Syngeneic graft-versus-host disease induced by cyclosporine--a reappraisal.

Cyclosporine-induced graft-versus-host disease has been described in lethally irradiated rats and mice following syngeneic bone marrow reconstitution. To further study this apparently CsA-restricted phenomenon, we followed reported protocols by administering CsA orally at 50 or 100 mg/kg/day to irradiated, syngeneically reconstituted C57B1/6 mice. No clinical evidence of GVHD was observed for more than 8 weeks after CsA discontinuation. Ear biopsies and circulating immunoglobulin levels 2-4 weeks after stopping CsA failed to demonstrate histological or serological evidence of GVHD, respectively, compared with mice allogeneically reconstituted with Balb/c marrow. To further follow a previous report, CsA 50 mg/kg/day orally or 10 mg/kg/day intraperitoneally was given to normal C57B1/6 mice prior to using their spleen or bone marrow cells for reconstitution of lethally irradiated syngeneic mice. Clinical monitoring and histological examination of recipients 2-5 weeks after reconstitution again failed to confirm GVHD. Thus our results were uniformly negative in attempting to reproduce syngeneic GVHD in mice. Existing data on rats and humans are reviewed, showing that syngeneic or autologous GVHD is not CsA-restricted and that the syndrome could be equated to the chronic form of GVHD found in rats and patients after allogeneic bone marrow transplantation.

Basal and lipopolysaccharide-inducible membrane alkaline phosphatase of lymphoid cells from mice with immune system dysfunctions.

The expression of membrane alkaline phosphatase (mAlPase) activity is an enzymatic marker of activated but not resting B cells which can be used on unseparated lymphoid cell suspensions. It is higher in lymphoid cell suspensions from mice with higher proportions of B cells (athymic mice) or with more activated B cells (autoimmune mice) than in those of control mice.

Bone marrow transfers in X-irradiated mice congenic at the lpr locus: some paradoxical effects.

MRL/l mice, which are homozygous at the lpr locus, can be inhibited in lpr phenotype expression (lymphadenopathy, accelerated death) by a transfer of MRL/n bone marrow cells following X-irradiation of the recipients (MRL/n bone marrow----X-irradiated MRL/l chimeras). Female MRL/l bone marrow----X-irradiated MRL/l chimeras express the lpr phenotype with a delay corresponding to the age at the time of cell transfer. However, the equivalent male chimeras resemble MRL/n bone marrow----X-irradiated MRL/l chimeras. When the reverse MRL/l bone marrow----X-irradiated MRL/n chimeras are constructed, one finds that whichever the sex is, the chimeras undergo a wasting disease looking like a graft-versus-host disease, with particularly a marked atrophy of the spleen. A similar GVH like disease is observed with C57Bl/6 lpr bone marrow----X-irradiated C57Bl/6 normal mice. These animals survive at least 5 months but manifest a spleen aplasia. When reconstituted with MRL/n bone marrow, MRL/l recipients develop higher levels of antinuclear and anti-ds/ss DNA antibodies than MRL/n recipients. This suggests that the 'lpr environment' of the host may have an influence on the development of B cell hyperactivity.

Membrane alkaline phosphatase activity: an enzymatic marker of B-cell activation.

The expression of membrane alkaline phosphatase (mAlPase) activity was studied on viable cells from mouse lymphoid organs. The low mAlPase activity level of ex vivo mouse spleen cells was markedly increased by in vitro culture in the presence of the direct B-cell mitogen lipopolysaccharide (LPS). This increase occurred nearly simultaneously with increased uptake of 3H-thymidine and an increased percentage of blasts in the culture. The T-cell-dependent B-cell pokeweed mitogen did not increase the mean level of mAlPase activity per cell, although there was an increase per culture. The T cell mitogen ConA did not cause an increase in mAlPase activity, although it was able to stimulate both cell proliferation and blast transformation. Several other mitogens and differentiating agents were tested, but did not detectably affect mAlPase expression. LPS high responder mouse strains C57BL/6 and CBA/J showed a higher LPS-induced mAlPase expression response to LPS than did LPS low responder strains BALB/c or CBA/N. These data suggest a preferential expression of mAlPase by stimulated cycling B cells. However, mAlPase expression appeared restricted to a subpopulation of cycling B cells and could not be elicited by every B-cell stimulus.

Cyclosporine facilitates B-cell membrane immunoglobulin capping.

The immunomodulatory molecule cyclosporine was found to cause an early acceleration of the capping of mouse B-cell membrane immunoglobulin. This was most marked when the capping process was slightly retarded by keeping the cells at 32 degrees. This is a sign that the drug can cause B-cell membrane alterations. At lower drug doses they might not be as easily detected, but are nevertheless sufficient to inhibit the activation of some B cells by membrane immunoglobulin clustering.

Increased membrane immunoglobulin capping of B cells from C57Bl/6 lpr/lpr and C57Bl/6 nu/nu mice.

