Periarteriolar Lymphocyte Sheath Research Articles

Stereological and Immunohistochemical Study of the Spleen in Hypothyroid Juvenile Rats

Abstract The purpose of this work was to investigate the influence of hypothyroidism on spleen tissue morphology and immune cell density in fourteen-day-old juvenile rats. Hypothyroidism in pups (n=10) was induced by administration of propylthiouracil (PTU) in drinking water (1.5 mg/L) to their mothers during pregnancy and period of lactation. Fourteen-day-old pups were sacrificed and the thyroid-stimulating hormone (TSH) serum concentration and thyroid activation index (Ia) were determined. Increased serum level of TSH and increased Ia showed that pups from PTU treated mothers were hypothyroid. White and red spleen pulp, marginal zone and connective tissue volume density has been assessed by using the stereological method. Using immunohistochemistry, the present CD3+ T lymphocytes, CD45RA+ B lymphocytes and CD68+ macrophages were quantified. A significant reduction of volume density of the periarteriolar lymphocyte sheath (VvPALS) and lymphatic follicles (Vvf) due to depletion of T and B lymphocytes respectively, was observed in the spleens of hypothyroid pups compared to controls. The volume density of the red pulp (Vvrp), marginal zone (Vvmz) and connective tissue (Vvct) was increased, as well as the number of CD68+ macrophages in the spleens of hypothyroid pups compared to controls. These results indicate that thyroid hormones might be important for normal development of both, specific and innate immune cells in the spleen during prenatal and early postnatal period.

Abstract 1254: Kaposi's Sarcoma herpesvirus K1's transcellular inhibition of Fas-mediated apoptosis

Abstract The long-term expression of human herpesvirus 8 (HHV-8) K1 produces hyperplasia of lymph nodes, splenomegaly, and lymphomas in mice. The mechanism of how K1 causes hyperplasia and lymphomas is not known. K1 is known to activate Akt and nuclear factor kappa B (NF-kB) through immunoreceptor tyrosine-based activation motif (ITAM) and may also bind to Fas receptor through its immunoglobulin (Ig) chain-like domain and interfere with apoptosis. We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by altered Fas signaling. Examination of mice expressing K1 via a ubiquitous promoter showed that 90% K1 transgenic mice (n=10) had developed lymphoid hyperplasia (at least 3 lymph nodes >3 mm) and 60% developed lymphomas after 18 months, while all (26) control nontransgenic mice remained free of lymph node hyperplasia, splenomegaly, and lymphoma. Some K1 mice developed liver or mesenteric tumors (4 of 10 mice). The spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-K1 transgenic control mice. Hematoxylin and eosin staining of spleen sections showed lymphocyte expansion in the periarteriolar lymphocyte sheath with disruption of normal spleen architecture. Anti-kappa and anti-lambda light chain antibodies revealed the presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but no foci were present in 4 control non-transgenic mice. Moreover, K1 protein was expressed in approximately 10% of splenic cells after staining with anti-K1 antibody 2H5. In vitro overexpression of an Ig domain-containing protein CD79b or treatment cells with K1 peptides revealed competition with K1-Fas binding in a dose-dependent manner and rate enhancement of Fas-mediated apoptosis. We have also shown that K1 suppressed Fas-mediated apoptosis, even in cells that did not express K1. Transfection of K1 into one pool of mouse cells protected against Fas-mediated apoptosis of a second pool of human Fas-transfected mouse cells indicating protection in trans. This analysis indicates a key role of K1 in suppression of Fas-mediated apoptosis which operates in a cis and trans protective role against apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1254.

Peptides Compete in Human Herpesvirus 8 K1-Fas Complexes Associated with Hyperplasia and Restore Apoptosis.

