie F. McHale, Olivier A. Harari, Diane Marshall and Dorian O. Haskard2

British Heart Foundation Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dysfunctional leukocyte-endothelial interactions are thought to play a key role in systemic lupus erythematosus pathogenesis. We questioned the importance of TNF-{alpha} and IL-1 for endothelial activation in MRL/lpr lupus-prone mice. Endothelial ICAM-1 and VCAM-1 expression increased significantly with disease evolution in kidney, heart, and brain, as shown by i.v. injected radiolabeled Ab uptake. Lung endothelial VCAM-1 also increased, while lung endothelial ICAM-1 did not rise above a high basal level. Immunoassays showed a significantly raised circulating level of TNF-{alpha} by 14 wk, with levels of circulating IL-1{alpha} and IL-1ß being additionally raised by 20 wk. With 14-wk-old MRL/lpr, anti-TNF-{alpha} antiserum inhibited expression of ICAM-1 and VCAM-1 by endothelial cells cultured with sera in vitro, and uptake of anti-ICAM-1 and anti-VCAM-1 mAb in lung, kidney, brain, and heart in vivo. In contrast, both anti-TNF-{alpha} and anti-IL-1 antisera were required for maximal inhibition in vitro and in vivo at 20 wk. These data indicate that TNF-{alpha} is largely responsible for the early up-regulation of endothelial ICAM-1 and VCAM-1, but that IL-1 enhances expression in late disease. Our observations provide novel insights of possible relevance to understanding endothelial activation in systemic lupus erythematosus, and highlight an approach that can be extended to dissecting other chronic inflammatory diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE)3 is a systemic autoimmune disease, characterized by widespread microvascular injury involving the kidneys, skin, lung, brain, joints, and other organs (1, 2). Of the mouse strains that develop autoimmune disease similar to human SLE, one of the best characterized is the MRL/MpJ-Faslpr mouse (MRL/lpr) (3). The lpr defect in these mice is due to a mutation in the Fas gene, a cell surface molecule of the TNF-receptor family involved in mediating apoptosis (4). Mononuclear cell infiltration of tissues, including lung, kidney, heart, and brain, is present as early as 8 wk after birth and increases steadily with age (5, 6, 7, 8). MRL/lpr mice die at between 7 and 9 mo of age, with renal failure being the principal cause of death (3).

There has been considerable progress over recent years in understanding the mechanisms by which leukocytes emigrate from the circulation into inflamed tissues. Thus, leukocyte recruitment typically involves a coordinated sequence of adhesion and activation events, consisting of rolling, firm adhesion, and transmigration into the tissues (9). ICAM-1 and VCAM-1 are members of the Ig superfamily and, when expressed by endothelial cells (EC), act respectively as ligands for leukocyte ß2 (i.e., ß2{alpha}L/LFA-1; ß2{alpha}M/Mac-1) and {alpha}4 (i.e., {alpha}4ß1/VLA-4; {alpha}4ß7/LPAM) integrins (9). Whereas ICAM-1 is involved in the ß2 integrin-mediated firm adhesion of leukocytes that are already rolling on endothelium, VCAM-1 may support both initial tethering and firm adhesion of cells expressing {alpha}4 integrins (e.g., monocytes and lymphocytes) (10, 11). Expression by EC of ICAM-1 and VCAM-1 is regulated by cytokines such as TNF and IL-1 (12).

Although the development of inflammatory lesions in both human and murine lupus is typically associated with leukocyte emigration into affected tissues, the mechanisms underlying this leukocyte redistribution are still unknown. However, EC expression of ICAM-1 and/or VCAM-1 has been demonstrated in MRL/lpr tissues (13, 14, 15, 16), suggesting an increased capacity of endothelium to support interactions with leukocytes. Furthermore, recent reports have shown that disease progression in MRL/lpr mice can be attenuated by anti-ICAM-1 mAb (17), or by back-crossing with ICAM-1 gene-targeted mice (18, 19). Although MRL/lpr mice are known to show increased expression of TNF-{alpha}, IL-1{alpha}, and IL-1ß (20, 21, 22, 23, 24, 25, 26, 27), the relative importance of these cytokines in stimulating EC expression of ICAM-1 and VCAM-1 during the course of the disease has not yet been determined.

In previous studies, our group has developed the use of i.v. injection of radiolabeled Abs to quantify lumenal expression of endothelial adhesion molecules in models of cutaneous inflammation in the pig (28, 29). The same technique has also been used to study expression of E- and P-selectins, ICAM-1, and VCAM-1 in models of inflammation in the rat (30, 31) and mouse (32, 33, 34). In this work, we report the application of this approach to dissecting the relative roles of TNF-{alpha} and IL-1 in the regulation of endothelial ICAM-1 and VCAM-1 expression during the development of disease in MRL/lpr mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Female BALB/c, MRL/lpr, and MRL/++ mice were purchased from Harlan Olac (Bicester, Oxon, U.K.). MRL/++ have the same genetic background as MRL/lpr mice, but lack the lpr mutation, and do not develop renal disease until the second year of life (35). All mice were kept in the same controlled climatic conditions, in filter-topped microisolator cages, with autoclaved bedding. Irradiated food and drinking water were made freely available. Urine samples were tested for protein using Hema-combistix (Bayer Diagnostics, Basingstoke, U.K.). Blood urea levels were estimated using Azostix (Bayer Diagnostics). All animals were housed and studied according to U.K. Home Office guidelines. Sentinel mice were housed alongside test animals and screened regularly for a standard panel of murine pathogens.

Abs and cell lines

The hybridoma lines for the rat anti-mouse mAb YN-1/1.7.4 (IgG2a, anti-ICAM-1), M/K 2.7 (IgG1, anti-VCAM-1), were obtained from the American Type Culture Collection (Manassas, VA). The hybridoma line for the rat anti-mouse anti-CD31 mAb EA3 (IgG2a) was a kind gift from Dr. Beat Imhof, University of Geneva. Irrelevant control mAbs (rat IgG1 and rat IgG2a) were raised against DNP and were a gift from Dr. David Gray (Imperial College School of Medicine, Hammersmith Hospital, London, U.K.). mAb were purified from tissue culture supernatants by protein G affinity chromatography (Pharmacia, Uppsala, Sweden). Sheep anti-mouse TNF-{alpha} antiserum was a gift from Dr. Tony Meagher, National Institute for Biological Standards and Control (Potters Bar, U.K.). Sheep anti-mouse IL-1{alpha} and IL-1ß antisera were gifts from Dr. Roberto Solari (Glaxo-Wellcome Laboratories, Stevenage, U.K.). Control sheep serum was purchased from Dakopatts (Glostrup, Denmark). The murine EC line bEnd-3 (36) was kindly supplied by Dr. M. Robinson (Celltech, Slough, U.K.) and grown in DMEM with 10% FCS, 50 U/ml penicillin (Life Technologies, Paisley, U.K.), 50 µg/ml streptomycin (Life Technologies), and 2 mM L-glutamine (ICN Biomedicals, Costa Mesa, CA) in 5% CO2 at 37°C.

