MRL/lpr lupus-prone mice show exaggerated ICAM-1-dependent leucocyte adhesion and transendothelial migration in response to TNF-
D. Marshall1,
J. P. Dangerfield,
V. K. Bhatia,
K. Y. Larbi,
S. Nourshargh and
D. O. Haskard
BHF Cardiovascular Medicine Unit, Imperial College Faculty of Medicine, National Heart and Lung Institute, Hammersmith Hospital, London W12 ONN, UK
1 Present address: Celltech R&D Ltd, 216 Bath Road, Slough SL1 4EN, UK
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Abstract
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Objective. Endothelial activation and dysfunctional leucocyteendothelial interactions are thought to play key roles in the pathogenesis of systemic lupus erythematosus (SLE). The object of this study was to investigate directly the effect of increased endothelial adhesion molecule expression on leucocyteendothelial cell interactions, using the MRL/lpr mouse model.
Methods. Leucocyte rolling, arrest and transendothelial migration were quantified in the cremaster muscle microcirculation of 20-week-old MRL/lpr mice, using intravital microscopy. Endothelial adhesion molecule expression was quantified using intravenously injected radiolabelled monoclonal antibodies.
Results. Basal expression of intercellular adhesion molecule 1 (ICAM-1) by cremaster endothelium was 2-fold greater in MRL/lpr than in MRL/++ mice (P<0.05). There was a 1.6-fold increase in expression of vascular adhesion molecule 1 (VCAM-1), but no increase in E-selectin or P-selectin expression. Following intrascrotal injection of saline, no difference was detected in leucocyteendothelial interactions between MRL/lpr and control MRL/++ mice. In contrast, intrascrotal injection of tumour necrosis factor
(TNF-
) (2 h test period) led to significantly increased numbers of adherent and extravasated leucocytes in MRL/lpr (5.98±0.71 and 5.45±0.34 leucocytes per 100 µm vessel segment respectively) compared with MRL/++ mice (3.63±0.26 and 2.97±0.24 respectively, each P<0.05). Treatment of TNF-
-stimulated mice with anti-ICAM-1 F(ab')2 (YN1) abolished the difference between MRL/lpr and MRL/++ mice, whereas a negative control anti-DNP F(ab')2 had no effect.
Conclusions. MRL/lpr lupus-prone mice show exaggerated ICAM-1-dependent leucocyteendothelial interactions in response to TNF-
. Increased leucocyteendothelial interactions due to endothelial priming could contribute to the clinical link between infection and flares of lupus disease activity.
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Introduction
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Over the last 20 yr, the molecular mechanisms of leucocyteendothelial cell interactions in inflammation have become increasingly understood [1]. Thus, initial rolling of leucocytes on endothelium is mediated largely by selectins. During rolling, the affinity and/or avidity of surface integrins (e.g.
Lß2,
Mß2,
4ß1) may be increased via the actions of chemokines or other stimuli. This leads to the arrest and subsequent transendothelial migration of leucocytes, mediated in large part through interactions between integrins and their immunoglobulin superfamily ligands [intercellular adhesion molecule (ICAM)-1, ICAM-2 and vascular cell adhesion molecule (VCAM)-1]. Localization of leucocyte trafficking into inflamed tissues is achieved in part through the capacity of endothelial cells to regulate surface expression of P-selectin, E-selectin, ICAM-1 and VCAM-1 in response to cytokines and other factors [2, 3].
Dysfunctional leucocyte interactions with endothelium and consequent microvascular injury may underlie much of the pathology of systemic lupus erythematosus (SLE). Potential endothelial-activating factors include circulating tumour necrosis factor
(TNF-
) [4, 5], interleukin (IL)-1ß [6], lymphocyte-expressed CD40-ligand (CD154) [7], complement C5b-9 [8, 9] and anti-phospholipid and/or anti-ß2 glycoprotein-1 antibodies [10, 11]. Furthermore, during active disease there is evidence for both leucocyte and endothelial cell activation [12, 13]. It has been suggested that microvascular injury results from mechanisms similar to those operating in a systemic Shwartzman reaction [14].
