Significant role of Fas ligand-binding but defective Fas receptor (CD95) in lymph node hyperplasia composed of abnormal double-negative T cells

Abstract

The functional differences between two mutations of the Fas (CD95) locus, Fas^lpr (lpr) and Fas^lprcg (lpr^cg), were investigated using bone marrow (BM) transplantation on the C3H mouse background. Both lpr/lpr and lpr^cg/lpr^cg BM transferred caused lymph node (LN) hyperplasia in lpr/+ and lpr^cg/+ recipients, although it was clearly smaller than that in lpr/lpr and lpr^cg/lpr^cg recipients of lpr/lpr and lpr^cg/lpr^cg BM. In addition, both BM induced significantly larger LN hyperplasia in lpr^cg/+ than lpr/+ recipients. Appearance of CD4⁻ CD8⁻[double negative (DN)] T cells in the periphery is the most consistent phenotype of Fas mutations. Importantly, the proportion of DN T cells was higher in larger LN hyperplasia in the order of lpr/+, lpr^cg/+ and lpr/lpr or lpr^cg/lpr^cg recipients. On the other hand, both lpr/lpr and lpr^cg/lpr^cg BM transferred into wild-type (+/+) mice caused marked LN atrophy. The former, but not the latter, induced wasting syndrome. Fasl^g1d (gld)-homozygous lpr/lpr BM transferred into +/+ mice elicited LN hyperplasia of the same extent as that in lpr/lpr mice transferred with lpr/lpr BM, but not wasting syndrome. Taken together with the fact that DN T cells massively express Fas ligand (FasL), this study implied that FasL overexpressed on DN cells may be involved in the accumulation of DN T cells in LN, LN atrophy and wasting syndrome, and that lpr^cg Fas, which can bind to Fas ligand but not transduce apoptosis signal into cells, may modulate these pathological conditions by interfering with the binding of FasL to Fas.

Introduction

It is well known that the two autosomal-recessive mutant genes, Fas^1pr (lpr) and Fasl^g1d (gld), induce practically the same autoimmune and lymphoproliferative syndromes in mice, albeit their location on different chromosomes,^1,2 the former being on chromosome 19³ and the latter on chromosome 1.⁴ The induced pathology has been characterized by marked lymphadenopathy dominated by CD4⁻ CD8⁻ B220⁺[double-negative (DN)] T cells, hypergammaglobulinaemia and lupus erythematosus-like autoantibody formation.^1,5 To explain the similarity between lpr and gld diseases, Allen et al.⁶ hypothesized that lpr and gld are mutants of interacting molecules, such as receptor and ligand. Support for this hypothesis comes from the molecular analyses in which lpr encodes a non-expressing allele of the apoptosis gene, Fas,⁷ while gld encodes a defective allelle of the Fas ligand gene, Fasl.⁸ Moreover, a new allele of the Fas locus was discovered and named Fas^1prcg (lpr^cg) because it can complement with gld to induce the same lymphadenopathy as does lpr or gld: double heterozygous lpr^cg/+ gld/+ mice develop significant lymphoid hyperplasia as a result of the accumulation of DN T cells.⁹ The lpr^cg has a single base substitution, resulting in an amino acid substitution in the intracytoplasmic death domain that is critical for apoptosis signalling.⁷ As a result, the cells with this defect can bind with FasL but are resistant to apoptosis via the FasL (CD95L)–Fas (CD95) pathway. Previous in vivo experiments using thymectomy, athymic nude mice and bone marrow transplantation have revealed that the following are prerequisite for full development of lymphadenopathy in Fas-defective mice: the thymus must be present, although its genotype is irrelevant;^10,11 and the lpr mutation must be present in both bone marrow (BM) and lymph nodes (LN).^12–14 Although DN T cells are enormously accumulated in LN and spleen, they do not proliferate actively there.¹⁵ It is thus considered that the liver of lpr/lpr mice may be the site of the proliferation of DN T cells in the periphery.¹⁶ Overall, lymphadenopathy may occur through the following process: abnormal lymphocytes escape deletion in the thymus, move into the liver, proliferate there and then home into lymphoid organs, leading to marked LN hyperplasia. In the LN homing of these abnormal cells, l-selectin (CD62L) plays a critical role as in that of normal T cells.¹⁷ Curiously, however, profound aplasia of lymphoid organs occurred in +/+ recipients of lpr/lpr or lpr^cg/lpr^cg BM cells.^{11, 12, 13, 14,18} This phenomenon was explained by massive up-regulation of FasL on DN T cells: DN T cells do not home into +/+ LN but rather destroy LN tissues by inducing apoptosis with FasL up-regulated on them.¹⁹ It is intriguing in this respect that lpr^cg/lpr^cg DN T cells homed into lpr^cg/+ LN in lpr^cg/+ mice transferred with lpr^cg/lpr^cg BM¹⁴ and additionally that lpr^cg/+ gld/+ DN T cells caused hyperplasia of lpr^cg/+ LN, but mostly atrophy of lpr/+ LN when these LN were implanted into double heterozygous lpr^cg/+ gld/+ mice.²⁰ This implies that the expression of lpr^cg Fas may have a significant influence on the homing of DN T cells into LN.