Immunohistochemistry

To localize the uptake in tissues of mAb that had been injected i.v., animals were given 20 µg of either YN-1, M/K 2.7, or control Ab i.v. and 5 min later perfused (as for radiolabeled Ab targeting below) with 2% paraformaldehyde-lysine-periodate (37) under terminal anesthesia. Tissue samples were collected, snap frozen, and subsequently stained to localize the binding of the primary Ab using biotinylated rabbit anti-rat Ig, streptavidin-biotin-HRP, and diaminobenzidine for detection.

Ab radiolabeling

All radioisotopes were purchased from Amersham International (Little Chalfont, U.K.). Abs were labeled with 125I, 111In, or 99mTc, as previously described (34).

Ab targeting

In a preliminary experiment, we established that the distribution of radiolabeled mAb to kidney, brain, lung, and heart 5 min after injection was not influenced by whether the Ab was labeled with 111In, 99mTc, or 125I. Accordingly, in additional experiments, animals received an injection via the tail vein of a mixture of specific and irrelevant control Abs differentially radiolabeled with these three isotopes. Syringes were weighed before and after injection to allow accurate determination of the injected dose (ID). Five minutes after injection, each animal was killed by i.p. injection of an overdose of pentobarbitone sodium (Rhone Merieux, Harlow, Essex, U.K.), and the vasculature was perfused with 20 ml PBS containing 10 U/ml of heparin (Leo Laboratories, Prince Risborough, U.K.), as described (34). Tissue samples were then collected and weighed, and their radioactivity was counted in an automated gamma counter. A standard aliquot of the injected solution was also counted to allow calculation of ID. After corrections for background, spillover between isotopes, and decay, the tissue uptake of each Ab in kidney, lung, heart, and brain was expressed as percentage of ID/g. The specific tissue uptake of each test Ab was then calculated by subtracting the uptake of irrelevant control mAb. In preliminary experiments in normal mice, we determined that the plasma concentration of radiolabeled mAb following i.v. injection was directly inversely related to body weight. Because the body weight of mice increased markedly from 6–20 wk in both BALB/c (18–26 g) and MRL/lpr (22–35 g) mice, in each mouse the specific tissue uptake of test Ab was normalized for body weight to give a concentration factor (CF). Thus, CF = (uptake of test mAb (% ID/g) - uptake of irrelevant control mAb (% ID/g)) x body weight in grams.

A previous study has indicated that the distribution in the mouse of anti-CD31 following i.v. injection can be used to estimate differences between organs in vascularity and endothelial cell surface area (33). Each mouse received 111In-labeled anti-DNP IgG2a control mAb (100 µg) and 100 µg 125I -labeled mAb EA3 anti-CD31 (100 µg), after which the anti-CD31 CF was calculated for lung, heart, kidney, and brain. The relative vascularity (RV) of each of the four organs was then established by dividing the anti-CD31 CF by the anti-CD31 CF for the organ with the lowest anti-CD31 uptake (i.e., brain). On this basis, we calculated an RV of 1, 8.6, 11.2, and 55.9 for brain, kidney, heart, and lung, respectively.

To measure endothelial expression of ICAM-1 and VCAM-1 in vivo, animals received an injection via the tail vein of a mixture (20 µg each) of 99mTc-labeled mAb YN-1/1.7.4, 111In-labeled mAb M/K 2.7, and 125I-labeled control anti-DNP Ab (either IgG1 or IgG2a), 5 min before the end of each experiment. The IgG1 or IgG2a anti-DNP Abs had identical in vivo distributions and were used interchangeably as the negative control, in view of the anti-VCAM-1 (IgG1) and anti-ICAM-1 (IgG2a) being of different isotypes. After calculation of the CF for anti-ICAM-1 and anti-VCAM-1, the values for each organ were divided by the RV for that organ to give a vascularity-corrected CF.

ELISAs for cytokine levels

Blood was aspirated from the left ventricle and allowed to clot at 4°C overnight. Serum was then separated and frozen at -20°C before assay of levels of TNF-{alpha}, IL-1{alpha}, and IL-1ß, using ELISA kits purchased from Genzyme (Cambridge, MA).

Stimulation of ICAM-1 and VCAM-1 expression with mouse sera

Mouse sera, which had been collected and stored as for ELISAs, were incubated at 1:2 in tissue culture medium with bEnd.3 EC for 8 h. After this, cells were analyzed for expression of ICAM-1 and VCAM-1 by flow cytometry, using FITC-conjugated rabbit anti-rat Ig (Dako, Carpenteria, CA) for detection. In some wells, EC were incubated with mouse sera in the presence of sheep anti-mouse TNF-{alpha} antiserum, and/or a combination of sheep antisera against mouse IL-1{alpha} and IL-1ß (i.e., anti-IL-1).

Inhibition of ICAM-1 and VCAM-1 expression in vivo with polyclonal antisera

In a previous study, we have characterized the ability of polyclonal rabbit antisera against TNF-{alpha}, IL-1{alpha}, and IL-1ß to inhibit EC expression of ICAM-1 and VCAM-1 in mouse skin following injection of the respective recombinant cytokine (34). In the present study, mice received an i.p. injection of anti-mouse TNF-{alpha} antiserum (100 µl), a combination of anti-IL-1{alpha} (50 µl) and anti-IL-1ß (50 µl) antisera (i.e., anti-IL-1), anti-TNF-{alpha} and anti-IL-1, or control sheep serum. To control for volume, each injection was made up to 200 µl with control sheep serum. Expression of ICAM-1 and VCAM-1 was measured by the radiolabeled mAb uptake technique 24 h after the i.p. injection of rabbit antisera.

Statistics

As the sample sizes in the experiments performed in this project were usually less than 10, a normal distribution was not assumed, and comparisons were therefore made using the Mann-Whitney U test. Differences between groups were described as significant when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To confirm that the MRL/lpr mice used in this study were developing autoimmune disease within a characteristic time scale, we monitored proteinuria and blood urea at various ages. No significant proteinuria or elevation of blood urea was observed in 6-wk-old MRL/lpr or in control BALB/c and MRL/++ mice up to 20 wk of age. By 14 wk, MRL/lpr mice had developed detectable proteinuria, and by 20 wk this had increased markedly. The blood urea was raised in 50% and 100% of MRL/lpr mice by 14 and 20 wk, respectively. Conventional histological staining of kidneys, lungs, hearts, and brains showed minimal abnormalities in MRL/lpr mice at 6 wk. By 14 wk, there was a definite mononuclear cell infiltration of kidney, lung, heart, and brain, and this was more marked at 20 wk. For the purposes of further investigations, we therefore chose to focus on mice at 6 wk (i.e., before overt disease), 14 wk (i.e., moderate disease), and 20 wk (i.e., severe disease).

