Birte Steiniger

The Perifollicular and Marginal Zones of the Human Splenic White Pulp: Do Fibroblasts Guide Lymphocyte Immigration?

We investigate the white pulp compartments of 73 human spleens and demonstrate that there are several microanatomical peculiarities in man that do not occur in rats or mice. Humans lack a marginal sinus separating the marginal zone (MZ) from the follicles or the follicular mantle zone. The MZ is divided into an inner and an outer compartment by a special type of fibroblasts. An additional compartment, termed the perifollicular zone, is present between the follicular MZ and the red pulp. The perifollicular zone contains sheathed capillaries and blood-filled spaces without endothelial lining. In the perifollicular zone, in the outer MZ, and in the T cell zone fibroblasts of an unusual phenotype occur. These cells stain for the adhesion molecules MAdCAM-1, VCAM-1 (CD106), and VAP-1; the Thy-1 (CD90) molecule; smooth muscle alpha-actin and smooth muscle myosin; cytokeratin 18; and thrombomodulin (CD141). They are, however, negative for the peripheral node addressin, the cutaneous lymphocyte antigen, CD34, PECAM-1 (CD31), and P- and E-selectin (CD62P and CD62E). In the MZ the fibroblasts are often tightly associated with CD4-positive T lymphocytes, whereas CD8-positive cells are almost absent. Our findings lead to the hypothesis, that recirculating CD4-positive T lymphocytes enter the human splenic white pulp from the open circulation of the perifollicular zone without crossing an endothelium. Specialized fibroblasts may attract these T cells and guide them into the periarteriolar T cell area.

Rat ependyma and microglia cells express class II MHC antigens after intravenous infusion of recombinant gamma interferon.

Recombinant rat gamma-interferon was administered to Lewis rats by continuous intravenous infusion. After a 3-day administration period, at various dosages, a constant pattern of class II major histocompatibility complex (MHC) antigen induction was found in the brains and cerebella. Immunohistological double staining for class II antigens and glial fibrillary acidic protein showed that the majority of newly induced cells were microglia. The endothelium of large blood vessels and ependymal cells also expressed class II antigens. These findings demonstrate that systemically raised interferon levels can affect MHC antigen expression in the brain. Astrocytes are obviously not the primary cell type to acquire class II reactivity, and thus potential antigen-presenting capacity, in this situation.

The species-specific structure of microanatomical compartments in the human spleen: strongly sialoadhesin-positive macrophages occur in the perifollicular zone, but not in the marginal zone.

The microanatomical structure of human and rat splenic white pulp is compared, with special emphasis on the localization of the marginal zone occupied by immunoglobulin M (IgM)+ IgD−/dull B lymphocytes and its specialized macrophages. Our study reveals that in contrast to rats, the marginal zone of humans primarily exists in the vicinity of primary and secondary splenic follicles and that it is almost absent around the periarteriolar T‐cell zones. We demonstrate that in humans there is an additional compartment, the perifollicular zone, located between the marginal zone and the red pulp. The perifollicular zone is a dynamic region of variable cellular and phenotypic composition, which can be regarded either as a part of the red pulp or of the follicles. In most cases the perifollicular zone appears as a compartment of the red pulp containing erythrocyte‐filled spaces which differ from the typical red pulp sinusoids. Similar to the splenic cords, the perifollicular zone mostly harbours scattered B and T lymphocytes. However, sometimes B lymphocytes clearly predominate in the perifollicular area. In addition, strongly sialoadhesin‐positive macrophages form sheaths around capillaries in the perifollicular zone. Such capillary sheaths are not observed in rats. In humans weakly sialoadhesin‐positive macrophages are also present in the perifollicular zone and in the red pulp. In some specimens sialoadhesin is, however, strongly expressed by a large number of dispersed perifollicular macrophages. Interestingly, in striking contrast to rats, the human marginal zone does not contain sialoadhesin‐positive macrophages and marginal metallophilic macrophages are also absent in humans. Thus, sialoadhesin‐positive macrophages and IgM+ IgD− memory B lymphocytes both share the marginal zone as a common compartment in rats, while they occupy different compartments in humans. We show that the human splenic marginal zone does not contain a marginal sinus and assume that in humans the perifollicular region is the compartment where antigen and recirculating lymphocytes enter the organ.

