A non-expressed transgene as a cell marker for the investigation of cellular traffic after splenic autotransplantation.

The investigation of cellular interactions during the regeneration of splenic transplants depends upon distinguishing host cells from graft derived cells. As a cell marker which, in contrast to many other marker systems, does not affect histocompatibility, the non-expressed transgene mMT-HGHRH-1 was introduced by mating into NMRI inbred mice to generate the transgenic mouse line BSM. By transplanting non-transgenic NMRI splenic tissue into transgenic BSM hosts, host derived cells could be identified in the transplants by in situ hybridization after regeneration. In a reciprocal approach, implant derived cells were detected in a transgenic transplant after 3 weeks of regeneration in a non-transgenic host. Relative amounts of transgenic cells in transplant cross-sections were estimated from the signal intensity of autoradiographs by densitometry and computer analysis. This approach could be broadly applied in studies of transplantation systems.

Regeneration of autotransplanted splenic tissue at different implantation sites.

Inbred animals (Lewis rats) were used to investigate the regeneration of autologously implanted splenic tissue at intra-omental and subcutaneous sites. Quantitative immunohistology with monoclonal antibodies against lymphocytes and macrophages was performed to analyse the cell density of red pulp (RP), periarteriolar lymphoid sheath (PALS), marginal zone (MZ) and follicle, 7-180 days after transplantation. Antigenic, allogeneic and mitogenic stimulation and Northern blotting were also performed. Transplant groups differed from spleen only in the reduced size of PALS; however, quantitative analysis demonstrated subtle differences between spleen and transplants. The cell density of B-cells and ED-1+ macrophages was reduced in the RP, Tsupp/cyt-cells were decreased and B-cells increased in PALS, and B-cells and Thelper-cells reduced in the MZ. No differences could be detected between the transplant groups. Flow-cytometric analysis of cell suspensions from spleen and transplants revealed a reduction of T-cells (OX-19+), MHC-I and transferrin-receptor-bearing cells in both transplant groups, and a decrease in the number of Thelper-cells and ED-3+ macrophages in subcutaneous transplants. Both transplant groups were defective regarding the allogeneic and pokeweed mitogen response. Aberration of the lipopolysaccharide response was restricted to subcutaneous transplants, which additionally showed abnormal expression of interferon-gamma, interleukin-5 and interleukin-6 mRNA. Thus, subtle alterations of the newly developed microenvironment and/or lymphocyte-homing may influence the regeneration of splenic tissue; the implantation site may represent an important parameter in functional reorganisation.

[Autotransplantation of the spleen in rats: development, function and cytokine expression in intra-omental and subcutaneous regeneration]. / Autotransplantation der Milz an Ratten: Entwicklung, Funktion und Cytokin-Expression in intraomentalen und subkutanen Regeneraten.

The implantation of splenic tissue at different implantation sites (intraomental and subcutaneous) into one animal (Lewis rats) results in the development of splenic nodules at both sites. In a quantitative immunohistological analysis of splenic compartments such as red pulp (RP), periarteriolar lymphoid sheaths (PALS), marginal zone (MZ) and follicles (F) the T-cell reduction was related to the T(helper) cells in the MZ and T(supp/cyt) cells in the PALS. In contrast, the cell density of B cells and ED-1+ macrophages in the PALS and T(supp/cyt) cells in the MZ was increased. Significant differences between the implantation sites were restricted to CD5+ cells (thymocytes and T cells) in the MZ and OX-33+ cells (B cells with LCAB antigen) in the PALS. The reorganisation of the compartments of subcutaneous implants showed a delay of one week as compared with omental ones. Functional assays like haemolytic plaque assay, mitogen stimulation and mixed lymphocyte assay elicited an analogous delay of the functional maturation of IgM-positive B cells, a reduced proliferation of both transplant groups after pokeweed mitogen (PWM) stimulation, a decreased response after lipopolysaccharide (LPS) stimulation in solely subcutaneous replants and no differences concerning the mitogens concanavalin A (ConA) and phytohaemagglutinin (PHA). Both transplant groups showed a significantly reduced allogeneic response. The results of the functional analysis and the abnormal mRNA expression of Il-5, Il-6 (Interleukin 5 and 6), GMCSF (Granulocyte-Macrophage-Colony-Stimulation-Factor) and IFN-gamma (Interferon gamma) in subcutaneous replants indicate subtle molecular alterations (independent of a spleen-like immunoarchitecture) at this site.(ABSTRACT TRUNCATED AT 250 WORDS)