Abstract 2965Poster Board II-941The long-term expression of human herpesvirus 8 (HHV-8) K1 produces hyperplasia of lymph nodes, splenomegaly, and lymphomas in mice. The mechanism of how K1 causes hyperplasia and lymphomas. The immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to activate of Akt and nuclear factor kappa B (NF-kB). However, we have recently shown that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survive a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by altered Fas signaling.Examination of mice expressing K1 via a ubiquitous promoter showed that K1 transgenic mice (n=10) had 90% developed lymphoid hyperplasia (at least 3 lymph nodes >3 mm) and 60% developed lymphomas after 18 months, while all (26) control nontransgenic mice remained free of lymph node hyperplasia, splenomegaly, and lymphoma. Some K1 mice developed liver or mesenteric tumors (4 of 10 mice). The spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-K1 transgenic control mice. Hematoxylin and eosin staining of spleen sections showed lymphocyte expansion in the periarteriolar lymphocyte sheath with disruption of normal spleen architecture. Anti-kappa and anti-lambda light chain antibodies revealed the presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but no foci were present in 4 control non-transgenic mice. Moreover, K1 protein was expressed in approximately 10% of splenic cells after staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein confers resistant to Fas-mediated apoptosis, splenic cells of 6-month-old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4±1% of splenocytes from K1 mice versus 17±2% of control splenocytes showed morphology of apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11±0.6% vs. 50±6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2-induced apoptosis compared with splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 out of 12 K1 transgenic (30%) and 13 out of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis.We mapped the region that K1 uses to bind to Fas to an immunoglobulin (Ig) chain-like domain by expressing deletion mutants of K1. Overexpression of an Ig domain-containing protein CD79b competed with K1-Fas binding in a dose-dependent manner. Two 20-amino acid peptides (N251, N253) representing the Ig domain of K1 competed with K1-Fas binding in immunoprecipitation/immunoblotting analysis. The N251 and N253 peptides (100 mM) enhanced anti-Fas antibody (CH-11, 50 ng/mL)-induced apoptosis of BJAB lymphoma cells that expressed K1 but not that of vector-transfected BJAB cells. K1 splenocytes incubated with N251 and N253 showed enhanced rates of Fas-mediated apoptosis over control peptide-treated K1, and peptides did not enhance the apoptosis rates of control non-K1 expressing splenocytes.This analysis indicates a key role of K1 in lymphoid hyperplasia and lymphoma and that K1 direct binding to Fas occurs in vivo, which is effectively targeted by competing peptides to restore apoptosis. Disclosures:No relevant conflicts of interest to declare.

Human Herpesvirus 8 Protein K1 Interference with Fas-Mediated Apoptosis Induces Clonal Growth Which Leads to Lymphoid Hyperplasia.

Background: Human herpesvirus 8 (HHV-8) infection is causally associated with the development of primary effusion lymphoma and Kaposi s sarcoma. A transmembrane protein of HHV-8, K1, is readily expressed in those tumors and the expression of K1 alone causes hyperplasia of lymph nodes and lymphomas in mice. The exact mechanism of how K1 causes hyperplasia and lymphomas in K1 expressing mice is not known. The cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to be involved in activation of Nuclear Factor kappa B (NF -B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by alterations of Fas signaling.Results: Gross examination of thoracic and abdominal cavities of transgenic mice with K1 expression driven by a ubiquitous promoter were sacrificed at 18 months of age revealed enlarged cervical, mediastinal, renal, mesenteric lymph nodes and spleens. Peyer s patches were also enlarged and readily visible on the outer surface of the ileum. Of 10 K1 mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 200 mg vs. 94 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with antikappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4 1% of splenocytes from K1 mice versus 17 2% of control splenocytes were undergoing apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11 0.6% vs. 50 6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2 induced apoptosis than splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 of 12 K1 transgenic (30%) and 13 of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis.Conclusion: These results over all confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation of this phenomenon. Interference of K1 with Fas-mediated apoptosis disrupts the normal life cycle of lymphocytes.

Human Herpesvirus 8 Protein K1 Interferes with Fas-Mediated Apoptosis Induces Clonal Growth and Lymphoid Hyperplasia

Background: Expression of Human herpesvirus 8 (HHV-8) K1 causes hyperplasia of lymph nodes and lymphomas in mice. The exact mechanism of how K1 causes hyperplasia and lymphomas in K1 expressing mice is not known. The cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to be involved in activation of Nuclear Factor kappa B (NF-k-B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by alterations of Fas signaling.Results: At 18 months, 10 K1 transgenic mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 ± 200 mg vs. 94 ± 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with anti-kappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4 ± 1% of splenocytes from K1 mice versus 17 ± 2% of control splenocytes were undergoing apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11 ± 0.6% vs. 50 ± 6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2 induced apoptosis than splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 of 12 K1 transgenic (30%) and 13 of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis. We mapped the region that K1 uses to bind to Fas as an immunoglobulin (Ig) chainlike domain by expressing deletion mutants of K1. Overexpression of an Ig domaincontaining protein CD79b competed with K1-Fas binding in a dose-dependent manner. Two 20-amino acid peptides (N251, N253) representing the Ig domain of K1 competed with K1-Fas binding in immunoprecipitation/immunoblotting analysis. The N251 and N253 peptides (100 mM) enhanced anti-Fas antibody (CH-11, 50 ng/mL)-induced apoptosis of BJAB lymphoma cells that expressed K1 but not that of vector-transfected BJAB cells. Ig-deleted K1 (K1dIg)-transfected mice were not protected (0/6), and K1- transfected mice were protected (7/10, P < 0.01) against the lethal effects of agonistic anti-Fas (Jo2) antibody. K1dIg expressed in mice did not form complexes with Fas, suggesting that the Ig domain is essential for K1-Fas binding and suppression of apoptosis.Conclusion: These results confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation. K1 blocks Fas-mediated apoptosis and competing peptides can reinstate apoptosis.

