The splenic marginal zone is absent in alymphoplastic aly mutant mice

aly is a unique spontaneous autosomal recessive mutation in mice that causes a deficiency in the systemic lymph nodes (LN) and Peyer's patches (PP). aly also induces abnormal histological findings in the spleen and a deficiency in humoral and cell-mediated immune functions, although lymphocytes of the aly/aly mouse show virtually normal immune responses in vitro. We studied the structure of the spleen of aly mice in detail by immunohistochemistry and electron microscopy. We found that the spleen of the aly/aly mouse was deficient in the expression of specific antigens for marginal metallophils (MM), marginal zone macrophages (MZM) and fibroblastic reticular cells (FRC), which are components of the marginal zone (MZ) of the spleen. Morphological analysis indicated that the aly/aly spleen is deficient in the structure of MZ, which may, in part, account for the severe immunodeficiency in the aly/aly mouse. We then performed reciprocal bone marrow transplantation experiments (BMT) between normal and aly mice and found a clear correlation between the formation of LN and the development of the splenic MZ in these mice; i.e. successful development of LN was invariably associated with the appearance of the MZ structure, whereas the failure of LN development was always associated with the absence of the development of MZ. These BMT results suggest that a common factor may regulate the generation of both LN and MZ of the spleen.     

Modulation of lymphocyte migration to the murine spleen after marginal zone macrophage phagocytosis of blood-borne particulate material

Our previous studies identified a role for MZM in the movement of lymphocytes into the splenic white pulp. Here we show that phagocytosis of colloidal carbon by marginal zone macrophages results in a splenic influx of B lymphocytes, and T lymphocytes of memory/activated phenotype, with concomitant upregulation of B Lymphocye Chemoattractant (BLC, CXCL13) mRNA, a chemokine acting on B and memory/activated T lymphocytes. The recruitment of B cells and activated T cells to the spleen after phagocytic uptake would allow an immune response against blood-borne pathogens to be quickly and effectively mounted by bringing together the two key cell types responsible for generating humoral immunity.     

SWAP-70 controls formation of the splenic marginal zone through regulating T1B-cell differentiation

T1 and T2 transitional B cells are precursors for marginal zone B cells (MZB), which surround splenic follicles. MZB are essential for marginal zone formation, are central to the innate immune response, and contribute to adaptive immunity. Differentiation, migration, and homing of MZB and their precursors remain to be fully understood. We show that SWAP-70, a RhoGTPase-interacting and F-actin-binding protein with functions in cell polarization, migration, and adhesion regulates MZB development and marginal zone formation. The percentage of MZB in spleen of Swap70(-/-) mice was reduced to about one-third of that found in WT mice. Swap70(-/-) T1 cells accumulated in integrin ligand(high) regions of the splenic red pulp and failed to efficiently develop into T2 cells. Adoptive transfer and mixed BM chimera experiments demonstrated this to be a B-cell intrinsic phenotype. T-cell-independent antibody production was not impaired, however, and thus suggests that this process does not require correct homing of MZB precursors. B-cell adhesion through α(L)β(2) and α(4)β(1) integrins was hyper-activated in vitro and on tissue sections, and S1P-stimulated chemokinesis of MZB was reduced in the absence of SWAP-70. Thus, SWAP-70 acts as a regulator of the adhesion process, particularly important for differentiation control of B-cell precursors and their contribution to splenic tissue formation.     

Interaction of SIGNR1 expressed by marginal zone macrophages with marginal zone B cells is essential to early IgM responses against Streptococcus pneumoniae

The spleen plays a pivotal role in the immune defense against encapsulated bacteria such as Streptococcus pneumoniae. Murine splenic marginal zone macrophages express the C-type lectin SIGNR1, which is crucial for the capture of S. pneumoniae from blood. In this study, we demonstrate that SIGNR1 is able to interact in vitro with the juxtaposing marginal zone B cell population, which is responsible for the production of the early IgM response against the S. pneumoniae-epitope phosphorylcholine. Strikingly, SIGNR1-deficient mice display a reduction in the marginal zone B cell population. In addition, ex vivo B cell stimulation assays demonstrate a decrease in phosphorylcholine specificity in the splenic B cell population derived from SIGNR1-deficient mice, whereas the total IgM response is unaffected. Therefore, we hypothesize that antigens are presented by SIGNR1 expressed by marginal zone macrophages to the developing marginal zone B cell population thereby influencing the repertoire of this B cell population, which is pivotal for the early immune response against encapsulated bacteria such as S. pneumoniae.     