Dynamics of monocytes/macrophages and T lymphocytes in acutely rejecting rat renal allografts

Abstract We examined the infiltration of acutely rejecting renal allografts (DA→LEW) by ED1+ and ED2+ macrophages and T lymphocytes at intervals of 24 h after transplantation. Donor and recipient macrophages were differentiated by MHC class II antigen expression in double-staining experiments with ED1. Proliferation was assayed after pulse-labelling with BrdU. We subdivided allograft infiltration into three consecutive phases: 1) During phase I on days 1 to 2 after allogeneic kidney transplantation, perivascular infiltrates developed that contained numerous donor and recipient macrophages. Allograft rejection could already be diagnosed 24 h after transplantation by perivascular infiltration of T lymphocytes, whereas T cells were rarely found in isografts. 2) Phase II of allograft rejection from day 3 to 4 was characterized by massive propagation of the infiltrate. About equal numbers of interstitial donor and recipient macrophages were counted. Both macrophages and T lymphocytes proliferated in situ and macrophages outnumbered T cells until complete rejection. 3) During phase III the allograft was destroyed. Large intravascular monocytes surprisingly expressed the ED2 antigen. In the interstitium of viable graft regions, the population of recipient macrophages grew, whereas the population of donor macrophages and of T lymphocytes decreased.

The splenic marginal zone in humans and rodents: an enigmatic compartment and its inhabitants

The role of the spleen in B memory cell development and maintenance is attracting increased attention. Studies in mice and rats have indicated that memory functions are associated with large B cells residing in the marginal zone (MZ) of the spleen. Although the cellular composition of the MZ is relatively well known in these species, controversies exist about the function of MZ B cells, their dependence on the presence of the spleen and the stage at which their development branches from that of recirculating follicular B cells. Additional confusion has arisen with respect to MZ B cells in humans, because the microscopic anatomy of the human splenic MZ differs decisively from that of rodents. Several recent publications indicate that the functional and migratory properties of human MZ B cells may be species-specific. The hypothesis derived from these publications and from our immunohistological observations implies that at least a major number of human splenic CD27+ MZ B cells are migratory. Phenotypic data suggest a recirculation pathway between the spleen and mucosal tissues in humans.

CD27+ B cells in human lymphatic organs: re‐evaluating the splenic marginal zone

The marginal zone of human spleens is regarded as an organ‐specific region harbouring sessile memory B cells. This opinion has arisen by extrapolating from results obtained in mice and rats. Detection of CD27+ B cells in situ now revealed similarities among the most superficial region of B‐cell follicles in human spleens, reactive lymph nodes, inflamed appendices, tonsils and terminal ilea. The follicular surface in these organs consists of small naïve immunoglobulin D (IgD)+ CD27– B cells predominating in an inner area and larger IgD+/– CD27+ B cells prevailing in a more superficial position. CD27+ B cells may, however, also occupy the entire follicular periphery around the germinal centre. Together with additional peculiarities this distribution indicates a fundamental microanatomical difference among the human and rodent splenic white pulp. We hypothesize that the follicular periphery represents a recirculation compartment both for naïve and memory/natural reactive B cells in all human secondary lymphatic organs. This assumption implies a difference in recirculation behaviour among human and rodent B memory cells.

Accumulating monocytes in the vasculature of rat renal allografts: phenotype, cytokine, inducible no synthase, and tissue factor mRNA expression.

Background. Necrotic patches and hemorrhagic lesions develop in the renal tissue between day 4 and day 5 after transplantation of fully allogeneic DA rat kidneys to LEW recipients.These lesions are at least in part due to destruction and obstruction of blood vessels. Damage of graft endothelial cells and blood coagulation are likely to be mediated by intravascular graft leukocytes. However, this cell population has not been thoroughly characterized before. Methods. We perfused untreated control kidneys, renal isografts, and allografts on day 4 after transplantation with phosphate-buffered saline/ethylenediaminetetraacetic acid to harvest leukocytes from both the blood stream as well as from the marginal intravascular pool. The mRNA expression of typical products of activated monocytes was analyzed in reverse-transcriptase polymerase chain reaction experiments. Graft monocytes were purified and their immunophenotype was investigated by flow cytometry. Results. Allograft rejection led to a 10-fold increase in the number of intravascular graft leukocytes compared to isografts. A mean number of about 100×106 leukocytes was harvested from a single allogeneic kidney, about 73% of these cells were monocytes and most of them displayed an activated phenotype. Compared to isografts, intravascular allograft leukocytes displayed an increased expression of tumor necrosis factor-&agr;, inducible NO synthase and tissue factor. Conclusions. Our study shows that large numbers of activated monocytes accumulate inside allograft vessels. As they express genes the products of which might damage the allograft by inducing cell death or thrombosis, we speculate that they directly participate in allograft destruction.