Autoantibody production in lpr/lpr gld/gld mice reflects accumulation of CD4 + effector cells that are resistant to regulatory T cell activity

In Fas/FasL-deficient mice anti-chromatin Ab production is T cell dependent and is not apparent until after 10 weeks of age. Early control of anti-chromatin antibodies may be due to the counterbalancing influence of Treg cells. Here we show that Treg cells block lpr/lpr gld/gld Th cells from providing help to anti-chromatin B cells in an in vivo transfer system. Interestingly, the percentage and absolute numbers of Foxp3 + Treg cells is elevated in BALB/c- lpr/lpr gld/gld mice and increases with age compared to BALB/c mice. The majority of Foxp3 expression is found in the B220 − CD4 + T cell population, and Foxp3-expressing cells are localized in the splenic PALS (periarteriolar lymphocyte sheath). Strikingly, although the lack of functional Fas/FasL does not affect the ability of Treg cells to block Th cell proliferation, Treg cells can block the IFN-γ differentiation of Th cells from BALB/c or young BALB- lpr/lpr gld/gld mice but not of pre-existing Th1 cells from older BALB/c- lpr/lpr gld/gld mice. Thus, we suggest autoantibody production is not caused by the lack of Treg cells but by a defect in activation-induced cell death that leads to the accumulation of T effector cells that are resistant to regulatory T cell activity.

Human Herpesvirus 8 Protein K1 Interference with Fas-Mediated Apoptosis Induces Clonal Growth Which Leads to Lymphoid Hyperplasia.

Background: Human herpesvirus 8 (HHV-8) infection is causally associated with the development of primary effusion lymphoma and Kaposi's sarcoma. A transmembrane protein of HHV-8, K1, is readily expressed in those tumors and the expression of K1 alone causes hyperplasia of lymph nodes and lymphomas in mice. The exact mechanism of how K1 causes hyperplasia and lymphomas in K1 expressing mice is not known. The cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to be involved in activation of Nuclear Factor kappa B (NFκ-B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by alterations of Fas signaling.Results: Gross examination of thoracic and abdominal cavities of transgenic mice with K1 expression driven by a ubiquitous promoter were sacrificed at 18 months of age revealed enlarged cervical, mediastinal, renal, mesenteric lymph nodes and spleens. Peyer's patches were also enlarged and readily visible on the outer surface of the ileum. Of 10 K1 mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 ± 200 mg vs. 94 ± 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with anti-kappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4±1% of splenocytes from K1 mice versus 17±2% of control splenocytes were undergoing apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11±0.6% vs. 50±6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2 - induced apoptosis than splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 of 12 K1 transgenic (30%) and 13 of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis.Conclusion: These results over all confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation of this phenomenon. Interference of K1 with Fas-mediated apoptosis disrupts the normal life cycle of lymphocytes.

Two-photon imaging in intact lymphoid tissue.

For imaging living tissue, the two-photon technique presents several advantages over conventional confocal fluorescence imaging, including reduced photobleaching, minimal phototoxicity, and the ability to resolve detail at greater depths. We have established a two-photon imaging method that permits five-dimensional(x y,z temission wavelength)ex vivo imaging in intact murine lymphoid organs of viable T lymphocytes, B lymphocytes, and antigen-presenting cells (APC) up to a depth of about 300 µm. Three-dimensional reconstruction of lymph nodes revealed B cells in shallow, discrete subcapsular follicles, T cells in interfollicular regions, reticular fiber networks, and migratory dendritic cells. Additionally, we have imaged lymphocyte activity within the periarteriolar lymphocyte sheath in the intact spleen. In both lymph node and spleen, distinct modes of B and T cell movement may reflect specific antigen search programs. In this paper we review the capabilities of our experimental system, and hope to demonstrate the importance of live tissue imaging studies at the cellular level.