In this study, we further investigated whether the expression of non-functional Fas with an ability to bind with FasL by lpr^cg acts favourably for the homing of DN T cells into LN using BM transplantation in congenic strains bearing lpr and lpr^cg on the same inbred background.

Materials and methods

Experimental animals

C3H/HeJ (+/+) mice were purchased from SLC (Hamamatsu, Japan). C3H/HeJ-gld/gld (gld/gld) and C3H/HeJ-lpr/lpr (lpr/lpr) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained in the animal facilities of the Institute of Medical Science, University of Tokyo (IMSUT). C3H/HeJ-lpr^cg/lpr^cg (lpr^cg/lpr^cg) mice, established by introducing lpr^cg into C3H/HeJ mice from CBA/KlJms-lpr^cg/lpr^cg by 12 repeated backcrosses,²¹ were used. Heterozygous lpr/+ and lpr^cg/+ mice were produced by mating +/+ to lpr/lpr and lpr^cg/lpr^cg mice, respectively. Double-homozygous C3H-lpr/lpr gld/gld (lpr/lpr gld/gld) mice, constructed from gld/gld and lpr/lpr mice,²² were used. All animal experiments were performed in accordance with the guidelines of the Animal Research Ethics of IMSUT.

BM transplantation

Recipient and donor mice (2–3 months of age) were matched in gender and age, and BM transfer was performed as described previously.¹³ Briefly, the recipients were given whole body irradiation of 850 rads from a ¹³⁷Caesium source (Gammacell 40; Atomic Energy of Canada, Ottawa, Canada) and were injected intravenously (i.v.) with 1×10⁷ viable BM cells (>95% viability in the Trypan blue exclusion test) prepared from femurs and tibias of donors 18–24 hr after irradiation. The BM-transferred mice were observed by palpation for swelling of subcutaneous LN for 6 months. At the end of this observation period they were deeply anaesthetized and killed by blood collection through heart puncture. Subcutaneous LNs (cervical, axillary, brachial and inguinal) were freed from the surrounding fatty tissue and weighed wet, separately. The total of their weights was designated LN weight and used for assessment of the degree of LN swelling. Serum was isolated from clotted blood and stored at −20° until use.