Apoptotic cell responses in the splenic marginal zone: a paradigm for immunologic reactions to apoptotic antigens with implications for autoimmunity

Apoptotic cells drive innate regulatory responses that result in tolerogenic immunity. This is a critical aspect of cell physiology as apoptotic cells expose potentially dangerous nuclear antigens on the surface in apoptotic blebs, and failure in their recognition, phagocytosis, or destruction can cause dramatic autoimmunity in experimental models and is linked to development and progression of systemic pathology in human. The marginal zone is a specialized splenic environment that serves as a transitional site from circulation to peripheral lymphoid structures. The marginal zone serves a key role in trapping of particulates and initiation of innate responses against systemic microbial pathogens. However in recent years, it has become clear the marginal zone is also important for initiation of immune tolerance to apoptotic cells, driving a coordinated response involving multiple phagocyte and lymphocyte subsets. Recent reports linking defects in splenic macrophage function to systemic lupus erythematosus in a manner analogous to marginal zone macrophages in lupus-prone mice provide an impetus to better understand the mechanistic basis of the apoptotic cell response in the marginal zone and its general applicability to apoptotic cell-driven tolerance at other tissue sites. In this review, we discuss immune responses to apoptotic cells in the spleen in general and the marginal zone in particular, the relationship of these responses to autoimmune disease, and comparisons to apoptotic cell immunity in humans.     

Putative role of marginal zone B cells in pathophysiological processes

The maintenance of inner integrity of an organism is founded on the proper performance of two immunity branches, innate and adaptive immune responses. Recently, it became apparent that subset of splenic B cells named marginal zone B cells (MZB cells) exhibits unique developmental and functional features that bridge these two immunity branches. Strategically positioned at the site where blood and lymph are filtered, MZB cells represent a population of sentinels that rapidly proliferate and differentiate into IgM plasmablast cells when encountered with blood-borne, thymus-independent (TI) Ags. Moreover, MZB cells have intrinsic capability to induce potent CD4⁺ helper T cell response and cytokine production upon stimulation with soluble antigens. Due to their ability to overcome a time gap prior the establishment of the full adaptive response towards pathogens, MZB cells connect and direct innate and adaptive immunity. An additional interesting characteristic of MZB cells is capacity to function as regulatory cells in autoimmune processes. MZB cells may also contribute to the control of autoimmunity via the induction of tolerance by apoptotic cells. Importantly, in the clear association with inflammation and autoimmunity, MZB cells may transform into MALT lymphoma, representing a concurrence point for the infection, immunity and malignancy. This paper presents an insight into the complex biology of marginal zone B cells and their role in intertwining and directing innate and adaptive immune processes at the physiological and pathological level.     

Development of nephritis but not sialadenitis in autoimmune-prone BAFF transgenic mice lacking marginal zone B cells

B cell-activating factor belonging to the TNF family (BAFF) is a B cell survival factor required for B cell maturation. BAFF transgenic (Tg) mice develop autoimmune disorders characterized by autoantibody production, which leads to nephritis and salivary gland destruction (sialadenitis), features reminiscent of systemic lupus erythematosus and Sj?gren's syndrome (SS), respectively. Disease in BAFF Tg mice correlates with the expansion of the marginal zone (MZ) B cell compartment and the abnormal presence of MZ-like B cells in the blood, LN and inflamed salivary glands, suggesting a role for these cells in BAFF-induced autoimmunity. Lymphotoxin-beta (LTbeta)-deficient mice show disrupted splenic architecture, lack MZ B cells and some peripheral LN, and are unable to mount T cell-dependent immune responses. BAFF Tg mice lacking LTbeta (LTbetaDelta-BTg) retained these defects, yet still developed nephritis associated with the presence of B-1 B cells in the kidneys. However, in contrast to old BAFF Tg mice, aging LTbetaDelta-BTg mice no longer developed sialadenitis. Thus, autoimmune disorders in BAFF Tg mice are possibly events coordinated by MZ and B-1 B cells at separate anatomical sites.     

