Georg Kraal

The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3.

In the present study, a set of three monoclonal antibodies is described, each of which recognizes cells of the monocyte-macrophage lineage in the rat. The tissue distribution, in particular in lymphoid organs, of each of the three monoclonals is determined by immunoenzyme histochemistry on cryostat sections, as well as on cell suspensions. Results show that ED1 recognizes a cytoplasmic antigen in monocytes and in most macrophages, free and fixed. ED2 and ED3 recognize membrane antigens of tissue macrophages, discriminating between distinct subpopulations of macrophages, each with a characteristic localization in the compartments of lymphoid organs. No other cell types except cells of the mononuclear phagocyte system are positive for any of the three monoclonals. Possible relations between the macrophages recognized by this set of monoclonals and dendritic cells are discussed.

Structure and function of the spleen.

The spleen combines the innate and adaptive immune system in a uniquely organized way. The structure of the spleen enables it to remove older erythrocytes from the circulation and leads to the efficient removal of blood-borne microorganisms and cellular debris. This function, in combination with a highly organized lymphoid compartment, makes the spleen the most important organ for antibacterial and antifungal immune reactivity. A better understanding of the function of this complex organ has been gained from recent studies, as outlined in this Review article.

Cloning of a novel bacteria-binding receptor structurally related to scavenger receptors and expressed in a subset of macrophages.

A novel murine plasma membrane protein has been identified in subpopulations of macrophages. It has an intracellular N-terminal domain, a transmembrane domain, and an extracellular region with a short spacer, an 89 Gly-Xaa-Yaa repeat-containing collagenous domain, and a C-terminal cysteine-rich domain. In situ hybridization and immunohistochemical staining have localized the protein to a subset of macrophages in the marginal zone of the spleen and the medullary cord of lymph nodes. No expression was observed in macrophages of liver or lung. Transfected COS cells synthesized a native trimeric plasma membrane protein that bound labeled bacteria and acetylated LDL, but not yeast or Ficoll. The results suggest that the novel protein is a macrophage-specific membrane receptor with a role in host defense, as it shows postnatal expression in macrophages, which are considered responsible for the binding of bacterial antigens and phagocytosis.

Nuclear Factor κB Signaling in Atherogenesis

Atherosclerosis is an inflammatory disease, characterized by the accumulation of macrophage-derived foam cells in the vessel wall and accompanied by the production of a wide range of chemokines, cytokines, and growth factors. These factors regulate the turnover and differentiation of immigrating and resident cells, eventually influencing plaque development. One of the key regulators of inflammation is the transcription factor nuclear factor &kgr;B (NF-&kgr;B), which, for a long time, has been regarded as a proatherogenic factor, mainly because of its regulation of many of the proinflammatory genes linked to atherosclerosis. NF-&kgr;B may play an important role in guarding the delicate balance of the atherosclerotic process as a direct regulator of proinflammatory and anti-inflammatory genes and as a regulator of cell survival and proliferation. Here we address recent literature on the function of NF-&kgr;B in inflammatory responses and its relation to atherosclerosis.

Cells in the marginal zone of the spleen.

The marginal zone of the spleen forms an intriguing area in which a variety of cell types are combined. Several of these cell types seem to have a fixed position in the marginal zone, such as the marginal zone macrophages, the marginal metallophilic macrophages at the inner border, and, to a lesser extent, the marginal zone B cells. For other cell types--T lymphocytes, small B cells, and dendritic cells--the marginal zone is only a temporary residence. It is this combination of relatively sessile cell populations and the continuous influx and passing of bloodborne immunocompetent cells that turn the marginal zone into a dynamic area, particularly apt for antigen processing and recognition. In no other lymphoid organ can such a unique combination of cells and functions be found. The opening of the arterial blood stream in the marginal sinuses results in a reduction of the velocity of the blood stream, and antigens are initially screened in the marginal zone. To this, extremely potent phagocytic cells, the marginal zone macrophages, are present which can take up and phagocytize large foreign particles, such as bacteria and effete red blood cells. Further filtration of the blood takes place in the filtration beds of the red pulp. The marginal zone macrophages express membrane receptors for bacterial polysaccharides which lead to efficient phagocytosis, probably even in the absence of prior opsonization. Antigenic fragments produced this way can be taken up by dendritic cells that enter the spleen by the blood as part of a mobile surveillance immune system. Dendritic cells present antigen to T cells in the outer area of the T cell-dependent PALS, leading to clustering and enrichment of antigen-specific T cells. Antigens in the marginal zone can also directly associate with memory B cells thought to reside here for longer times, having intimate contact with the marginal zone macrophages. B memory cells then migrate into the PALS and present antigen to T cells. The marginal zone therefore functions not only as an area of initial filtration and phagocytosis of antigens from the blood, but also as a site of lymphocyte emigration. Some of the incoming T and B lymphocytes in the recirculating pool enter the white pulp from the marginal zone. The underlying force and selective molecular mechanisms that guide this migration are unknown. Both B and T lymphocytes recirculate through the outer PALS area on their way to the follicles and the inner PALS, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice

Atherosclerosis is now generally accepted as a chronic inflammatory condition. The transcription factor NF-kappaB is a key regulator of inflammation, immune responses, cell survival, and cell proliferation. To investigate the role of NF-kappaB activation in macrophages during atherogenesis, we used LDL receptor-deficient mice with a macrophage-restricted deletion of IkappaB kinase 2 (IKK2), which is essential for NF-kappaB activation by proinflammatory signals. These mice showed increased atherosclerosis as quantified by lesion area measurements. In addition, the lesions were more advanced and showed more necrosis and increased cell number in early lesions. Southern blotting revealed that deletion of IKK2 was approximately 65% in macrophages, coinciding with a reduction of 50% in NF-kappaB activation, as compared with controls. In both groups, the expression of differentiation markers, uptake of bacteria, and endocytosis of modified LDL was similar. Upon stimulation with LPS, production of TNF was reduced by approximately 50% in IKK2-deleted macrophages. Interestingly, we also found a major reduction in the anti-inflammatory cytokine IL-10. Our data show that inhibition of the NF-kappaB pathway in macrophages leads to more severe atherosclerosis in mice, possibly by affecting the pro- and anti-inflammatory balance that controls the development of atherosclerosis.

Macrophage subset repopulation in the spleen: differential kinetics after liposome-mediated elimination.

Different macrophage subsets can be discriminated in the well defined compartments of the mouse spleen by specialized functions and the presence of specific surface determinants. Red pulp macrophages, marginal zone macrophages, and marginal metallophilic macrophages are eliminated simultaneously within 24 hr by a single injection with liposome‐entrapped dichloromethylene diphosphonate (DMDP). After such elimination, these subsets show a striking difference in their kinetics of reappearance: Red pulp macrophages are back in normal numbers after 1 week, the marginal metallophilic macrophages take 2 weeks to regain fully their position at the border of the marginal zone and periarteriolar lymphocyte sheath, but it takes over 1 month for complete reappearance of the marginal zone macrophages. Marginal zone lymphocytes, also affected by treatment with the liposome‐entrapped drug, reappeared in the marginal zone within 2 weeks, indicating that marginal zone macrophages are not required for their localization and/or retention there. Approximately 2 weeks after treatment, all cells in the spleen have returned to normal numbers with the exception of marginal zone macrophages, which can be found only sporadically at that time. The results indicate that these macrophage subpopulations must have different precursor requirements. The differential reappearance of the macrophages creates the possibility of studying lineage analysis and will help to unravel the precise function of the marginal zone macrophages and marginal metallophilic macrophages in particular.

A conduit system distributes chemokines and small blood-borne molecules through the splenic white pulp.

Access to the splenic white pulp is restricted to lymphocytes and dendritic cells. Here we show that movement of molecules from the blood into these confined areas is also limited. Large molecules, such as bovine serum albumin (68 kD), immunoglobulin G (150 kD), and 500 kD dextran are unable to enter the white pulp, whereas smaller blood-borne molecules can directly permeate this compartment. The distribution is restricted to a stromal network that we refer to as the splenic conduit system. The small lumen of the conduit contains collagen fibers and is surrounded in the T cell areas by reticular fibroblasts that express ER-TR7. It also contains the chemokine CCL21. Conversely, in B cell follicles the B cell–attracting chemokine CXCL13 was found to be associated with the conduit and absence of ER-TR7+ fibroblasts. These results show heterogeneity of reticular fibroblasts that enfold the conduit system and suggest that locally produced chemokines are transported through and presented on this reticular network. Therefore, the conduit plays a role in distribution of both blood-borne and locally produced molecules and provides a framework for directing lymphocyte migration and organization of the splenic white pulp.

The macrophage receptor MARCO.

MARCO (macrophage receptor with collagenous structure) belongs to the class A scavenger receptor molecules. The structure and function of the molecule is described. Although it is expressed on subsets of macrophages, it can be upregulated on other macrophages after bacterial infection. The strategic position of MARCO-expressing cells in lymphoid organs suggests an important role for this bacteria-binding molecule in removal of pathogens.

Repopulation of Macrophages in Popliteal Lymph Nodes of Mice After Liposome-Mediated Depletion

Macrophages lining the subcapsular sinus (SCS) and those located in the medulla of popliteal lymph nodes (PLN) of mice were eliminated after subcutaneous (s.c.) injection of dichloromethylene diphosphonate (CI2MDP)‐containing liposomes. No effect of liposome‐entrapped CI2MDP could be seen on nonphagocytic cells, e.g., interdigitating cells (IDC) and B‐ and T‐lymphocytes. One month after injection the eliminated subsets of macrophages were still absent. After 2 mo a small number of macrophages had reappeared along the SCS and in the medulla of the PLN of a few animals. Complete repopulation of the PLN with macrophages was observed only after 5 mo.