Endothelial expression of ICAM-1 and VCAM-1

Standard immunohistochemical staining of kidney, lung, heart, and brain showed that in each organ there was increased expression of ICAM-1 by EC as the MRL/lpr mice aged and also by several extravascular cell types (not shown). On the other hand, staining of 20-wk-old MRL/lpr mouse tissues with rabbit anti-rat Ig following an injection i.v. of anti-ICAM-1 or anti-VCAM-1 mAb showed rat Ig localized specifically on endothelium, as for example in kidney (Fig. 1Go). In view of the difficulties of quantifying immunohistochemical staining, we then utilized a radiolabeled Ab-targeting technique to directly measure changes in endothelial ICAM-1 and VCAM-1 expression over the course of the disease.



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  		FIGURE 1. Kidney localization of anti-ICAM-1 and anti-VCAM-1 Abs following i.v. injection. Twenty-week-old MRL/lpr mice received an i.v. injection 5 min before sacrifice of either anti-ICAM-1 Ab (A, x400), anti-VCAM-1 Ab (B, x200), or control IgG (C, x200). The kidneys were snap frozen and immunostained with a rabbit anti-rat Ig to detect mAb localized within the tissue. Brown staining indicates the endothelial localization of anti-ICAM-1 and anti-VCAM-1 Abs.

 
We initially studied mice at 6 wk of age (i.e., before the onset of disease), to determine whether there are intrinsic differences between organs of MRL/lpr and BALB/c in the expression by endothelium of ICAM-1 and VCAM-1. Mice were injected i.v. with a mixture of 99mTc-labeled anti-ICAM-1, 111In-labeled anti-VCAM-1, and 125I-labeled nonbinding control mAb, after which the CF for each Ab in heart, lung, brain, and kidney was calculated and then corrected by dividing by the RV for that organ (Fig. 2Go). Although no significant differences were observed between the two strains at this age, the vascularity-corrected uptake of anti-ICAM-1 mAb in lung was ~3-fold greater than in kidney, heart, and brain, whereas uptake of anti-VCAM-1 mAb was 4- to 5-fold greater in kidney compared with the other organs.



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  		FIGURE 2. Vascularity-corrected organ uptake of anti-ICAM-1 and anti-VCAM-1 mAb in 6-wk-old BALB/c and MRL/lpr mice. Six-week-old BALB/c (open bars) or MRL/lpr (filled bars) mice received an i.v. injection of 99mTc-labeled anti-ICAM-1 mAb YN-1, 111In-labeled anti-VCAM-1 mAb M/K 2.7, and 125I-labeled control IgG mAb. The CF for the uptake of anti-ICAM-1 mAb (top) and anti-VCAM-1 mAb (bottom) were corrected for vascularity by dividing by the RV obtained by the distribution of anti-CD31 mAb (see Materials and Methods). Data points are mean ± SD of six mice/group. Similar organ differences were observed in six other experiments with BALB/c mice at different ages (6–20 wk) and two other experiments with 6-wk-old MRL/lpr mice.

 
No significant age-related changes in Ab uptake were detected in BALB/c mice from 6–20 wk of age (data not shown). Furthermore, no differences in anti-ICAM-1 or anti-VCAM-1 mAb uptake were observed when 20-wk-old BALB/c mice were compared with 20-wk-old MRL/++ mice (data not shown). In contrast, marked age-related changes were detectable in MRL/lpr mice, with significantly increased uptake of both anti-ICAM-1 and anti-VCAM-1 in heart and brain at 14 wk and additionally in kidney at 20 wk (Fig. 3Go). For example, the mean anti-ICAM-1 uptake in heart was 1.8- and 2.6-fold greater at 14 and 20 wk, respectively, compared with uptake at 6 wk, while mean uptake of anti-VCAM-1 was 2.9- and 8.1-fold greater than baseline at these ages. Although uptake of anti-VCAM-1 was significantly increased in lung at 20 wk, this was not the case for anti-ICAM-1.



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  		FIGURE 3. Age-related increases in organ uptake of anti-ICAM-1 and anti-VCAM-1 mAb in MRL/lpr mice. MRL/lpr mice aged 6 (open bars), 14 (shaded bars), or 20 (filled bars) wk received an i.v. injection of 99mTc-labeled anti-ICAM-1 mAb YN-1, 111In-labeled anti-VCAM-1 mAb M/K 2.7, and 125I-labeled control IgG mAb. After 5 min, organs were removed, weighed, and counted. Data points are the mean ± SD of five mice per group, and for each organ were obtained by dividing individual values by the mean CF for that organ at 6 wk. It can be seen that organ uptake of anti-ICAM-1 and anti-VCAM-1 mAb increased with age. *, p < 0.05 compared with 6-wk-old MRL/lpr mice; +, p < 0.05 compared with 14-wk-old mice.

 
Levels of circulating TNF-{alpha} and IL-1 in MRL/lpr mice

We next investigated whether the increase in endothelial ICAM-1 and VCAM-1 detectable in vivo was paralleled by increased serum levels of TNF-{alpha}, IL-1{alpha}, and IL-1ß. The circulating cytokine levels detectable by ELISA in 6-wk-old animals were similar in MRL/lpr, BALB/c, and MRL/++ mice, and levels in the two control strains did not change with age (Fig. 4Go). By 14 wk, the concentration of immunoreactive TNF-{alpha} in the serum of the MRL/lpr mice was 148.3 ± 39.2 pg/ml (mean ± SD, n = 4), which was significantly greater (p < 0.05) than at 6 wk (37.2 ± 19.3 pg/ml), and there was a further significant increase by 20 wk (242.3 ± 103 pg/ml). Levels of IL-1{alpha} and IL-1ß were the same at 6 and 14 wk (IL-1{alpha}, 5.74 ± 1 pg/ml at 6 wk and 5.65 ± 2.3 pg/ml at 14 wk; IL-1ß, 61.9 ± 14.6 pg/ml at 6 wk and 75.9 ± 24.9 pg/ml at 14 wk), but had risen significantly by 20 wk (49.3 ± 20.3 pg/ml and 148 ± 60.8 pg/ml, respectively, both p < 0.05 compared with 14 wk).