The MRL/lpr mouse, in which there is a spontaneous mutation in fas, is a well-established model of lupus [1517]. In a previous study we demonstrated that MRL/lpr mice show increased endothelial expression of ICAM-1 and VCAM-1 in most organs examined (heart, brain, kidney, lung) in parallel with the development of disease. Furthermore, this was related to the appearance in the circulation of bioactive TNF-
, IL-1
and IL-1ß [18]. Because in most organs we were unable to find evidence for increased expression of P- and E-selectin [19], we proposed that the ongoing increased expression of integrin ligands may represent a state of endothelial priming that may amplify early leucocyteendothelial cell interactions occurring upon superimposed stimulation. In this paper we report experiments that examined this possibility directly, using intravital microscopy.
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Methods
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Animals
Male MRL/lpr and MRL/++ mice were purchased from Harlan Olac (Bicester, UK). 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 [20]. All mice were kept in the same controlled climatic conditions, in filter-topped micro-isolator cages with autoclaved bedding. Irradiated food and drinking water were made freely available. All animals were housed and studied according to UK Home Office guidelines. Sentinel mice were housed alongside test animals and screened regularly for a standard panel of murine pathogens. Animals were studied at 20 weeks of age, at which time, in our hands, MRL/lpr mice have established lupus-like disease [18].
Adhesion molecule expression
Expression of endothelial adhesion molecules in the cremaster muscle was quantified by measuring uptake of radiolabelled antibodies, as described previously [18]. Antibodies used were anti-P-selectin monoclonal antibody (mAb) RB40 (anti-P-selectin, a kind gift from Professor D. Vestweber, University of Munster, Germany), anti-E-selectin mAb MES-1 (kindly supplied by Dr D. Brown, Celltech R&D, Slough, UK), anti-VCAM-1 mAb MK2.7 and anti-ICAM-1 mAb YN-1. Both mAb MK2.7 and mAb YN-1/1.7.4 were obtained from hybridomas purchased from the American Type Culture Collection, Manassas, VA, USA. A rat anti-dinitrophenol (DNP) mAb (a kind gift from Professor D. Gray, Edinburgh University, Edinburgh, UK) was used as a control for non-specific in vivo antibody uptake.
Intravital microscopy
Intravital microscopy on the mouse cremaster muscle was performed as described previously, with the observer blind to the identity of the mice [21]. Briefly, mice were injected intrascrotally (i.s.) with recombinant mouse TNF-
(300 ng in 400 µl saline/mouse) or saline alone. Two hours later animals were anaesthetized by intraperitoneal administration of ketamine (100 mg/kg) and xylazine (10 mg/kg) and placed on a custom-built heated (37°C) microscope stage, where the surgical procedure was carried out. Following incision of the scrotum, one testis was gently withdrawn to allow the cremaster muscle to be incised and pinned out flat over the window in the microscope stage. The cremaster muscle was kept warm and moist by continuous application of warmed Tyrode balanced salt solution. The effects of inhibiting ICAM-1 were assessed by i.v. injection of F(ab')2 fragments of anti-mouse ICAM-1 mAb YN-1/1.7.4 or F(ab')2 fragments of isotype-matched irrelevant control mAb (anti-DNP) 2 h prior to TNF stimulation.
Postcapillary cremasteric venules (2040 µM in diameter) were viewed on an upright fixed-stage microscope (Axioskop FS; Carl Zeiss, Welwyn Garden City, UK) fitted with water-immersion objectives. Images were then captured using a colour video camera (Model KY-F55BE; JVC) and stored by videocassette recorder (Model MD830E; Panasonic, Bracknell, UK). Rolling cells were defined as cells moving slower than the flowing erythrocytes and rolling flux was then quantified as the number of rolling cells moving past a fixed point on the venular wall per minute for 5 min. Firmly arrested leucocytes were defined as cells that remained stationary for at least 30 s within a 100-µm segment of a venule. Extravasated leucocytes were defined as cells in the perivenular tissue adjacent to but remaining within a distance of 50 µm of a 100-µm vessel segment under study. In each animal, three to five vessel segments and three to four vessels were quantified and averages taken.
