Characterization of thoracic duct cells that transfer polyarthritis.

Polyarthritis may result from the haematogenous distribution of arthritogenic effector lymphocytes that emerge in the efferent lymph and pass through the thoracic duct (TD) to the circulation. We therefore examined whether TD cells collected from rats in the late prodrome of adjuvant-induced arthritis (AA) could transfer polyarthritis adoptively and whether these cells included a subpopulation of arthritogenic cells that could be identified phenotypically. Unfractionated TD cells collected from donor rats 9 days after adjuvant inoculation were injected intravenously into normal syngeneic recipients in numbers equivalent to the overnight harvest from a single donor. TD cell subpopulations, equivalent in number to proportions in the same inoculum, were prepared by negative selection. Unfractionated TD cells transferred polyarthritis without in vitro stimulation or conditioning of recipient animals. Abrogation of arthritogenicity by depletion of alpha/beta TCR(+) cells showed that the polyarthritis was transferred by T cells. Negatively selected CD4(+) but not CD8(+) TD cells transferred AA. An arthritogenic subpopulation of CD4(+) T cells, enriched by either negative or positive selection, expressed the activation markers CD25 (IL-2 receptor alpha), CD71 (transferrin receptor), CD134 (OX40 antigen) and MHC class II. Cells expressing these markers were more numerous in TD lymph from arthritic rats than in lymph from normal rats and they included the majority of large CD4(+) T cells. Thus, arthritogenic effector T cells bearing activation markers are released into the central efferent lymph in the late prodrome of AA. Recruitment of these arthritogenic cells to synovium probably determines the polyarticular pattern of AA.

Recruitment of mononuclear leucocytes to osteoarthritic human synovial xenografts in the ears of SCID mice.

A system has been established to assess the recruitment of 99mTc-hexamethylpropylene amine oxamine (99mTc-HMPAO)-labelled PBMC and [125I]iododeoxyuridine-labelled Con A stimulated lymphoblasts to allogeneic human synovial xenografts in the ears of SCID mice. Successful engraftment of osteoarthritic synovium was achieved in approximately 90% of cases and a connection between the human microvasculature of the xenograft and the circulation of the mouse was shown. Cells were delivered to the xenograft by a system of regional vascular perfusion, thus avoiding the major murine vascular beds. The accumulation of 99mTc-HMPAO-labelled PBMC in mouse ears was monitored in real time. Direct injection of xenograft-bearing ears with recombinant human TNF-alpha, 7 h prior to perfusion, increased the accumulation of both PBMC and lymphoblasts in cytokine-injected ears compared to contralateral control-injected ears. Autoradiography revealed the presence of [125I]iododeoxyuridine-labelled lymphoblasts associated with human microvasculature within the xenograft. However, the increased accumulation of lymphoblasts in cytokine-injected ears occurred in the tissues surrounding the xenograft, where lymphoblasts were associated more often with murine than human vessels. Although the system described offers advantages over similar models, the propensity for mouse endothelium to interact with human leucocytes is likely to be a generic disadvantage for models of human leucocyte recruitment to xenografts in immunodeficient mice.

Apoptosis is a prominent feature of acute anterior uveitis in the Fischer 344 rat.

AIMS: To examine the hypothesis that apoptosis of infiltrating cells contributes to spontaneous resolution of uveitis in clinically relevant rodent models. METHODS: Experimental melanin induced uveitis (EMIU) was induced in Fischer 344 rats by immunisation with 250 microg bovine ocular melanin. Endotoxin induced uveitis (EIU) was induced by injection of 200 microg Escherichia coli lipopolysaccharide. Formalin fixed, paraffin embedded ocular cross sections were stained by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labelling (TUNEL) to identify apoptotic cells. Indirect immunoperoxidase staining of paraformaldehyde lysine periodate fixed tissue cross sections was used to demonstrate expression of inducible nitric oxide synthase (iNOS). RESULTS: TUNEL positive mononuclear cells were observed in the anterior uvea during both EMIU and EIU at all selected time points. However, whereas the majority of mononuclear cells appeared apoptotic from the outset of disease, neutrophils were notably TUNEL negative at all time points examined. Many infiltrating neutrophils expressed iNOS. CONCLUSION: Apoptosis occurs early in the course of rat EMIU and EIU, and may contribute to resolution of these diseases. In general, infiltrating mononuclear cells die rapidly, while neutrophils survive, producing inducible nitric oxide synthase which may contribute to disease pathogenesis.