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  		FIGURE 4. Serum cytokine levels in MRL/lpr, MRL/++, and BALB/c mice. Serum was collected at 6, 14, and 20 wk of age and tested by ELISA for immunoreactive TNF-{alpha} (A), IL-1{alpha} (B), and IL-1ß (C). Data points are mean + SD (n = 6 per group). Raised levels of TNF-{alpha} were detectable in MRL/lpr mice by 14 wk, and raised levels of IL-1{alpha} and IL-1ß by 20 wk. *, p < 0.05 compared with 6-wk-old MRL/lpr.

 
Mouse sera were incubated with bEnd-3 EC in vitro, to determine their ability to stimulate ICAM-1 and VCAM-1 expression. Sera from BALB/c mice (6–20 wk) or 6-wk-old MRL/lpr mice did not cause any up-regulation of ICAM-1 and VCAM-1 expression (Fig. 5GoA). In contrast, 14- or 20-wk-old MRL/lpr sera led to a readily detectable up-regulation of EC expression of both adhesion molecules. In the case of 14-wk-old serum, up-regulation of ICAM-1 and VCAM-1 expression could be almost completely inhibited by anti-TNF-{alpha} antiserum, while anti-IL-1 had no effect (Fig. 5GoB). In contrast, anti-TNF-{alpha} together with anti-IL-1 led to greater inhibition of ICAM-1 and VCAM-1 expression than anti-TNF-{alpha} alone when EC were incubated with serum from 20-wk-old MRL/lpr mice (Fig. 5GoC).



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  		FIGURE 5. Effect of MRL/lpr serum on expression of ICAM-1 and VCAM-1 by cultured endothelial cells. bEnd-3 cells were incubated for 8 h with TNF-{alpha} (10 ng/ml) or with pooled sera (n = 6/group) from MRL/lpr mice or BALB/c mice aged 6 (A), 14 (B), or 20 (C) wk, in the presence or absence of neutralizing antisera to TNF-{alpha} and/or IL-1. Following this, EC were analyzed by flow cytometry for expression of ICAM-1 and VCAM-1. Anti-TNF-{alpha} was sufficient to inhibit adhesion molecule expression induced by 14-wk-old MRL/lpr mouse serum, while anti-TNF-{alpha} and anti-IL-1 together were needed to inhibit adhesion molecule expression induced by serum from 20-wk-old MRL/lpr mice. Experiments shown in A–C were performed on separate EC cultures. Mean fluorescence intensities for ICAM-1 and VCAM-1 following stimulation of parallel cultures with TNF-{alpha} (10 ng/ml) were 12.6 and 6.32 (B), and 5.33 and 1.92 (C). The data shown are representative of three experiments for each time point.

 
Effect of anti-cytokine antisera on ICAM-1 and VCAM-1 expression in vivo

The results of the analysis of cytokines in MRL/lpr sera suggested that at 14 wk the cytokine that might be responsible for stimulating increased endothelial expression of ICAM-1 and VCAM-1 at 14 wk was TNF-{alpha}, but that by 20 wk IL-1{alpha} and/or IL-1ß might also play a role. Experiments were therefore performed to directly analyze the requirement for these cytokines in vivo, using anti-cytokine antisera injected i.p. 24 h before injecting radiolabeled Abs (Fig. 6Go). No effects of anti-cytokine antisera were detected in 6-wk MRL/lpr mice, other than significant inhibition (p < 0.01) of anti-ICAM-1 mAb uptake by lung endothelium when mice were pretreated with anti-IL-1. Because inhibition of IL-1 had similar effects in control 14-wk-old BALB/c mice, it seems likely that the high endothelial expression of ICAM-1 in mouse lung is maintained normally by the local generation of IL-1{alpha} and/or IL-1ß.



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  		FIGURE 6. The effect of antisera to TNF-{alpha} and/or IL-1 on the uptake of anti-ICAM-1 and anti-VCAM-1 mAb. BALB/c mice at 14 wk and MRL/lpr mice at 6, 14, and 20 wk received an i.p. injection of either control antiserum, anti-TNF-{alpha}, anti-IL-1, or anti-TNF-{alpha} and anti-IL-1 together. Twenty-four hours later, each mouse received an i.v. injection of 99mTc- labeled anti-ICAM-1 mAb YN-1, 111In-labeled anti-VCAM-1 mAb M/K 2.7, and 125I control IgG mAb. After 5 min, organs were removed, weighed, and counted. Data are expressed as the percentage of the specific tissue uptake seen in mice treated with control antiserum, and values are the mean ± SD of six animals per group for uptake of anti-ICAM-1 (open bars) and anti-VCAM-1 (filled bars). *, p < 0.05 compared with animals that received control antiserum; +, p < 0.05 compared with animals that received anti-TNF-{alpha} alone.

 
In 14-wk-old MRL/lpr mice, the uptake of both anti-ICAM-1 and anti-VCAM-1 mAb in heart, kidney, brain, and lung was significantly reduced by pretreatment with anti-TNF-{alpha} antiserum. Anti-IL-1 had a weak but significant (p < 0.05) inhibitory effect on anti-ICAM-1 mAb uptake in the heart, and had the same marked inhibitory effect on anti-ICAM-1 mAb uptake in lung as that seen at 6 wk. In contrast, anti-IL-1 had no effect on anti-ICAM-1 mAb uptake in brain and kidney and failed to inhibit significantly the uptake of anti-VCAM-1 mAb in any organ. Inhibition of both TNF-{alpha} and IL-1 did not lead to any further reduction in ICAM-1 and VCAM-1 expression over that seen with anti-TNF-{alpha} alone in heart, kidney, and brain or over that seen in lung with anti-IL-1 alone.

By 20 wk of age, the degree of inhibition of anti-ICAM-1 mAb uptake by anti-TNF-{alpha} alone was generally reduced compared with that seen at 14 wk, such that there was no longer statistically significant inhibition of anti-ICAM-1 mAb uptake in kidney, brain, or lung. However, ICAM-1 expression was significantly reduced in all four organs by inhibiting TNF-{alpha} and IL-1 together, when compared with animals that received control antisera. Furthermore, in contrast to 14-wk-old animals, the combination of anti-TNF-{alpha} and anti-IL-1 led to a further significant reduction (p < 0.05) in ICAM-1 expression in kidney compared with mice that received anti-TNF-{alpha} alone. As in 6- and 14-wk-old mice, ICAM-1 expression in lung was significantly reduced by inhibiting IL-1. In contrast to anti-ICAM-1 mAb uptake, inhibiting TNF-{alpha} led to similar reductions in VCAM-1 expression at 20 wk as at 14 wk, and inhibiting IL-1 led to no extra effect.