Enhancement of disease by neutralizing antiviral antibodies in the absence of primed antiviral cytotoxic T cells

The effects of neutralizing antibodies on the disease course in mice infected with the noncytopathic lymphocytic choriomeningitis virus (LCMV) were evaluated. Whereas non-neutralizing antisera exhibiting high enzyme - linked immunosorbent assay titers had no effect on T cell responses and their consequences, neutralizing antisera modulated them variably. Neutralizing antibodies were able to prevent lethal choriomeningitis after intracerebral infection with a neurotropic LCMV-isolate (ARMSTRONG) although they could not control local virus replication. The same antibodies exhibited little or no protective effect on choriomeningitis induced by LCMV-WE, a viscerotrope isolate. Surprisingly, these antibodies rendered mice much more susceptible to choriomeningitis after intracerebral infection with LCMV DOCILE, a very rapidly spreading lymphocyto-viscerotrope virus; in this situation antibodies prevented overwhelming infection which causes deletion of immunopathogenic cytotoxic T cell responses. Thus preexisting neutralizing antiviral antibodies had little influence on local virus spread in peripheral tissues but they reduced hematogenic spread and infection of antigen-presenting cells; thereby they influenced the primary cytotoxic T cell (CTL) response and indirectly modulated the extent of T cell-mediated immunopathology in peripheral organs. These results may explain why vaccines inducing neutralizing antibodies but no CTL may enhance an immunopathological disease caused by challenge infection with a noncytopathic virus.     

Modulation of Lymphocyte Migration to the Murine Spleen after Marginal Zone Macrophage Phagocytosis of Blood-Borne Particulate Material

Our previous studies identified a role for MZM in the movement of lymphocytes into the splenic white pulp. Here we show that phagocytosis of colloidal carbon by marginal zone macrophages results in a splenic influx of B lymphocytes, and T lymphocytes of memory/activated phenotype, with concomitant upregulation of B Lymphocye Chemoattractant (BLC, CXCL13) mRNA, a chemokine acting on B and memory/activated T lymphocytes. The recruitment of B cells and activated T cells to the spleen after phagocytic uptake would allow an immune response against blood-borne pathogens to be quickly and effectively mounted by bringing together the two key cell types responsible for generating humoral immunity.     

Natural antibodies target virus-antibody complexes to organized lymphoid tissue

Natural antibodies (NA) specific for infectious pathogens are found at low titer (usually <1:40) in the serum of healthy, non-immunized, individuals. Therefore, NA are part of the first line of defence against blood borne microorganisms. They directly neutralize viral infections or lyse pathogens by activating the complement cascade. In addition, recent studies highlighted their role in the pooling of infectious pathogens and other antigens to the spleen. This prevents infection of vital target organs and enhances the induction of adaptive immune responses. Specific T and B-cell responses are exclusively induced in highly organized secondary lymphoid organs including lymph nodes and the spleen. As a consequence, mice with disrupted microorganisation of lymphoid organs have defective adaptive immunity. In addition, some pathogens including lymphocytic choriomeningitis virus (LCMV), Leishmania and HIV developed strategies to destroy the splenic architecture in order to induce an acquired immunosuppression and to establish persistent infection. NA antibodies enhance early neutralizing antibodies in the absence of T help mainly by targeting antigen to the splenic marginal zone. In addition, by activating the complement cascade, NA enhance T cell and T-cell dependent B-cell responses. Therefore, natural antibodies are an important link between innate and adaptive immunity.     

Are murine marginal-zone macrophages the splenic white pulp analog of high endothelial venules?