Determination of anti-double stranded DNA antibodies

Immunoglobulin G (IgG) antibodies (Abs) against double-stranded DNA (dsDNA) were determined by using an enzyme-linked immunosorbent assay (ELISA). Briefly, microtitre plates (Becton-Dickinson, Mountain View, CA) were coated with 50 µg/ml of poly-l-lysine (Sigma, St Louis, MO) in phosphate-buffered saline (PBS). After washing, the plates were incubated with 2 µg/ml of calf thymus DNA (Sigma) in PBS at 37° overnight to dry up. The antigen-coated plates were first incubated with 100-fold diluted sera and then with alkaline phosphatase-conjugated anti-mouse IgG (Cappel, Malvern, PA) with washing between both incubations. The colour was developed with 1 mg/ml p-nitrophenylphosphate (Sigma) in 50 mm cabonate-bicarbonate buffer (pH 9·7) supplemented with 2 mm MgCl₂, and the absorbency at 405 nm was measured (Immuno Reader; Nippon InterMed, Tokyo, Japan) to express the antibody titre as A₄₀₅ units.

Flow cytometry

Swelled LN were mechanically dissociated in minimal essential medium (MEM), containing 3% fetal calf serum (FCS), to prepare a single-cell suspension. Direct and indirect methods were used for immunofluorescence staining. For direct assay, 10⁶ cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat F(ab′)₂ anti-mouse immunoglobulin M (IgM) Ab (Tago Inc., Burlingame, CA) for 30 min at 4°. For indirect assay, 10⁶ cells were incubated with hybridoma supernatant containing anti-Thy-1 (AT83), -CD4 (GK-1·5), -CD8 (53–6·7), -B220 (Ly-5) (3A1) or -CD44 (Ly-24) (9F3) Abs. After washing, the cells were incubated with FITC-conjugated goat anti-rat IgG Ab (Tago Inc.) for 30 min at 4°. Control cells were treated with FITC-conjugated reagent alone. After washing, the stained cells were analysed with a flow cytometer and the data were collected using a logarithmic amplification.

Histology

Tissue samples obtained from LN, liver and kidney were fixed in 10% formalin solution in PBS, embedded in paraffin, sectioned at 3 µm, and stained with haematoxylin and eosin for histological examination.

Statistics

The data were statistically evaluated using the Student's t-test and the differences were considered significant at a P-value of <0·05.

Results and discussion

Many reports have demonstrated that lpr/lpr or lpr^cg/lpr^cg BM cells transferred into +/+ mice fail to cause lymphoid hyperplasia, instead causing marked lymphoid aplasia and wasting disease.^{11–14,18,23–27} In accordance with this, LN atrophy with fibrosis occurred in all +/+ mice transferred with lpr/lpr or lpr^cg/lpr^cg BM (Table 1). Moreover, lpr/lpr and lpr^cg/lpr^cg LN swelled predominantly owing to the accumulation of DN T cells when implanted into +/+ mice transferred with lpr^cg/lpr^cg BM.¹⁴ Therefore, DN T cells are produced from Fas-defective BM stem cells, even in the +/+ mouse environment. In addition, FasL expression was strikingly increased in lpr and lpr^cg-induced DN T cells.¹⁹ Thus, FasL on DN T cells caused the degeneration of LN tissues, resulting in LN aplasia through its Fas-mediated cytotoxic activity against radioresistant Fas-expressing cells of +/+ hosts. In support of this, LN underwent hyperplasia instead of atrophy in +/+ mice transferred with double-homozygous lpr/lpr gld/gld BM in which DN T cells express defective FasL at high levels, and lpr/lpr and lpr^cg/lpr^cg BM caused the same extent of LN hyperplasia in lpr/lpr and lpr^cg/lpr^cg recipients devoid of functional Fas (Table 1). In addition, lpr/lpr, but not lpr/lpr gld/gld DN T cells induced apoptosis of Fas-transfected tumour cells in vitro (data not shown)²⁸ indicating that FasL on the former is functional but that on the latter non-functional.

Table 1. Swelling of lymph nodes (LN) and spleen in recipients of various genotypes implanted with lpr/lpr, lpr^cg/lpr^cg and lpr/lpr gld/gld bone marrow (BM) on a C3H background.