Regulation of gastrointestinal growth in fetal sheep by luminally administered insulin-like growth factor-I.

Fetuses swallow large volumes of amniotic fluid. Absence of swallowing results in gastrointestinal tract (GIT) growth deficits. While it is not yet known to what extent the growth factors present in amniotic fluid are involved in GIT ontogeny, milk-derived growth factors are considered to be important for neonatal growth. Our experiment tested the hypothesis that a luminal growth factor (insulin-like growth factor-I, IGF-I) can sustain or promote GIT growth in utero in a model of gastrointestinal tract growth retardation. Ten-day infusion of either human recombinant IGF-I or vehicle into twin fetal sheep at 80 days gestation via an indwelling esophageal catheter resulted in altered GIT growth. Weight of the forestomach and small intestine increased. Significant histological changes were noted in the proximal small intestine, i.e. the region most exposed to the luminal infusion. Mucosal tissues were reduced in size. While the enterocytes in the proximal small intestine were generally more mature with regard to the ontogeny of the apical endocytic complex (which is responsible for uptake and transport of whole peptides), there were also many abnormal cytological features present. These included the development of large lysosomal-like inclusion bodies and many surfactant-like particles within the apical cytoplasm. Plasma IGF-I levels were on average 20% higher in treated siblings, suggesting that luminal IGF-I crossed the fetal gut and entered blood. IGF-II levels were not significantly affected. These observations are consistent with the suggestion that growth factors, which are present in swallowed amniotic fluid, influence fetal ontogeny.

Failure of short-term luminal IGF-I to protect against atrophy in a model of fetal esophageal atresia.

Short-term luminal infusion in utero (3 days) of insulin-like growth factor I (IGF-I) failed to protect the fetal small intestine against atrophy induced by ablation of swallowing. Human recombinant IGF-1 (or vehicle) was infused into the duodenum of fetal sheep at 125 days' gestation for 3 days (day 1, 0.025 mg; day 2, 0.25 mg: day 3, 2.5 mg). Fetal swallowing was prevented by esophageal ligation, and a carotid catheter was implanted for blood sampling. There were no changes in body growth of in major organ growth. Small intestinal (SI) weight (corrected for body weight) was significantly lower for IGF-I treated fetuses. Villus height decreased significantly in proximal regions. Villus enterocyte cellularity was reduced significantly in the proximal regions. The percentage of crypt cells labeled with a 4-hour pulse of tritiated thymidine (as assessed by autoradiography) decreased significantly in the proximal SI only, from 16.14% (1.06% SEM) to 13.28% (1.05% SEM) (P < .05). Plasma levels of IGF-1 increased in the treated fetuses by an average of 76%. IGF-1 immunoreactivity was detected in the apical endocytic complex of enterocytes from proximal SI. This study shows that wasting of fetal intestinal tissues in the absence of enteral input cannot be prevented by IGF-1 delivered luminally.

Patterns of corneal graft rejection in the rabbit and reversal of rejection with monoclonal antibodies.

The purpose of this study was to determine whether the local administration of monoclonal antibodies could reverse rabbit corneal graft rejection. To provide a rational basis for the choice of monoclonal antibodies as potential immunosuppressive agents, the phenotypes of cells infiltrating rejecting rabbit corneal allografts were examined by immunohistochemistry. About half the leukocytes accumulating in these grafts bore an immunodominant T cell marker, over two-thirds carried MHC class II antigens, and about one-fifth carried myeloid cell markers. A kinetic study of the cell population appearing in rabbit aqueous during corneal graft rejection was performed by examination of repetitive anterior chamber taps taken over a ten-day period; again, the major components were T cells, MHC class II antigen-positive cells and myeloid cells. Monoclonal antibodies L11/135 (directed against a peripheral T cell determinant), 2C4 (directed against a monomorphic MHC class II antigen), and LION 2 (directed against a myeloid antigen) were chosen for intracameral injection into rabbits with rejecting corneal grafts. Each animal received a total of 50-100 micrograms of antibody in two injections at 3-4-day intervals. L11/135 and LION 2 reversed rejection in 5/9 and 8/12 animals, respectively, in the absence of any other immunosuppression; 2C4 was without effect. We suggest that monoclonal antibody therapy in corneal transplantation deserves further attention.