The entry of lymphocytes into the spleen, in contrast to lymph nodes, does not involve high endothelial venule (HEV) interaction. The precise point of entry, as well as the mechanism by which lymphocytes enter the lymphoid areas of the spleen, remains controversial. We examined in detail the effect of two agents, pertussis toxin (PT) and the sulfated polysaccharide fucoidan, on splenic lymphocyte entry and positioning. These have previously been shown to interfere with lymphocyte extravasation across HEV. PT prevents lymphocyte extravasation, but not binding, to HEV, whereas fucoidan prevents binding and thus subsequent extravasation. Studies presented here show that pretreatment of murine lymphocytes with PT does not numerically affect entry into spleen, but profoundly alters lymphocyte positioning within the spleen. When fluorescently labeled, PT-treated lymphocytes are injected intravenously, they initially accumulate in the marginal zone, in apparent association with the layer of marginal zone macrophages (MZM?) which form a shell around the white pulp. They fail to traverse this layer into the white pulp, and subsequently localize in the red pulp. In contrast, untreated cells initially appear in the marginal zone, then continue to migrate into the white pulp after traversing the MZM? layer. The localization of PT-pretreated lymphocytes adjacent to the MZM? layer is disrupted by intravenous administration of fucoidan. Using a flow cytometric assay of aggregation between MZM? and lymphocytes, we confirmed that fucoidan is also able to inhibit this association in vitro, whereas PT has no effect on this interaction. We propose that MZM? in the mouse are the splenic analog of HEV, forming the port of entry of lymphocytes into the white pulp of the spleen.     

Complement-mediated follicular localization of T-independent type-2 antigens: the role of marginal zone macrophages revisited.

In this study we demonstrate a hitherto undescribed phenomenon, namely that thymus-independent type-2 antigens (TI-2 Ag) localize in splenic follicles within 1 h after administration. The follicular localization of 2,4,6-trinitrophenyl (TNP)-Ficoll was not antibody mediated. In addition in case of high-dose administration we observed a relatively large amount of TI-2 Ag in marginal zone macrophages. However, after low-dose administration we observed a preferential localization of TNP-Ficoll in the splenic follicles. Detection of TNP-haptenated Ag in cryostat sections of murine spleens was performed with a high-affinity TNP-specific monoclonal antibody conjugated to beta-galactosidase. Within minutes after injection the TI-2 Ag localized in the marginal zone, attached to marginal zone macrophages and B cells. Twenty minutes after injection the Ag was also detected in the follicles and gradually accumulated there until 7 h after injection. Thereafter, the amount of follicular Ag gradually decreased but was still detectable up to 14 days after immunization. The follicular localization of TNP-Ficoll was complement dependent in contrast to the binding to and uptake by marginal zone macrophages. Double staining revealed that Ag was bound by macrophages, B cells and follicular dendritic cells. Haptenated thymus-dependent (TD) Ag localized exclusively in the red pulp macrophages. In vivo macrophage elimination drastically increased the amount of TNP-Ficoll in the follicles, and enhanced the humoral immune response at low doses of Ag. Moreover, complement deprivation of mice abrogated the localization of TI-2 Ag in the follicles, and led to a decreased humoral TI-2 immune response. In conclusion, we demonstrate for the first time that TI-2 Ag localize in follicles. Moreover, the presented results provide further evidence that B cells and follicular localized Ag play an important role in the induction of humoral TI-2 immune responses.     

Specific ICAM-3 grabbing nonintegrin-related 1 (SIGNR1) expressed by marginal zone macrophages is essential for defense against pulmonary Streptococcus pneumoniae infection

The dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) homolog, SIGN-related 1 (SIGNR1) is a pathogen receptor expressed by splenic marginal zone and peritoneal macrophages, and is essential for clearance of Streptococcus pneumoniae by phagocytosis after intraperitoneal infection. Here, we identified an important in vivo function for SIGNR1 in S. pneumonia infection induced via its natural entrance route. Upon intranasal infection with S. pneumoniae, SIGNR1-deficient mice did not clear bacteria from lung and blood, and displayed severely enhanced inflammatory parameters compared to the wild-type mice. However, SIGNR1 is not expressed by alveolar macrophages, suggesting that another mechanism than a decrease in phagocytosis is responsible for this difference. Natural anti-phosphorylcholine IgM produced by marginal zone B cells is essential for protection against infection with S. pneumoniae. Strikingly, during infection, SIGNR1-deficient mice failed to produce a rapid anti-phosphorylcholine IgM response. Marginal zone macrophages have been suggested to capture antigens for presentation to marginal zone B cells. We demonstrate that marginal zone macrophages from SIGNR1-deficient mice in contrast to wild-type mice are not able to capture pneumococci from blood, suggesting that SIGNR1 on marginal zone macrophages captures S. pneumoniae for antigen presentation to and activation of marginal zone B cells, resulting in an anti-phosphorylcholine IgM response.     