Statistics
As the sample sizes in the experiments were usually less than 10, a normal distribution was not assumed. Comparisons were therefore made using the MannWhitney U-test. Differences between groups were described as significant when P<0.05.
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Results
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In this study we analysed leucocyteendothelial interactions in 20-week-old MRL/lpr mice in comparison with age-matched MRL/++ mice. By 20 weeks, MRL/lpr mice have established murine lupus, as demonstrated clinically by proteinuria.
In order to determine whether there are increased leucocyteendothelial cell interactions in diseased MRL/lpr mice, we counted the number of leucocytes rolling and arresting on endothelium in the cremaster muscle. Two hours after injection of saline, the numbers of rolling and arrested cells were similar in MRL/lpr mice to those in MRL/++ control mice, indicating that basal leucocyte interactions in this tissue are not significantly different (Fig. 1
). Furthermore, no statistically significant difference in leucocyte rolling was detected in either strain 2 h after injection of TNF-
(300 ng). In contrast, TNF-
led to a significant increase in leucocyte arrest (to 5.98±0.71 leucocytes per 100 µm vessel segment; mean±S.E.M., P<0.05) and in leucocyte emigration into the perivascular tissue (to 5.45±0.34 leucocytes per 100 µm vessel segment; P<0.01) in MRL/lpr mice but not in MRL/++ controls. The TNF-
-induced responses in MRL/lpr mice corresponded to increases in arrest and transmigration of 87 and 184% respectively.
To determine whether enhanced leucocyteendothelial interactions could be related to altered expression of endothelial adhesion molecules, we compared expression of P-selectin, E-selectin, ICAM-1 and VCAM-1 in the two strains. As shown in Fig. 2
, there was no detectable difference between MRL/lpr and MRL/++ mice in cremaster muscle expression of P-selectin and E-selectin. On the other hand, endothelial ICAM-1 expression was significantly increased in MRL/lpr mice (2.00±0.40-fold; mean±S.E.M., P<0.05). VCAM-1 expression was also increased (1.63±0.29-fold), but this did not reach statistical significance. In a further experiment, we failed to show any increase in expression of P-selectin, E-selectin, ICAM-1 or VCAM-1 in either strain 2 h after i.s. injection of TNF-
(300 ng) (data not shown).

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FIG. 2. ICAM-1 expression is increased in the cremaster of MRL/lpr mice compared with MRL/++ mice. Mice received an i.v. injection of technetium-99m (99mTc)-labelled anti-P-selectin mAb, indium-111 (111In)-labelled anti-E-selectin mAb and iodine-125 (125I)-labelled control mAb, or an i.v. injection of 99mTc-labelled anti-ICAM-1 mAb, 111In-labelled anti-VCAM-1 mAb and 125I-labelled control mAb. Five minutes after mAb injection, the animals were killed and specific antibody uptake in cremaster tissue was determined. The figure shows the ratio of specific antibody uptake per gram in MRL/lpr cremaster to that in MRL/++ cremaster muscle. Data are mean and S.D. (n=10 per group). *P<0.05 compared with MRL/++ animals.
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Having determined that ICAM-1 expression was increased in unstimulated MRL/lpr cremaster endothelium, we tested the importance of ICAM-1 for TNF-
-stimulated leucocyte arrest and extravasation, using neutralizing anti-ICAM-1 mAb YN-1/1.7.4. As shown in Fig. 3
, pretreatment with F(ab')2 fragments of anti-ICAM-1 (150 µg/mouse) had no detectable effect on leucocyte rolling but abolished the enhanced leucocyte arrest (P<0.05) and transmigration (P<0.01) attributable to TNF-
in MRL/lpr mice.
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Discussion
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In this study we found that leucocyteendothelial interactions in the cremaster microcirculation of 20-week-old MRL/lpr mice were similar to those of control mice under basal conditions, but that leucocyte arrest and transmigration were significantly augmented 2 h after stimulation with TNF-
. Furthermore, TNF-
-stimulated leucocyte arrest and transmigration were abolished by anti-ICAM-1 mAb, suggesting a critical role for ß2 integrin interactions with ICAM-1.