Monocytes in the rat: Phenotype and function during acute allograft rejection

Summary: Cells of the monocyte/macrophage system originate from the bone marrow, reach the organs via the blood, immigrate through post‐capillary venules and further differentiate into organ‐specific tissue macrophages. In rats and other species, activated monocytes/macrophages aggravate autoimmune reactions, rejection of non‐vascularized allografts and chronic allograft rejection. It is very likely that they also contribute to acute allograft destruction. So far it has been impossible to distinguish the function of monocytes from that of macrophages, because cell phenotypes and their alterations upon activation are ill‐defined. We have thus begun to characterize the ex vivo phenotype and function of rat monocytes in the normal state and during renal allograft rejection. Monocytes are recovered from both the central and the marginal blood pool by perfusing either the recipients circulation or the allograft vasculature. Rat monocytes have a unique surface phenotype. During allograft rejection or after infusion of interferon‐γ they up‐regulate class II MHC molecules, CD161 (NKR‐P1A), CD62L and CD8, while CD4 and CD43 are down‐modulated. Activated perfusate monocytes exert increased in vitro cytotoxicity against tumour targets, which differs from that of NK cells. We speculate that activated monocytes contribute to kidney allograft destruction by directly damaging endothelial cells or by promoting intravascular coagulation.

Rat monocytes up-regulate NKR-P1A and down-modulate CD4 and CD43 during activation in vivo: monocyte subpopulations in normal and IFN-gamma-treated rats.

LEW rats were treated intravenously with recombinant rat interferon‐γ (IFN‐γ) for 3 days to achieve intravascular accumulation, proliferation, and activation of monocytes. Monocytes, defined by their expression of the ED1, ED9, and Ox41 antigens, were recovered from the vasculature by perfusion with PBS/EDTA, subsequently depleted of erythrocytes and granulocytes by Percoll density gradient centrifugation, and analyzed by flow cytometry and immunocytology. In untreated and control‐infused specified pathogen‐free (SPF) rats, lymphocytes and monocytes formed overlapping cell populations with respect to size and internal granularity. At least two intravascular monocyte subsets, probably central and marginating cells, were distinguished by their size and differential expression of CD43, CD4, CD11a, CD18, and L‐selectin. It is interesting to note that a fraction of the monocytes in normal and control‐infused animals carried the NKR‐P1A molecule. IFN‐γ treatment provoked a duplication of monocyte size and granularity. Both the number of positive monocytes and the level of expression of NKR‐P1A strongly increased after IFN‐γ infusion, whereas CD43 (leukosialin) and CD4 were impressively down‐regulated. NKR‐P1A+ L‐selectin+CD43low CD4‐ monocytes also occur in the vasculature of rats during immune reactions in vivo. We speculate that these cells are involved in organ damage and that their number is controlled by activation‐induced cell death within the vessels. J. Leukoc. Biol. 62: 741–752; 1997.

High-yield purification of rat monocytes by combined density gradient and immunomagnetic separation

Satisfactory purification of rodent monocytes in suspension has not been achieved up to now because in rats and mice these cells occur as a minor population of peripheral blood leukocytes overlapping with lymphocytes in size and density. We describe a two-step procedure for the isolation of monocytes from rat blood with high yield and purity. This method permits the recovery of more than 90% of monocytes collected by perfusion of the vasculature and avoids loss of major subpopulations. Percoll density gradient centrifugation of perfusate cells is combined with subsequent indirect immunomagnetic depletion of lymphocytes using an antibody cocktail. The method described produces more than 90% pure rat monocytes.