In summary, these data suggest that TNF-{alpha} has a driving role in promoting EC ICAM-1 and VCAM-1 expression during the onset of lupus-like disease in MRL/lpr mice, and that IL-1{alpha} and/or IL-1ß contribute to ICAM-1 but not VCAM-1 expression in late disease (i.e., at 20 wk).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although the importance of endothelium for the generation of inflammatory responses is now widely appreciated, there is little information available on how endothelial activation changes during the course of chronic inflammatory multisystem disease states, nor on the mechanisms involved. These questions can now be addressed using radiolabeled Abs to quantify surface expression of endothelial Ags in multiple organs in vivo (28, 29, 30, 31, 32, 33, 34). In this investigation, we have used this approach to dissect the kinetics and mechanisms of endothelial adhesion molecule expression during the course of the lupus-like disease of MRL/lpr mice.

We have been able to demonstrate that there are age-related increases in expression of ICAM-1 and/or VCAM-1 in heart, lung, brain, and kidney of MRL/lpr mice in parallel with the development of disease. These increases are superimposed upon levels of basal expression that differ between organs, but are similar in 6-wk MRL/lpr mice (i.e., before disease onset) to those seen in normal BALB/c mice. Thus, lung has a relatively high basal level of ICAM-1 expression, whereas VCAM-1 is prominent in kidney. A high uptake of anti-ICAM-1 mAb in lung was previously noted in the rat by Panes et al. (31). It is of interest that the lungs of MRL/lpr mice show a particular susceptibility to mononuclear cell infiltration (6), which is reduced by back-crossing MRL/lpr against ICAM-1 gene-targeted mice (18, 19). It is therefore possible that the high expression of ICAM-1 by lung endothelium renders this organ particularly susceptible to the expression of disease. On the other hand, the age-related increase in VCAM-1 expression superimposed upon the relatively high basal expression of VCAM-1 in kidney may provide an alternative adhesion pathway to explain why ICAM-1 deficiency was found not to prevent mononuclear cell infiltration of the kidney in MRL/lpr mice (18, 19).

Increased endothelial expression of ICAM-1 and/or VCAM-1 could clearly be due to factors acting in a paracrine fashion locally and/or to the presence of circulating factors able to activate endothelium generally. Because the uptake of anti-ICAM-1 in both BALB/c and MRL/lpr lungs was significantly inhibited by a combination of anti-IL-1{alpha} and IL-1ß, it seems likely that the high basal expression of endothelial ICAM-1 in mouse lung is due to the local generation of IL-1{alpha} and/or IL-1ß, perhaps in response to inhaled environmental stimuli. Moreover, IL-1-mediated basal stimulation of lung endothelial ICAM-1 expression may explain why the lung was the only one of the four organs in which an age-related increase in anti-ICAM-1 uptake was not detected. In 14-wk MRL/lpr mice, it is also possible that there may be local production of IL-1{alpha} and/or IL-1ß in the heart, because uptake of anti-ICAM-1 mAb was inhibited, albeit weakly, by anti-IL-1 antisera and there was no evidence for increased levels of circulating IL-1{alpha} or IL-1ß at that age. On the other hand, both the direct serum cytokine measurements and the inhibitory effects of anti-TNF-{alpha} antiserum in vitro strongly suggest that the generalized increase in ICAM-1 and VCAM-1 expression at 14 wk is at least in part related to the effects of increased circulating biologically active TNF-{alpha}. Using the same argument, circulating IL-1{alpha} and/or IL-1ß appear to contribute to increased ICAM-1 expression by 20 wk.

Whereas antisera against IL-1{alpha} and IL-1ß demonstrated that IL-1 contributes to the generalized ICAM-1 expression in MRL/lpr mice by 20 wk, we were unable to find any evidence for the in vivo regulation of VCAM-1 by IL-1, either basally in the lung or during the progression of disease in MRL/lpr. Although anti-IL-1 antisera did reveal that the IL-1{alpha} and/or IL-1ß present in 20-wk-old MRL/lpr sera contributed in vitro to the up-regulation of VCAM-1 by bEnd-3 cells, differences are known to exist between cultured EC in their capacity to express VCAM-1 in response to IL-1 (38). Furthermore, our observations in MRL/lpr mice support other in vivo work that has indicated that TNF-{alpha} is a more important agonist for EC VCAM-1 expression than IL-1 (29). It is possible that the impaired capacity of IL-1 to stimulate VCAM-1 expression is related to the presence of an IL-1-mediated transcriptional repressor in EC (39).

Determining the origin of the increased circulating TNF-{alpha}, IL-1{alpha}, and IL-1ß was beyond the aims of the present study. Previous investigations have reported raised levels of these and other cytokines in numerous tissues in MRL/lpr mice (20, 21, 22, 23, 24, 25, 26, 27), perhaps stimulated by autoantibody or immune-complex deposition (40). Although our data suggest that TNF-{alpha} is of primary importance for the up-regulation of EC ICAM-1 and VCAM-1 expression in MRL/lpr mice, and that IL-1 contributes to ICAM-1 expression in late disease, it is also possible that additional mechanisms may be involved. For example, endothelium might be stimulated by the surface deposition of activated complement components (41). Moreover, autoantibodies found in SLE against phospholipids, DNA, and other endothelial cell Ags have been shown to be capable of directly inducing EC adhesion molecule expression in vitro (42, 43).

Although our data indicate that there is an age-related increase in endothelial expression of ICAM-1 and VCAM-1 in MRL/lpr mice, this should not be seen as implying a generalized increase in leukocyte adhesion to endothelium and a subsequent nonselective transmigration of leukocytes into the tissues. In fact, our parallel unpublished experiments investigating expression of E- and P-selectins in MRL/lpr mice have shown that these EC adhesion molecules are not up-regulated with age to the same degree as ICAM-1 and VCAM-1, and this might be expected to impair neutrophil-EC interactions in most tissues. In the absence of endothelial selectins, two particular mechanisms might contribute to the development of the mononuclear cell tissue infiltrate that characterizes the tissues of MRL/lpr mice. First, selectin-independent leukocyte-EC contacts are formed normally during leukocyte passage through capillaries in lung (44), and probably also in other tissues. Increased expression of ICAM-1 and VCAM-1 in pulmonary and glomerular capillary beds might therefore be capable of promoting selectin-independent leukocyte recruitment into these susceptible organs. Second, the selective expression of {alpha}4 integrins on mononuclear cells, together with the capacity of {alpha}4 integrins to mediate the initial tethering of circulating leukocytes as well as their stable adhesion to endothelium (10, 11), may make {alpha}4 integrin-VCAM-1 interactions particularly important in this model, particularly in kidney, which we found to express VCAM-1 relatively highly. Further studies are therefore required to understand in detail the functional contributions of increased endothelial ICAM-1 and VCAM-1 expression in MRL/lpr mice and the relative roles of the two molecules in ongoing leukocyte-EC interactions.