Using i.v.-injected radiolabelled antibodies to quantify endothelial adhesion molecules, we found that the cremaster muscle microcirculation of MRL/lpr and MRL/++ mice had similar expression of P-selectin and E-selectin. This result is consistent with our previous work, in which we failed to find evidence for increased P-selectin in heart, brain, kidney and lung and for E-selectin expression in heart, brain and lung [19]. On the other hand, we were previously able to detect a small but significant increase in E-selectin in MRL/lpr kidney [19], and Hickey et al. [22] have recently reported evidence for increased P- and E-selectin expression in MRL/lpr skin, indicating a degree of organ heterogeneity in terms of endothelial selectin expression in this model. Nevertheless, our failure to demonstrate differences between the two strains in terms of leucocyte rolling flux is entirely consistent with the similarity in levels of expression of the two endothelial selectins in the cremaster microcirculation, although it remains possible that differences in rolling velocity might have been observed had this been quantified.
In contrast to P-selectin and E-selectin, we found evidence for an increase in endothelial ICAM-1 and VCAM-1 expression in the MRL/lpr cremaster muscle microcirculation, as in other tissues [18]. Under basal conditions this did not translate to an increase in the number of leucocytes arresting on endothelium, possibly because of an absence of local factors capable of activating integrin function. On the other hand, we found significantly augmented ICAM-1-dependent arrest and transmigration in MRL/lpr mice 2 h after stimulation with TNF-
.
TNF-
had no effect on leucocyteendothelial cell interactions in the MRL/++ strain, although there was a small but statistically insignificant reduction in the number of leucocytes rolling. The failure of TNF-
to stimulate leucocyte adhesion in MRL/++ mice is consistent with our previous work with C57BL/6 mice, in which a 2-h test period led to no significant increase in leucocyteendothelial cell interactions, while a 4-h test period gave very marked effects [21]. Indeed, these previous data led to the choice of dose and test period of TNF-
employed in the present study.
In interpreting the effect of TNF-
on leucocyte adhesion and transmigration in MRL/lpr mice, it is important to stress that the effect is likely to be mediated primarily on leucocyte integrin function rather than the expression of adhesion molecules by endothelial cells. Thus, in TNF-
-stimulated cremaster muscle there was no increased endothelial cell adhesion molecule expression detectable using the mAb technique, and no evidence for increased leucocyte rolling. On the other hand, TNF-
is able to induce rapid lymphocyte function-associated antigen-1 (LFA)-1-dependent and protein synthesis-independent neutrophil adhesion and extravasation in mouse cremasteric venules in vivo [21, 23], and is capable of directly stimulating mouse neutrophils in vitro [21, 24].
It is of interest to compare our results using anti-ICAM-1 mAb with the observations of Ley and colleagues [25, 26], in which ICAM-1 appeared not to be critical for TNF-
-stimulated leucocyte firm adhesion in cremasteric venules of C57BL/6 mice. In contrast to these observations, our data suggest that ICAM-1 is critical for TNF-
-enhanced leucocyteendothelial cell interactions in the MRL/lpr mice, supporting the concept that in this mouse strain the microvascular endothelium is primed through increased expression of ICAM-1.
A further question relates to the nature of the leucocytes interacting with endothelium. Detailed electron microscope analysis of TNF-
-stimulated cremasteric venules of C57BL/6 mice has shown that the only leucocytes entering the tissues within 4 h are neutrophils [21, 24], and this is likely to be the case in MRL/++ mice. The question of whether this is also the case in MRL/lpr mice will need further study. It remains possible that the increased arrest and transmigration of leucocytes in response to TNF-
could be at least partly due to the presence of a large population of CD4/CD8 double-negative T cells in MRL/lpr mice [27, 28].
In conclusion, our observations are consistent with a model of leucocyteendothelial cell interactions in MRL/lpr mice in which the microvasculature is primed through increased expression of ICAM-1 and VCAM-1. In the absence of increased local expression of selectins and leucocyte activating factors, there may be little augmentation of leucocyte adhesion to endothelium under basal conditions. Upon superimposed stimulation, however, leucocyte-activating factors such as TNF-
may be more effective in stimulating arrest because of pre-existing increased integrin ligand availability. This in turn may increase the potential of dysfunctional leucocyteendothelial cell interactions to cause microvascular injury, as proposed previously [29].