Macrophage subsets harbouring Leishmania donovani in spleens of infected BALB/c mice: localization and characterization.

The purpose of the current study was to characterize parasite-containing cells located in spleens of BALB/c mice infected with Leishmania donovani. In particular, expression of MHC class II molecules by these cells was examined to determine whether they could potentially act as cells capable of immunostimulating Leishmania-reactive CD4+ T lymphocytes. To this end, an immunohistological analysis of spleens taken at various time points after infection was undertaken. Using this approach, we observed, in the red pulp, the formation of small cellular infliltrates containing heavily infected macrophages that could be stained with the monoclonal antibodies MOMA-2 and FA/11. All of them expressed high levels of MHC class II molecules. Parasites were also detected in the white pulp, especially in MOMA-2+, FA/11+ and MHC class II+ macrophages of the periarteriolar lymphocyte sheath and in MOMA-2+ marginal zone macrophages. Infected cells were further characterized by fluorescence microscopy after their enrichment by adherence. All infected mononuclear cells recovered by this procedure could be stained with MOMA-2 and FA/11 and thus very probably belonged to the mononuclear phagocyte lineage. Furthermore, all of them strongly expressed both MHC class II as well as H-2M molecules, regardless of the time points after infection. Analysis of the parasitophorous vacuoles (PV) by confocal microscopy showed that these compartments were surrounded by a membrane enriched in lysosomal glycoproteins lamp-1 and lamp-2, in macrosialin (a membrane protein of prelysosomes recognized by FA/11) and in MOMA-2 antigen. About 80% of the PV also had MHC class II and H-2M molecules on their membrane. Altogether, these data indicate that in the spleens of L. donovani-infected mice, a high percentage of amastigotes are located in macrophages expressing MHC class II molecules and that they live in PV exhibiting properties similar to those of PV detected in mouse bone marrow-derived macrophages exposed to a low dose of interferon gamma (IFN-gamma) and infected in vitro.

Loss of N-Glycolylneuraminic Acid in Human Evolution: IMPLICATIONS FOR SIALIC ACID RECOGNITION BY SIGLECS

The common sialic acids of mammalian cells are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans are an exception, because of a mutation in CMP-sialic acid hydroxylase, which occurred after our common ancestor with great apes. We asked if the resulting loss of Neu5Gc and increase in Neu5Ac in humans alters the biology of the siglecs, which are Ig superfamily members that recognize sialic acids. Human siglec-1 (sialoadhesin) strongly prefers Neu5Ac over Neu5Gc. Thus, humans have a higher density of siglec-1 ligands than great apes. Siglec-1-positive macrophages in humans are found primarily in the perifollicular zone, whereas in chimpanzees they also occur in the marginal zone and surrounding the periarteriolar lymphocyte sheaths. Although only a subset of chimpanzee macrophages express siglec-1, most human macrophages are positive. A known evolutionary difference is the strong preference of mouse siglec-2 (CD22) for Neu5Gc, contrasting with human siglec-2, which binds Neu5Ac equally well. To ask when the preference for Neu5Gc was adjusted in the human lineage, we cloned the first three extracellular domains of siglec-2 from all of the great apes and examined their preference. In fact, siglec-2 had evolved a higher degree of recognition flexibility before Neu5Gc was lost in humans. Human siglec-3 (CD33) and siglec-6 (obesity-binding protein 1) also recognize both Neu5Ac and Neu5Gc, and siglec-5 may have some preference for Neu5Gc. Others showed that siglec-4a (myelin-associated glycoprotein) prefers Neu5Ac over Neu5Gc. Thus, the human loss of Neu5Gc may alter biological processes involving siglec-1, and possibly, siglec-4a or -5.

CXCR5-deficient mice develop functional germinal centers in the splenic T cell zone.