There are a number of clinical immunocytochemical studies showing increased endothelial expression of ICAM-1 and VCAM-1 in SLE tissues, including skin (45), skeletal muscle (46), and kidney (47). Moreover, patients with SLE tend to have raised circulating levels of soluble VCAM-1 and, to a lesser extent, soluble ICAM-1 (48, 49). Because there is evidence that blood levels of TNF-{alpha} are raised in human SLE (50, 51, 52), it is possible that similar mechanisms of endothelial cell activation to those seen in MRL/lpr mice might operate in the human disease.

Because IL-1 production by several cell types is itself driven by TNF-{alpha} (53, 54, 55), it is possible that pretreating the mice with anti-TNF-{alpha} for longer than 24 h might have resulted in a lowering of circulating IL-1 levels and greater inhibition of adhesion molecule expression than was seen with the protocol we employed. If so, our study might be interpreted as predicting a beneficial therapeutic effect of inhibiting TNF-{alpha} in SLE through the down-regulation of endothelial activation and adhesion molecule expression. However previous studies in lpr and NZB/W lupus-prone mouse models have given conflicting results on the overall contribution of TNF-{alpha} to disease progression, concluding that TNF-{alpha} protects (56, 57) or accelerates disease (58, 59). With anti-TNF-{alpha} mAb entering clinical practice for the treatment of rheumatoid arthritis and inflammatory bowel disease (60, 61, 62), our data indicate that further research on the possible therapeutic efficacy of inhibiting TNF-{alpha} in human SLE would be worthwhile.

In summary, we have found that the development of lupus-like disease in MRL/lpr mice is related to systemic TNF-{alpha}-mediated endothelial activation and ICAM-1 and VCAM-1 expression, and that these changes are superimposed upon differences between organs in the basal expression of the two adhesion molecules. Besides suggesting a possible therapeutic benefit of modulating the endothelial cell response to cytokines in SLE, our study gives insight into the diversity in endothelial adhesion molecule expression between organs, which is likely to impact significantly on the tissue distribution of multisystem inflammatory diseases. We believe that the approach described in this study provides an important precedent for dissecting mechanisms underlying the expression of adhesion molecules and other endothelial Ags in other models of chronic inflammation.


   Acknowledgments
 
We thank Drs. David Gray, Tony Meagher, Beat Imhof, and Roberto Solari for their generous gifts of recombinant cytokines and Abs. We are very grateful to Drs. Sussan Nourshargh, Clive Landis, and Justin Mason for helpful discussions during the preparation of the manuscript.


   Footnotes
 
1 J.F.M. was a Medical Research Council Clinical Research Training Fellow. O.A.H. was a Wellcome Trust Clinical Research Training Fellow. D.O.H. was supported by a Medical Research Council Realising our Potential award and holds a British Heart Foundation professorial award. Back

2 Address correspondence and reprint requests to Dr. Dorian O. Haskard, British Heart Foundation Cardiovascular Medicine Unit, NHLI, Imperial College School of Medicine, Hammersmith Hospital, London W12 ONN, U.K. E-mail address: Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; CF, concentration factor; EC, endothelial cell; ID, injected dose; RV, relative vascularity. Back