			Mean weight±SE (mg)
Genotype of BM implanted	Genotype of recipients	No. of recipients	Subcutaneous LN	Spleen
lpr/lpr	+/+	6*	Atrophic	63±8
	lpr/lpr	6	326±27†	237±30§§
	lprcg/lprcg	6	382±42‡	198±9¶¶
	lpr/+	8	80±5	96±8§§
lprcg/lprcg	lprcg/+	8	203±26	112±10¶¶
	+/+	6	Atrophic	79±5
	lpr/lpr	6	357±58¶	166±32
	lprcg/lprcg	6	425±39**	210±19
	lpr/+	8	142±21¶,††,‡‡	181±18
	lprcg/+	8	214±25**,††	213±37
lpr/lpr gld/gld	+/+	6	308±31	213±35

^*One of six mice died of wasting disease before the end of observation. Therefore, LN and spleen were examined in 5 mice.

^{†,§,**,§§,¶¶}Significant difference between two groups at P<0·001.

^‡,¶Significant difference between two groups at P<0·005.

^††Significant difference between two groups at P<0·05.

^‡‡Significant difference between two groups at P<0·02.

Notably, one of six +/+ mice transferred with lpr/lpr BM, but none of six +/+ mice with lpr^cg/lpr^cg BM died of graft-versus-host (GVH)-like wasting syndrome (Table 1), and the former were relatively wasting but the latter normal in appearance during the last 2 months of observation. Fas is expressed in the liver, gastrointestinal tract and skin,²⁹ which are organs involved in wasting disease. However, any special pathological lesions were not observed (except for slight-to-moderate infiltration of lymphoid cells in the liver and kidney) even in +/+ mice transferred with lpr/lpr BM (data not shown). In a previous study,¹³+/+ mice transferred with lpr^cg/lpr^cg BM also did not develop clear GVH-like wasting syndrome on the CBA mouse background. In addition, wasting disease was not observed, albeit development of conspicuous lymphadenopathy, in +/+ recipients of gld/gld lpr/lpr BM. Although it is necessary to elucidate the causes for wasting of animals, the absence of wasting syndrome in +/+ recipients of lpr^cg/lpr^cg BM, in contrast to those of lpr/lpr BM may be concerned with non-functional Fas on DN T cells, which can bind to FasL but not transduce the death signal into cells. Non-functional Fas on lpr^cg DN T cells might inhibit competitively the binding of FasL massively expressed on themselves to functional Fas on +/+ host cells and attenuate the cytotoxic activity of FasL against host cells. In this respect, it is important that lpr^cg/lpr^cg LN cells exerted a weaker cytotoxic activity against Fas⁺ tumour cells than lpr/lpr LN cells.²⁸ On the other hand, it was noticeable that no wasting signs were observed in any of the lpr/+ mice transferred with lpr/lpr BM. Flow cytometry demonstrated that lpr/+ cells were 50% lower in Fas expression than +/+ cells (data not shown). This suggests that 50% reduced expression of functional Fas might make lpr/+ cells resistant to FasL-induced apoptosis by decreasing apoptosis signalling into cells, resulting in evasion of wasting events. In accordance with this, GVH-like wasting disease appeared significantly later in MRL-lpr/+ than MRL-+/+ mice transferred with MRL-lpr/lpr BM, although it was not reported whether LN underwent hyperplasia or atrophy in the former.¹² Therefore, it is probable that the MRL background genes may also play an important role in induction of GVH-like wasting syndrome, as in glomerulonephritis. Overall, it may be possible that FasL, when abnormally expressed on lymphoid cells, wastes Fas⁺ tissues and organs by apoptosis through the FasL–Fas pathway. This wasting process may be suppressed by the reduction of Fas expression and the presence of non-functional Fas.