Virus-specific T cell response prevents lymphoma development in mice infected by intrathymic inoculation of Moloney leukaemia virus (M-MuLV).

Previous work on mice neonatally injected with Moloney-murine leukaemia virus (M-MuLV) had shown that T cell lymphoma development correlates with virus infection of lymphoreticular cells (T and B lymphocytes and macrophages) as well as with a lack of generation of virus-specific cytotoxic T lymphocytes (CTL) due to clonal deletion of CTL precursors. In the present report, viral antigen expression and T cell response in mice injected as adults with M-MuLV intrathymus (i.t.) was investigated. Only thymic and splenic T lymphocytes from these mice express virus-induced antigens since they were lysed by virus-specific CTL, and stained by anti-M-MuLV fluorescent serum. In addition, the percentage of M-MuLV-infected T cells increased with increasing post- inoculation times. However, these mice could mount a strong cellular immune response against M-MuLV-infected cells, as detected by massive mixed leucocyte tumour cell culture and by evaluation of virus- specific CTL precursor frequency. Finally, i.t. injected mice were not viraemic and did not develop lymphomas during an observation period of 12-15 months. These data, in contrast with the recent hypothesis that T cell lymphoma development depends on a chronic stimulation of virus-specific T lymphocytes, indicate that the cellular immune response is sufficient for prevention of neoplastic transformation, despite a persistent viral infection of the thymus and peripheral T lymphocytes.     

Morphological and immunohistochemical changes in splenic macrophages of pigs infected with classical swine fever

Classical swine fever (CSF) was induced in 20 pigs by inoculation with a virulent strain of CSF virus to determine sequential changes (2, 4, 7, 10 and 14 days post-inoculation) in the number and morphology of splenic macrophages (red pulp and lymphoid marginal zone) and thus to assess the role of these cells in the pathogenesis of the disease. The first splenic cells to be infected with CSF virus were macrophages in the marginal zone followed by other macrophage populations. The initial phase of CSF was associated with an increase in splenic macrophage numbers in the marginal zone and a decrease in the red pulp. Subsequently, the numbers in the red pulp increased. The study suggested that infection, mobilization and apoptosis of splenic macrophages play an important role in the spread of CSF virus in vivo. Moreover, the secretory changes that occurred in macrophages in the initial phase of the infection suggested that macrophages release chemical mediators capable of modulating pathogenesis.     

Of mice and men: cytotoxic T cells and AIDS pathogenesis

CD8+ cytotoxic T lymphocytes (CTLs) represent a first line of defense against HIV infection, although their precise role in disease pathogenesis remains enigmatic. They play an important part in viral control but may also contribute to disease progression through destruction of CD4+ helper T cells. The role of CTLs in lymphocytic choriomeningitis virus (LCMV) infection in mice has been studied extensively, and the effects of CTL activity on host and virus are well defined. Although LCMV is not a retrovirus, it shares salient features with HIV, including a wide tropism, a capacity to persist, and genetic instability. The diseases caused by LCMV and HIV are linked by common immune effector mechanisms and, potentially, immunopathologies. Understanding the well-characterized immune responses in LCMV infection may therefore cast light on the role of CTLs in HIV disease.     

Impact of macrophage and dendritic cell subset elimination on antiviral immunity, viral clearance and production of type 1 interferon

We report herein that vesicular stomatitis virus (VSV) induced a concurrent primary Th1 (T helper 1) and Th2 cytokine response detectable ex vivo. Liposome-encapsulated clodronate-mediated elimination of CD8- marginal dendritic cells (DCs) and splenic macrophages (m Phi), but not CD8+ interdigitating DCs, prior to infection resulted in a markedly diminished chemokine and Th1 (IL-2, interferon-gamma) cytokine response, although the Th2 response (IL-4) remained relatively intact. Repopulation with marginal DCs and marginal metallophilic macrophages (MMM) restored Th1 cytokine profiles but did not restore chemokine responsiveness or reduce VSV-induced morbidity/mortality. Chemokine competency returned approximately 4 weeks post-depletion, which correlated temporally with repopulation of the spleen with marginal zone macrophages (MZM) and red pulp macrophages (RPM). Unexpectedly, virus-induced morbidity persisted for over 1 month post-depletion and was associated with virus dissemination and distinctive histological lesions in the liver. Depletion of interferon-producing plasmacytoid dendritic cells did not account for virus-induced morbidity because serum levels of type I interferon were not diminished in Cl2MBP-liposome-treated mice. Thus, distinct m Phi subsets are critical for chemokine production and viral clearance, and, in their absence, VSV disseminates even in the presence of high titers of interferon.     