The chemokine receptor CXCR5 is thought to be essential for the migration of B cells into the network of follicular dendritic cells in the spleen. However, as shown here, B cells and follicular dendritic cells do co-localize, albeit aberrantly, even in the absence of CXCR5. In mice lacking CXCR5 both cell types are found in a broad ring around the sinuses of the marginal zones. Upon immunization with the T cell-dependent antigen 2-phenyl-oxazolone, ectopic germinal centers develop in the periarteriolar lymphocyte sheath. A network of follicular dendritic cells forms in the vicinity of the central arteriole within which the antigen-activated B cells proliferate. The analysis of the expressed V gene repertoire revealed that during B cell proliferation, hypermutation is activated and V region genes accumulate somatic mutations. The pattern of somatic mutations suggests that affinity selection may occur. This analysis confirms that in CXCR5-deficient mice, the organization of splenic primary follicles is severely impaired. However, within the T cell zone a micro-environment is built up, which provides all requirements needed for the affinity maturation to take place.

Antigen detection in vivo after immunization with different presentation forms of rabies virus antigen, II. Cellular, but not humoral, systemic immune responses against rabies virus immune-stimulating complexes are macrophage dependent.

In this paper we describe the effect of depletion of splenic macrophages on the uptake, and immune response against, different formulations of rabies virus antigen. Splenic macrophages were removed by intravenous injection with clodronate liposomes. beta-propiolacton inactivated rabies virus (RV-BPL) and immune-stimulating complexes (iscom) containing these antigens were given to macrophage-depleted and control mice. In the absence of phagocytic cells in the spleen, antigen is still trapped in the red pulp and to a lesser extent in the peri-arteriolar lymphocyte sheaths (PALS) for both antigen formulations. The localization pattern in the main area of immune response induction, namely the follicles, was unaltered after macrophage depletion. Functionally, the depletion of splenic and liver macrophages had no influence on the induction of specific antibody responses in both RV-BPL or RV-iscom immunized mice, even though the latter presentation form was clearly associated with specific localization in the marginal metallophillic macrophages. In RV-BPL immunized mice, macrophage depletion had no influence on proliferative T-cell responses. However, macrophage-depleted mice that were immunized with RV-iscom showed a significant decrease in proliferative T-cell responses. These results confirm existing ideas on the spleen as a physical filter rather than an induction site for humoral responses and shed new light on the efficient role of iscoms as antigen-presenting moieties in relation to their specific in vivo localization patterns and partial macrophage dependency.

Analysis of germinal centres in the immune response to oxazolone.

In a primary T-cell dependent immune response B-cell proliferation is observed in two areas of the spleen. In the first days after immunisation expansion of B-cell clones can be observed in the periarteriolar lymphocyte sheath. Here the development of plasma cells seems to take place. Then, around day 3 to day 4, antigen activated B-cells start to proliferate in the primary follicles. This leads to the formation of the germinal centre within which memory cells develop. The analysis of germinal centre B-cells at different timepoints after immunisation has shown that between days 10 and 14 somatic mutations rapidly accumulate in the variable regions of the antibody molecules (Berek et al, 1991). There is intraclonal diversification of the B-cell repertoire in the geminal centres (Jacob et al., 1991). We have now isolated DNA from single germinal centres of mice immunised with the hapten 2-phenyl-oxazolone (phOx). The data demonstrate that the affinity maturation of the primary immune response takes place in the germinal centres.

In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. I. In vivo expression of CD40 ligand, cytokines, and antibody production delineates sites of cognate T-B cell interactions.

T-B cell interactions have a central role in the development of antibody responses. Upon activation, T helper (Th) cells express the ligand for CD40, gp39, which is essential for Th cell-dependent B cell activation. The cytokines produced by activated Th cells have a regulatory role in B cell differentiation. In this study, we investigated, using immunohistochemical techniques, the in vivo time course and localization of gp39 expression and cytokine production in relation to the specific antibody production. Both the immunization with keyhole limpet hemocyanin (KLH), a thymus-dependent (TD) antigen, and trinitrophenyl (TNP)-Ficoll, a thymus-independent type 2 (TI-2) antigen, induced Th cells to express gp39. The expression of gp39 was restricted to Th cells in the outer periarteriolar lymphocyte sheaths (outer-PALS) and around the terminal arterioles (TA). Incidentally, gp39+ Th cells were found in the corona of follicles, whereas gp39+ cells were never found in the germinal centers or marginal zones of the spleen. Maximum frequencies of gp39+ cells were observed 3 and 4 d after primary and secondary immunization with KLH. After injection of TNP-Ficoll, a marked increase in gp39+ cells was observed, confirming previous observations that activated T cells are involved in TI-2 antibody responses. Analysis of the in vivo cytokine production revealed that interleukin 2 (IL-2)-, IL-4- and interferon gamma (IFN-gamma)-producing cells (IFN-gamma-PC) developed according to similar kinetics as observed for gp39+ cells. IL-2-PC and IL-4-PC were present in higher frequencies as were IFN-gamma-PC in the immune response against TNP-KLH. Double staining experiments revealed gp39+ Th cells producing IL-2, IL-4, or IFN-gamma, suggesting that these cells were involved in both the initial activation as well as the differentiation process of B cells into antibody-forming cells. Dual immunohistochemical analysis revealed gp39+ T cells and cytokine-PC in close proximity to antigen-specific, antibody-forming B cells. In conclusion, this study shows that in vivo gp39 is expressed on activated Th cells after immunization with TD and TI-2 antigens. Furthermore, the time course and compartmentalization of gp39+ expression, cytokine production and antibody formation after immunization suggest that cognate T-B cell interactions and T cell-regulated B cell differentiation occur in the outer-PALS and around the TA of the spleen.