Received for publication March 24, 1999. Accepted for publication July 19, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Isenberg, D. A., A. C. Horsfall. 1998. Systemic lupus erythematosus. ed. Oxford Textbook of Rheumatology 2nd Ed.1146. Oxford Medical Publications, Oxford.
  2. Belmont, H. M., S. B. Abramson, J. T. Lie. 1996. Pathology and pathogenesis of vascular injury in systemic lupus erythematosus: interactions of inflammatory cells and activated endothelium. Arthritis Rheum. 39:9.[Medline]
  3. Andrews, B. S., R. A. Eisenberg, A. N. Theofilopoulos, S. Izui, C. B. Wilson, P. J. McConahey, E. D. Murphy, J. B. Roths, F. J. Dixon. 1978. Spontaneous murine lupus-like syndromes: clinical and immunopathological manifestations in several strains. J. Exp. Med. 148:1198.[Abstract/Free Full Text]
  4. Watanabe-Fukunaga, R., C. I. Brannan, N. G. Copeland, N. A. Jenkins, S. Nagata. 1992. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314.[Medline]
  5. Cohen, P. L., R. A. Eisenberg. 1991. Lpr and gld: single gene models of systemic autoimmunity and lymphoproliferative disease. Annu. Rev. Immunol. 9:243.[Medline]
  6. Sunderrajan, E. V., W. N. McKenzie, T. R. Lieske, J. L. Kavanaugh, S. R. Braun, S. E. Walker. 1986. Pulmonary inflammation in autoimmune MRL/Mp-lpr/lpr mice: histopathology and bronchoalveolar lavage evaluation. Am. J. Pathol. 124:353.[Abstract]
  7. Moyer, C. F., J. D. Strandberg, C. L. Reinisch. 1987. Systemic mononuclear-cell vasculitis in MRL/Mp-lpr/lpr mice: a histologic and immunocytochemical analysis. Am. J. Pathol. 127:229.[Abstract]
  8. Vogelweid, C. M., G. C. Johnson, C. L. Besch-Williford, J. Basler, S. E. Walker. 1991. Inflammatory central nervous system disease in lupus-prone MRL/lpr mice: comparative histologic and immunohistochemical findings. J. Neuroimmunol. 35:89.[Medline]
  9. Springer, T. A.. 1995. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu. Rev. Physiol. 57:827.[Medline]
  10. Jones, D. A., L. V. McIntyre, C. W. Smith, L. J. Picker. 1994. A two-step adhesion cascade for T cell/endothelial cell interactions under flow conditions. J. Clin. Invest. 94:2443.
  11. Alon, R., P. D. Kassner, M. Woldemar Carr, E. B. Finger, M. E. Hemler, T. A. Springer. 1995. The integrin VLA-4 supports tethering and rolling in flow on VCAM-1. J. Cell Biol. 128:1243.[Abstract/Free Full Text]
  12. Pober, J. S., R. S. Cotran. 1990. Cytokines and endothelial cell biology. Physiol. Rev. 70:427.[Free Full Text]
  13. Wuthrich, R. P., A. M. Jevnikar, F. Takei, L. H. Glimcher, V. E. Kelley. 1990. Intercellular adhesion molecule-1 (ICAM-1) expression is up-regulated in autoimmune murine lupus nephritis. Am. J. Pathol. 136:441.[Abstract]
  14. Wuthrich, R. P.. 1992. Vascular cell adhesion molecule-1 (VCAM-1) expression in murine lupus nephritis. Kidney Int. 42:903.[Medline]
  15. Wang, W., S. Kobayashi, T. Uede. 1992. Dysregulated expression of cellular adhesion molecules in autoimmune-prone mice. J. Dermatol. 19:831.[Medline]
  16. Hayashi, Y., N. Haneji, K. Yanagi, H. Higashiyama, H. Yagita, H. Hamano. 1995. Prevention of adoptive transfer of murine Sjogren’s syndrome into severe combined immunodeficient (SCID) mice by antibodies against intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1). Clin. Exp. Immunol. 102:360.[Medline]
  17. Brey, R. L., A. A. Amato, K. Kagan-Hallet, C. B. Rhine, C. L. Stallworth. 1997. Anti-intercellular adhesion molecule-1 (ICAM-1) antibody treatment prevents central and peripheral nervous system disease in autoimmune-prone mice. Lupus Please verify journal title; it is not listed in our sources. 6:645.
  18. Lloyd, C. M., J.-A. Gonzalo, D. J. Salant, J. Just, J.-C. Gutierrez-Ramos. 1997. Intercellular adhesion molecule-1 deficiency prolongs survival and protects against the development of pulmonary inflammation during murine lupus. J. Clin. Invest. 100:963.[Medline]
  19. Bullard, D. C., P. D. King, M. J. Hicks, B. Dupont, A. L. Beaudet, K. B. Elkon. 1997. Intercellular adhesion molecule-1 deficiency protects mrl/mpj-fas(lpr) mice from early lethality. J. Immunol. 159:2058.[Abstract]
  20. Boswell, J. M., M. A. Yui, S. Endres, D. W. Burt, V. E. Kelley. 1988. Novel and enhanced IL-1 gene expression in autoimmune mice with lupus. J. Immunol. 141:118.[Abstract]
  21. Boswell, J. M., M. A. Yui, D. W. Burt, V. E. Kelley. 1988. Increased tumor necrosis factor and IL-1ß gene expression in the kidneys of mice with lupus nephritis. J. Immunol. 141:3050.[Abstract]
  22. Murray, L. J., R. Lee, C. Martens. 1990. In vivo cytokine gene expression in T cell subsets of the autoimmune MRL/Mp-lpr/lpr mouse. Eur. J. Immunol. 20:163.[Medline]
  23. Hamano, H., I. Saito, N. Haneji, Y. Mitsuhashi, N. Miyasaka, Y. Hayashi. 1993. Expressions of cytokine genes during development of autoimmune sialadenitis in MRL/lpr mice. Eur. J. Immunol. 23:2387.[Medline]
  24. Prud’homme, G. J., D. H. Kono, A. N. Theofilopoulos. 1995. Quantitative polymerase chain reaction analysis reveals marked overexpression of interleukin-1ß, interleukin-1 and interferon-{gamma} mRNA in the lymph nodes of lupus-prone mice. Mol. Immunol. 32:495.[Medline]
  25. Tsai, C.-Y., T.-H. Wu, S.-F. Huang, K.-H. Sun, S.-C. Hsieh, S.-H. Han, H.-S. Yu, C.-L. Yu. 1995. Abnormal splenic and thymic IL-4 and TNF{alpha} expression in MRL-lpr/lpr mice. Scand. J. Immunol. 41:157.[Medline]
  26. Yokoyama, H., B. Kreft, V. R. Kelley. 1995. Biphasic increase in circulating and renal TNF-{alpha} in MRL-lpr mice with differing regulatory mechanisms. Kidney Int. 47:122.[Medline]
  27. Lemay, S., C. Mao, A. K. Singh. 1996. Cytokine gene expression in the MRL/lpr model of lupus nephritis. Kidney Int. 50:85.[Medline]
  28. Keelan, E. T. M., S. T. Licence, A. M. Peters, R. M. Binns, D. O. Haskard. 1994. Characterization of E-selectin expression in vivo using a radiolabelled monoclonal antibody. Am. J. Physiol. 266:H279.
  29. Harrison, A. A., C. J. Stocker, P. T. Chapman, Y. T. Tsang, T. Y. Huehns, R. H. Gundel, A. M. Peters, K. A. Davies, A. J. George, M. K. Robinson, D. O. Haskard. 1997. Expression of VCAM-1 by vascular endothelial cells in immune- and non-immune inflammatory reactions in the skin. J. Immunol. 159:4546.[Abstract]
  30. Mulligan, M. S., A. A. Vaporciyan, M. Miyasaka, T. Tamatani, P. A. Ward. 1993. Tumor necrosis factor {alpha} regulates in vivo intrapulmonary expression of ICAM-1. Am. J. Pathol. 142:1739.[Abstract]
  31. Panes, J., M. A. Perry, D. C. Anderson, A. Manning, B. Leone, G. Cepinskas, C. L. Rosenbloom, M. Miyasaka, P. R. Kvietys, D. N. Granger. 1995. Regional differences in constitutive and induced ICAM-1 expression in vivo. Am. J. Physiol. 38:H1955.
  32. Eppihimer, M. J., B. A. Wolitzky, D. C. Anderson, M. A. Labow, D. N. Granger. 1996. Heterogeneity of expression of E- and P-selectins in vivo. Circ. Res. 79:560.[Abstract/Free Full Text]