Nodal marginal zone B cell lymphoma with prominent follicular colonization with deletion of chromosome 13

Nodal marginal zone B cell lymphoma is a rare type of malignant lymphoma and appears to be heterogeneous. Here we report a 60-year-old woman with stage I splenic type of nodal marginal zone B cell lymphoma with prominent follicular colonization. She was treated only by radiation therapy, and remained free of disease on examination for 4 years. The lymph node cells showed an abnormal chromosome of deletion 13, although neither bone marrow cells nor peripheral blood cells demonstrated the same abnormal chromosome. This type of chromosomal abnormality has not been previously reported and may be related to good prognosis in the present case.     

Roles of a macrophage receptor with collagenous structure (MARCO) in host defense and heterogeneity of splenic marginal zone macrophages

Class A type I and type II macrophage scavenger receptors (MSR-A) and a macrophage receptor with collagenous structure (MARCO) are trimeric membrane glycoproteins mediating the uptake of chemically modified low density lipoproteins. MSR-A is expressed constitutively in several tissue macrophages and in liver sinusoidal endothelial cells, whereas MARCO is expressed constitutively in splenic marginal zone macrophages and in macrophages and endothelial cells in the lymphatic medullary sinuses of lymph nodes. The administration of LPS, zymosan, BCG, or L. monocytogenes to mice resulted in marked and transient MARCO expression and in the upregulation of MSR-A expression in the liver and spleen. In osteopetrotic (op) mutant mice defective in the production on M-CSF, ER-TR9-positive marginal zone macrophages and MOMA-1-positive marginal metallophilic macrophages were absent, whereas MARCO-expressing marginal zone macrophages were present, indicating the heterogeneity of marginal zone macrophages. Intravenous administration of BCG resulted in marked accumulation of BCG bacilli in the both marginal zone macrophages and marginal metallophilic macrophages in littermate control mice. In contrast, BCG bacilli were incorporated almost exclusively by MARCO-expressing marginal zone macrophages in op/op mice. These results indicate that MARCO is not only expressed constitutively in specific macrophage subpopulations but is also induced by various bacterial antigens and plays a role in host defense against bacteria.     

Protection of CD3 delta knockout mice from lymphocytic choriomeningitis virus-induced immunopathology: implications for viral neuroinvasion

For a virus to establish a neuronal infection, it must spread from its primary site of infection to the central nervous system (CNS) before immune-mediated clearance occurs. Lymphocytic choriomeningitis virus (LCMV) is a murine pathogen that can result in persistent neuronal infection in newborn mice and in adults that lack CD8(+) T cells. To determine the neuroinvasive capacity of LCMV in the presence of an existent, but compromised, cytotoxic T lymphocyte response, the course of LCMV infection was examined in mice that possess 10% of the normal complement of T lymphocytes, due to the lack of the CD3 delta (delta) subunit of the T cell receptor complex (CD3 delta KO mice). Unlike immunocompetent mice that produced a massive immune response that caused death by 6-7 days postinfection, CD3 delta KO mice mounted a weak response and survived. The presence of viral antigen gradually shifted from the class I MHC-positive meninges and ependyma to class I MHC-deficient CNS neurons 10-30 days postinoculation. The infected CD3 delta KO mice developed a delayed T cell response that suppressed virus replication in peripheral tissues but not in the CNS; subsequent adoptive transfer experiments supported the hypothesis that the lack of clearance from neurons was due to sequestration of LCMV in an immune-privileged cell type. Based on these results, we propose that a critical parameter in the pathogenesis of neurotropic viruses is the rate of immune activation; individuals with impaired T cell responses may be more vulnerable to persisting CNS infections.