Noticeably, LN did not become atrophic but rather swelled to a significant degree in lpr/+ and lpr^cg/+ mice transferred with lpr/lpr or lpr^cg/lpr^cg BM (Table 1). lpr/+ and lpr^cg/+ LN cells have wild-type or functional Fas in haploid, although the latter also have non-fuctional Fas in haploid. Therefore, the reduced expression of functional Fas by 50% might make lpr/+ and lpr^cg/+ LN resistant to the apoptosis induced with FasL massively expressed on DN T cells, and the LN might escape from atrophy and allow lymphoid cells, including even abnormal DN T cells, to home into them. Intriguingly, lpr^cg/+ LN hyperplasia was significantly larger than lpr/+ LN hyperplasia in the recipients of lpr/lpr or lpr^cg/lpr^cg BM (Table 1). Therefore, non-functional Fas present at the same molar ratio as functional Fas on lpr^cg/+ cells might inhibit the binding of FasL on DN T cells to functional Fas on lpr^cg/+ cells, making lpr^cg/+ LN more resistant to FasL-induced apoptosis than lpr/+ LN. As a result, lpr^cg/+ LN underwent significantly larger hyperplasia than lpr/+ LN. In addition, significantly larger hyperplasia of lpr/+ LN was induced after transfer of lpr^cg/lpr^cg BM than lpr/lpr BM. lpr^cg/lpr^cg LN cells were less cytotoxic against Fas⁺ tumour cells than lpr/lpr LN cells.²⁸ Therefore, it was suggested that non-functional Fas expressed on lpr^cg DN T cells might also inhibit the binding of FasL on themselves to functional Fas on lpr/+ cells. Thus, we speculated that non-functional Fas might interfere with the binding of FasL on DN T cells to functional Fas on LN cells, which resulted in less severe tissue damage and more efficient LN homing of lymphoid cells. Consistent with this speculation, lpr and lpr^cg-homozygous LN underwent significantly larger hyperplasia than heterozygous LN in recipients of either lpr/lpr BM or lpr^cg/lpr^cg BM (Table 1).

Lymphoid cells accumulated in hyperplastic LN were characterized by flow cytometry. The number of cells (×10⁻⁶) per mg of tissue was not significantly different among swelled LN of various genotypes: 1·29±0·06 (mean±SE) (n=4), 1·35±0·10 (n=4) and 1·42±0·08 (n=4) in lpr/+, lpr^cg/+ and lpr/lpr LN from lpr/lpr BM recipients, and 1·39±0·10 (n=4), 1·40±0·10 (n=4) and 1·45±0·09 (n=4) in lpr/+, lpr^cg/+ and lpr^cg/lpr^cg LN from lpr^cg/lpr^cg BM recipients, respectively. Moreover, the significantly swelled LN showed practically the same cellularity in histology. Thus, the LN weight differences mentioned above reflected the differences in number of cells accumulated in LN. Noticeably, however, partial degeneration with the same histological features as those of the atrophic +/+ LN from lpr/lpr and lpr^cg/lpr^cg BM recipients was occasionally found in insignificantly or slightly swelled lpr/+ LN from lpr/lpr BM recipients. This might explain the slightly lower cell content in them compared with the other LN. The results of the flow cytometry analyses are shown in Table 2. As CD4⁺ CD8⁺ double-positive cells are not present in the periphery in normal and Fas-mutant mice,³⁰ CD4⁺ or CD8⁺ cells are considered to be single positive. Thus, the content of single-positive T cells decreased in parallel with the increase of LN weight in the order of lpr/+, lpr^cg/+ and lpr/lpr or lpr^cg/lpr^cg LN in lpr/lpr BM or lpr^cg/lpr^cg BM recipients (Tables 1 and 2). Conversely, the content of DN T cells, estimated approximately by [Thy-1⁺ cells−(CD4⁺ + CD8⁺) cells], increased in this order. Consistent with this, the proportion of lymphoid cells bearing CD44, a surface molecule reported to be expressed on abnormal DN T cells,³¹ increased along with the increase of LN weights in the same order (Table 2). Thus, it is plausible that the FasL–Fas pathway is involved in the homing of lymphoid cells into LN. As expected, lpr/+, lpr^cg/+, lpr/lpr and lpr^cg/lpr^cg spleens were clearly larger than +/+ spleens in both lpr/lpr and lpr^cg/lpr^cg BM recipients (Table 1). However, lpr/+ and lpr^cg/+ spleens were significantly less hyperplastic than the respective homozygous spleens in lpr/lpr BM recipients alone. Therefore, the FasL–Fas pathway appeared to play a less important role in the homing of lymphoid cells into spleen than LN, probably as a result of the different homing mechanism of lymphoid cells between them.