  33. Henninger, D. D., J. Panes, M. Eppihimer, J. Russell, M. Gerritsen, D. C. Anderson, D. N. Granger. 1997. Cytokine-induced VCAM-1 and ICAM-1 expression in different organs in the mouse. J. Immunol. 158:1825.[Abstract]
  34. McHale, J., O. A. Harari, D. Marshall, D. O. Haskard. 1999. Vascular endothelial cell expression of ICAM-1 and VCAM-1 at the onset of eliciting contact hypersensitivity response in mice: evidence for a dominant role of TNF{alpha}. J. Immunol. 162:1648.[Abstract/Free Full Text]
  35. Kelley, V. E., J. B. Roths. 1985. Interaction of mutant lpr gene with background strain influences renal disease. Clin. Immunol. Immunopathol. 37:220.[Medline]
  36. Hahne, M., U. Jäger, S. Isenmann, R. Hallmann, D. Vestweber. 1993. Five tumor necrosis factor-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes. J. Cell Biol. 121:655.[Abstract/Free Full Text]
  37. McLean, I. W., P. K. Nakane. 1974. Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22:1077.[Abstract]
  38. Swerlick, R. A., K. H. Lee, L.-J. Li, N. T. Sepp, S. W. Caughman, T. J. Lawley. 1992. Regulation of vascular cell adhesion molecule 1 on human dermal microvascular endothelial cells. J. Immunol. 149:698.[Abstract]
  39. Gille, J., R. A. Swerlick, T. J. Lawley, S. W. Caughman. 1996. Differential regulation of vascular cell adhesion molecule-1 gene transcription by tumor necrosis factor {alpha} and interleukin-1{alpha} in dermal microvascular endothelial cells. Blood 87:211.[Abstract/Free Full Text]
  40. Vissers, M. C., J. C. Fantone, R. Wiggins, S. L. Kunkel. 1989. Glomerular basement membrane-containing immune complexes stimulate tumor necrosis factor and interleukin-1 production by human monocytes. Am. J. Pathol. 134:1.[Abstract]
  41. Tedesco, F., M. Pausa, E. Nardon, M. Introna, A. Mantovani, A. Dobrina. 1997. The cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity. J. Exp. Med. 185:1619.[Abstract/Free Full Text]
  42. Simantov, R., J. M. LaSala, S. K. Lo, A. E. Gharavi, L. R. Sammaritano, J. E. Salmon, R. L. Silverstein. 1995. Activation of cultured vascular endothelial cells by anti-phospholipid antibodies. J. Clin. Invest. 96:2211.
  43. Lai, K. N., J. C. Leung, K. B. Lai, K. C. Wong, C. K. Lai. 1996. Up-regulation of adhesion molecule expression on endothelial cells by anti-DNA autoantibodies in systemic lupus erythematosus. Clin. Immunol. Immunopathol. 81:229.[Medline]
  44. Mizgerd, J. P., B. B. Meek, G. J. Kutkoski, D. C. Bullard, A. L. Beaudet, C. M. Doerschuk. 1996. Selectins and neutrophil traffic: margination and Streptococcus pneumoniae-induced emigration in murine lungs. J. Exp. Med. 184:639.[Abstract/Free Full Text]
  45. Belmont, H. M., J. Buyon, R. Giorno, S. Abramson. 1994. Up-regulation of endothelial cell adhesion molecules characterizes disease activity in systemic lupus erythematosus. Arthritis Rheum. 37:376.[Medline]
  46. Pallis, M., D. K. Robson, D. O. Haskard, R. J. Powell. 1993. Distribution of cell adhesion molecules in peripheral muscle from patients with SLE. Ann. Rheum. Dis. 52:667.[Abstract/Free Full Text]
  47. Yokoyama, H., M. Takaeda, T. Wada, S. Ohta, Y. Hisada, C. Segawa, K. Furuichi, K. Kobayashi. 1997. Glomerular ICAM-1 expression related to circulating TNF-{alpha} in human glomerulonephritis. Nephron 76:425.[Medline]
  48. Wellicome, S., P. Kapahi, J. C. Mason, Y. Lebranchu, H. Yarwood, D. O. Haskard. 1993. Detection of a circulating form of vascular cell adhesion molecule-1: raised levels in rheumatoid arthritis and systemic lupus erythematosus. Clin. Exp. Immunol. 92:412.[Medline]
  49. Kling, E., S. Bieg, M. Boehme, W. A. Scherbaum. 1993. Circulating intercellular adhesion molecule 1 as a new activity marker in patients with systemic lupus erythematosus. Clin. Invest. 71:299.[Medline]
  50. Maury, C. P. J., A.-M. Teppo. 1989. Tumor necrosis factor in the serum of patients with systemic lupus erythematosus. Arthritis Rheum. 32:146.[Medline]
  51. Studnicka-Benke, A., G. Steiner, P. Petera, J. S. Smolen. 1996. Tumor necrosis factor {alpha} and its soluble receptors parallel clinical disease and autoimmune activity in systemic lupus erythematosus. Br. J. Rheumatol. 35:1067.[Abstract/Free Full Text]
  52. Gabay, C., N. Cakir, F. Moral, P. Roux-Lombard, O. Meyer, J. M. Dayer, T. Vischer, H. Yazici, P. A. Guerne. 1997. Circulating levels of tumor necrosis factor soluble receptors in systemic lupus erythematosus are significantly higher than in other rheumatic diseases and correlate with disease activity. J. Rheumatol. 24:303.[Medline]
  53. Dinarello, C. A., J. G. Cannon, S. M. Wolff, H. A. Bernheim, B. Beutler, A. Cerami, I. S. Figari, M. A. Palladino, J. V. O’Connor. 1986. Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1. J. Exp. Med. 163:1433.[Abstract/Free Full Text]
  54. Libby, P., J. M. Ordovas, K. R. Auger, A. H. Robbins, L. K. Birinyi, C. A. Dinarello. 1986. Endotoxin and tumor necrosis factor induce interleukin 1 gene expression in adult human vascular endothelial cells. Am. J. Pathol. 124:179.[Abstract]
  55. Brennan, F. M., A. Jackson, D. Chantry, R. Maini, M. Feldman. 1989. Inhibitory effect of TNF{alpha} antibodies on the synovial cell interleukin-1 production in rheumatoid arthritis. Lancet i:244.
  56. Jacob, C. O., H. O. McDevitt. 1988. Tumor necrosis factor-{alpha} in murine autoimmune "lupus" nephritis. Nature 331:356.[Medline]
  57. Gordon, C., G. E. Ranges, J. S. Greenspan, D. Wofsy. 1989. Chronic therapy with recombinant tumor necrosis factor-{alpha} in autoimmune NZB/NZW F1 mice. Clin. Immunol. Immunopathol. 52:421.[Medline]
  58. Brennan, D. C., M. A. Yui, R. P. Wuthrich, V. E. Kelley. 1989. Tumor necrosis factor and IL-1 in New Zealand Black/White mice: enhanced gene expression and acceleration of renal injury. J. Immunol. 143:3470.[Abstract]
  59. Zhou, T., C. K. Edwards, P. Yang, Z. Wang, H. Bluethmann, J. D. Mountz. 1996. Greatly accelerated lymphadenopathy and autoimmune disease in lpr mice lacking tumor necrosis factor receptor I. J. Immunol. 156:2661.[Abstract]
  60. Feldmann, M., M. J. Elliott, J. N. Woody, R. N. Maini. 1997. Anti-tumor necrosis factor-{alpha} therapy of rheumatoid arthritis. Adv. Immunol. 64:283.[Medline]
  61. Targan, S. R., S. B. Hanauer, S. J. van Deventer, L. Mayer, D. H. Present, T. Braakman, K. L. De Woody, T. F. Schaible, P. J. Rutgeerts. 1997. A short term study of chimeric monoclonal antibody cA2 to tumor necrosis factor {alpha} for Crohn’s disease. N. Engl. J. Med. 337:1029.[Abstract/Free Full Text]
  62. Stack, W. A., S. D. Mann, A. J. Roy, P. Heath, M. Sopwith, J. Freeman, G. Holmes, R. Long, A. Forbes, M. A. Kamm. 1997. Randomized controlled trial of CDP571 antibody to tumor necrosis factor {alpha} in Crohn’s disease. Lancet 349:521.[Medline]



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