When the capping of membrane immunoglobulin on spleen B cells from normal C57Bl/6 mice (B6) is taken as reference, a faster capping rate is found for cells of age-matched B6 mice which are congenic at the lymphoproliferation (lpr) or nude (nu) loci. Though both congenic strains can be characterized by an abnormal T-lineage cell content, the nature of the abnormality itself is very different since B6 nudes lack thymus-processed/influenced lymphocytes whereas B6 mice with the lpr phenotype suffer from an invasion of all lymphoid organs with cells of a particular T-cell subset. Moreover, the more "normal" capping rate of B cells from the double congenic B6 mice (nu/nu, lpr/lpr) is intriguing. Since other mice homozygous at the lpr locus (MRL-1) or at the nu locus (BALB/c nude) also cap faster than their congenic controls (MRL-n and BALB/c, respectively), the observed effects do not appear to depend on a peculiarity of the B6 genetic background. If the faster capping of B cells of nu congenic and of lpr congenic mice had a common origin, it might be that T cells would control in some way the mobility of B-cell membrane immunoglobulins: both congenic mice have in their spleen a very low proportion of mature T cells together with a very high proportion of prethymic/thymic immature T-cell types, either of which might affect B-cell behavioral responses to membrane immunoglobulin clustering.

The C57B1/6 nu/nu, lpr/lpr mouse. III. Autoimmunity status.

C57B1/6 mice homozygous at the lpr (lymphoproliferation) locus display evident lymphadenopathy (in our B6 colony, primordially cervical lymph node enlargement) and autoimmunity (various autoantibodies). Four groups of mice corresponding to the diverse combinations of the lpr gene and the nu (nude, athymic) gene on the B6 genetic background have been compared for signs of lymphadenopathy. It occurred in all tested B6 +/+, lpr/lpr mice and none of the other groups (B6 nu/nu, +/+; B6 nu/nu, lpr/lpr and B6 +/+, +/+). B cell hyperactivity/autoimmunity was also evaluated by serum antibody analyses: higher serum immunoglobulin levels, anti-nuclear antibodies, anti-native DNA, anti-single stranded DNA, rheumatoid-like factors (anti-rabbit IgG), and natural antibodies against dinitrophenol and trinitrophenol haptens and their non cross reactive carriers: bovine serum albumin, hen egg albumin and keyhole limpet haemocyanin. Interestingly the levels of serum immunoglobulins and of some specific antibodies were somewhat higher in B6 nu/nu, lpr/lpr than in 'normal' B6 nu/nu, +/+, though they remained much lower than in B6 +/+, lpr/lpr animals. This suggests that the lpr gene may express its influence on the level of B cell activity in the absence of T lineage cells that would have normally matured in a thymus and that this effect of the lpr gene does not require the massive proliferation of T lineage cells observed in B6 +/+, lpr/lpr mice.

The C57BL/6 nu/nu lpr/lpr mouse. I. Expression of the 'lpr phenotype' in the C57BL/6 genetic background.

The C57BL/6 lpr/lpr mice develop within 3-6 moths a series of abnormalities of their immune system which characterize the 'lpr phenotype' and allow them to be distinguished easily from normal C57BL/6 as well as from C57BL/6 nu/nu: lymphadenopathy, hyperimmunoglobulinemia, high anti-nuclear antibody titers and, less regularly, anti-whole DNA antibody. Though C57BL/6 nu/nu occasionally present auto-antibody too, the combined use of three or four of the aforementioned parameters for each tested animal allows an easy detection of animals presenting the lpr phenotype. In our C57BL/6 lpr/lpr colony, the lymphadenopathy does not seem to start by chance in any lymphoid organ but shows a very strong preferential occurrence in the cervical lymph nodes. These parameters of the lpr phenotype have been used to trace the lpr genes and to construct the C57BL/6 nu/nu lpr/lpr as described in the companion paper.

The C57BL/6 nu/nu lpr/lpr mouse. II. Pedigree and preliminary characteristics.

A short-cut procedure is described which allows to construct new mouse strains which are double congenic for the nu gene (nude, athymic) and the lpr gene (lymphoproliferation). Since both nu and lpr genes are recessive and that the lack of thymus impairs the expression of the lpr phenotype in euthymic animals, the homozygosity of nude animals for the lpr gene was based on the results of progeny analyses. A C57BL/6 nu/nu lpr/lpr (B6 nu/nu, lpr/lpr) was made in four generations starting from C57BL/6 nu/nu (B6 nu/nu) and C57BL/6 lpr/lpr (B6 lpr/lpr). This possibility stems from an absence of close linkage of the nu and lpr genes. The emergence of the nude phenotype at the different steps of the pedigree was less frequent than expected if the nu and lpr genes were completely independent. However, this is probably due to environmental factors causing a negative selection of nude littermates. The B6 nu/nu lpr/lpr represents a new important tool for the study of how the thymus modulates the lpr gene controlled acceleration of autoimmunity. Preliminary data indicate that the lpr phenotype (massive lymphadenopathy and splenomegaly accompanied by severe auto-immune disease with features of systemic lupus erythematosus) appears when a thymus is grafted to the B6 nu/nu lpr/lpr.