In summary, we have established for the first time that MRL/lpr lupus-prone mice have enhanced ICAM-1-dependent leucocyte adhesion to endothelium and transmigration into the tissues upon inflammatory cytokine stimulation. If the same holds true in human lupus, this could provide a link between intercurrent infections and activity of disease. Furthermore, as previous studies using ICAM-1-deficient mice have shown a critical role for ICAM-1 in the evolution of disease in the MRL/lpr model [30, 31], ß2-integrinICAM-1 interactions are a possible target for the treatment of acute flares in SLE.
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Acknowledgments
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This work was supported by a project grant from the Arthritis Research Campaign.
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Notes
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Correspondence to: D. Haskard, BHF Cardiovascular Medicine Unit, Imperial College School of Medicine, Hammersmith Hospital, London W12 ONN, UK. E-mail: d.haskard{at}ic.ac.uk 
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References
|
---|
- Springer TA. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu Rev Physiol 1995;57:82772.[CrossRef][ISI][Medline]
- Pober JS, Cotran RS. Cytokines and endothelial cell biology. Physiol Rev 1990;70:42751.[Free Full Text]
- Mantovani A, Garlanda C, Introna M, Vecchi A. Regulation of endothelial cell function by pro- and anti-inflammatory cytokines. Transplant Proc 1998;30:423943.[CrossRef][ISI][Medline]
- Maury CPJ, Teppo A-M. Tumor necrosis factor in the serum of patients with systemic lupus erythematosus. Arthritis Rheum 1989;32:14650.[ISI][Medline]
- Studnicka-Benke A, Steiner G, Petera P, Smolen JS. Tumour necrosis factor alpha and its soluble receptors parallel clinical disease and autoimmune activity in systemic lupus erythematosus. Br J Rheumatol 1996;35:106774.[CrossRef][ISI][Medline]
- Liou LB. Serum and in vitro production of IL-1 receptor antagonist correlate with C-reactive protein levels in newly diagnosed, untreated lupus patients. Clin Exp Rheumatol 2001;19:51523.[ISI][Medline]
- Koshy M, Berger D, Crow MK. Increased expression of CD40 ligand on systemic lupus erythematosus lymphocytes. J Clin Invest 1996;98:82637.[Abstract/Free Full Text]
- Magro CM, Crowson AN, Harrist TJ. The use of antibody to C5b-9 in the subclassification of lupus erythematosus. Br J Dermatol 1996;134:85562.[ISI][Medline]
- Tedesco F, Pausa M, Nardon E, Introna M, Mantovani A, Dobrina A. The cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity. J Exp Med 1997;185:161927.[Abstract/Free Full Text]
- Simantov R, LaSala JM, Lo SK et al. Activation of cultured vascular endothelial cells by anti-phospholipid antibodies. J Clin Invest 1995;96:22119.[ISI][Medline]
- Del Papa N, Guidali L, Sala A et al. Endothelial cells as target for antiphospholipid antibodies. Human polyclonal and monoclonal anti-beta 2-glycoprotein I antibodies react in vitro with endothelial cells through adherent beta 2-glycoprotein I and induce endothelial activation. Arthritis Rheum 1997;40:55161.[Medline]
- Molad Y, Buyon J, Anderson DC, Abramson SB, Cronstein BN. Intravascular neutrophil activation in systemic lupus erythematosus (SLE): Dissociation between increased expression of CD11b/CD18 and diminished expression of L-selectin on neutrophils from patients with active SLE. Clin Immunol Immunopathol 1994;71:2816.[CrossRef][ISI][Medline]
- Belmont HM, Abramson SB, Lie JT. Pathology and pathogenesis of vascular injury in systemic lupus erythematosus. Interactions of inflammatory cells and activated endothelium. Arthritis Rheum 1996;39:922.[ISI][Medline]