Table 2. Surface markers of lymphoid cells from enlarged lymph nodes (LN) of bone marrow (BM)-transferred mice.

		Percentage of cells with surface markers*
Genotype of BM transferred	Genotype of recipient	sIg	B220	Thy-1	CD4	CD8	CD44
lpr/lpr	lpr/+	18·2±2·4	32·3±7·9	75·0±3·0	37·9±6·1	11·3±2·3	40·1±8·3
	lprcg/+	14·7±2·0	58·4±3·9	78·3±0·9	19·9±5·1	11·8±4·0	67·3±5·3
	lpr/lpr	11·9±2·3	74·9±1·1	80·8±1·1	12·2±0·4	4·2±0·1	78·8±1·0
lprcg/lprcg	lpr/+	21·2±4·2	42·5±4·6	76·1±4·5	43·1±2·3	3·6±1·2	57·5±7·3
	lprcg/+	20·6±0·7	45·1±4·3	82·0±1·2	30·1±2·2	13·2±2·0	61·8±2·6
	lprcg/lprcg	18·8±4·4	67·2±6·5	74·2±3·6	24·6±3·3	3·8±0·4	78·5±5·4

^*Mean±SE for four mice in each group.

Anti-dsDNA levels in lpr/+ and lpr^cg/+ mice carrying lpr/lpr or lpr^cg/lpr^cg BM were significantly higher than those in +/+ mice carrying +/+ BM, tended to be higher than those in +/+ mice transferred with lpr/lpr or lpr^cg/lpr^cg BM, and were as high as those in lpr/lpr and lpr^cg/lpr^cg mice carrying the respective BM (Table 3), indicating that autoantibody-producing cells can survive well when functional Fas was reduced by 50% or more. It is noted relative to this that the expression of lpr in the heterozygous state accelerated the autoimmune process in MRL mice.^32–34

Table 3. Serum anti-double-stranded DNA antibody levels in bone marrow (BM)-transferred mice.

Genotype of BM transferred	Genotype of recipient	No. of mice examined	Anti-DNA antibody level*
+/+	+/+	6	0·064±0·011†
lpr/lpr	+/+	5	0·135±0·019§,‡‡
	lpr/lpr	6	0·327±0·061**,‡‡
	lpr/+	8	0·215±0·076
	lprcg/+	8	0·234±0·071‡
lprcg/lprcg	+/+	6	0·217±0·082
	lprcg/lprcg	6	0·458±0·085††
	lpr/+	8	0·447±0·087¶
	lprcg/+	8	0·423±0·093§
lpr/lpr gld/gld	+/+	6	0·440±0·074††

^*Mean±SE is expressed as A₄₀₅ units.

^{‡,§,¶,**,††}Significant difference at P<0·05, 0·01, 0·005, 0·002 and 0·001, respectively compared with †.

^‡‡Significant difference at P <0·05 between two groups.

Many distinct mutations at the Fas locus have been identified in human patients with a lymphoproliferative syndrome and/or autoimmune disorders.^35–39 Interestingly, they were very frequently heterozygous, unlike lpr and lpr^cg in mice, and occurred in the intracellular domain. The LN homing of DN T cells allowing autoreactive cells to expand more in lpr and lpr^cg-heterozygous states, and the possible alleviation of wasting syndrome by decreased expression of Fas and non-functional lpr^cg Fas, observed in this study, imply that functional and non-functional Fas may interact with FasL in a complicated manner to cause distinct pathological conditions in human patients.