- Belmont HM, Buyon J, Giorno R, Abramson S. Up-regulation of endothelial cell adhesion molecules characterizes disease activity in systemic lupus erythematosus: the Shwartzman phenomenon revisited. Arthritis Rheum 1994;37:37683.[ISI][Medline]
- Andrews BS, Eisenberg RA, Theofilopoulos AN et al. Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains. J Exp Med 1978;148:1198215.[Abstract]
- Moyer CF, Strandberg JD, Reinisch CL. Systemic mononuclear-cell vasculitis in MRL/Mp-lpr/lpr mice. A histologic and immunocytochemical analysis. Am J Pathol 1987;127:22942.[Abstract]
- Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 1992;356:3147.[CrossRef][ISI][Medline]
- McHale JF, Harari OA, Marshall D, Haskard DO. TNFalpha and IL-1 sequentially induce endothelial ICAM-1 and VCAM-1 expression in MRL/lpr lupus-prone mice. J Immunol 1999;163:39934000.[Abstract/Free Full Text]
- Harari O, Marshall D, McHale J, Ahmed S, Haskard DO. Limited endothelial E- and P-selectin expression in MRL/lpr lupus-prone mice. Rheumatology 2001;40:88995.[Abstract/Free Full Text]
- Kelley VE, Roths JB. Interaction of mutant lpr gene with background strain influences renal disease. Clin Immunol Immunopathol 1985;37:2209.[ISI][Medline]
- Thompson RD, Noble KE, Larbi KY et al. Platelet-endothelial cell adhesion molecule-1 (PECAM-1)-deficient mice demonstrate a transient and cytokine-specific role for PECAM-1 in leukocyte migration through the perivascular basement membrane. Blood 2001;97:185460.[Abstract/Free Full Text]
- Hickey MJ, Bullard DC, Issekutz A, James WG. Leukocyteendothelial cell interactions are enhanced in dermal postcapillary venules of MRL/fas(lpr) (lupus-prone) mice: roles of P- and E-selectin. J Immunol 2002;168:472836.[Abstract/Free Full Text]
- Thorlacius H, Vollmar B, Guo Y et al. Lymphocyte function antigen 1 (LFA-1) mediates early tumour necrosis factor alpha-induced leucocyte adhesion in venules. Br J Haematol 2000;110:4249.[CrossRef][ISI][Medline]
- Christofidou-Solomidou M, Nakada MT, Williams J, Muller WA, DeLisser HM. Neutrophil platelet endothelial cell adhesion molecule-1 participates in neutrophil recruitment at inflammatory sites and is down-regulated after leukocyte extravasation. J Immunol 1997;158:48728.[Abstract]
- Ley K, Allietta M, Bullard DC, Morgan S. Importance of E-selectin for firm leukocyte adhesion in vivo. Circ Res 1998;83:28794.[Abstract/Free Full Text]
- Foy DS, Ley K. Intercellular adhesion molecule-1 is required for chemoattractant-induced leukocyte adhesion in resting, but not inflamed, venules in vivo. Microvasc Res 2000;60:24960.[CrossRef][ISI][Medline]
- Ford MS, Young KJ, Zhang Z, Ohashi PS, Zhang L. The immune regulatory function of lymphoproliferative double negative T cells in vitro and in vivo. J Exp Med 2002;196:2617.[Abstract/Free Full Text]
- Watanabe D, Suda T, Hashimoto H, Nagata S. Constitutive activation of the Fas ligand gene in mouse lymphoproliferative disorders. EMBO J 1995;14:128.[Abstract]
- Guadagno TM, Ohtsubo M, Roberts JM, Assoian RK. A link between cyclin A expression and adhesion-dependent cell cycle progression. Science 1993;262:15725.[ISI][Medline]
- Bullard DC, King PD, Hicks MJ, Dupont B, Beaudet AL, Elkon KB. Intercellular adhesion molecule-1 deficiency protects mrl/mpj-fas(lpr) mice from early lethality. J Immunol 1997;159:205867.[Abstract]
- Lloyd CM, Gonzalo J-A, Salant DJ, Just J, Gutierrez-Ramos J-C. Intercellular adhesion molecule-1 deficiency prolongs survival and protects against the development of pulmonary inflammation during murine lupus. J Clin Invest 1997;100:96371.[Abstract/Free Full Text]
Submitted 23 August 2002;
Accepted 19 December 2002