Structure and histochemical organization of the spleen of Agama stellio (Sauria: Agamidae) and Chalcides ocellatus (Sauria: Scincidae).

The spleen of Agama stellio is composed mainly of red pulp; the white pulp is poorly developed, and its clusters are scattered throughout the organ and contain lymphocytes, reticular cells, and some plasma cells. The red pulp consists of clear reticular cells intermingled with blood cells, sinusoids, and pigment cells. The spleen of Chalcides ocellatus is encapsulated by connective tissue and is composed of white and red pulp. The white pulp consists of lymphoid tissue that surrounds the central arterioles, forming the periarteriolar lymphocyte sheath (PALS). The red pulp is composed of a system of venous sinuses and cords. The results of various histochemical procedures designed to demonstrate mucosubstances, proteins, and nucleic acids indicate that the spleen in these species resembles the mammalian spleen. © 1993 Wiley-Liss, Inc.

Quantitative assessment of a change of hemosiderin deposition with age in splenic compartments of rats.

Hemosiderin deposition was quantitatively estimated in paraffin sections stained using Perls' iron reaction in the spleens of untreated male rats aged from 0 to 28 months. Hemosiderin was deposited in the periarteriolar lymphocyte sheath (PALS), lymph follicles, and marginal zone, as well as in the red pulp which was the main compartment of hemosiderin deposition. Special attention was paid to the PALS and marginal zone. Marked hemosiderin deposition was evident from 4 months, although only few hemosiderin-laden macrophages appeared in the first 3 months. Hemosiderin deposition peaked at 12 months in the red pulp and at 20 months in the white pulp and marginal zone. The deposition in the white pulp and marginal zone was about 1/9 to 1/4 of that in the red pulp. A few hemosiderin-laden macrophages were found in the lymph follicles after 2 months. In the periarterial region of the splenic hilus, deposition of hemosiderin granules was observed even at 1 month when there was little hemosiderin deposition elsewhere. Since no arteries open there, the presence of hemosiderin-laden macrophages in the PALS and lymph follicles suggested movement of some macrophages from the red pulp and/or the marginal zone. In addition, hemosiderin deposition in the PALS increased with age after it decreased in the other compartments.

Ｇａｎｄｙ‐Ｇａｍｎａ結節の再検討

Gandy-Gamna nodules and focal hemosiderin depositions in the spleens obtained from 4 patients with idiopathic portal hypertension (IPH), 5 with liver cirrhosis (LC), and one with extrahepatic portal vein obstruction (EHO) were examined histologically. In all patients except one with IPH, full-scale, large, cross-cut sections stained by the Perls iron reaction were examined. In one patient of each disease group, serial sections were also used for three-dimensional observation. The Gandy-Gamna nodules are caused by hemorrhage and connective tissue proliferation in the periarteriolar lymphocyte sheath (PALS). Hemosiderin deposition extended to the trabecula via the lymphatic flow in the PALS, which is the reverse of that of blood in the arteriole. Although calcium deposition is observed later, it is not considered to be essential to the formation of a Gandy-Gamna nodule. Three-dimensional observation revealed that Gandy-Gamna nodules are not spherical, but spindle-shaped or branched, as a result of their formation along the central arterioles; however, they give the appearance of being two-dimensionally round in a single section.Gandy-Gamna nodules are concluded to be characteristic of the spleen of patients with portal hypertension. The definition of Gandy-Gamna nodules as nodules composed of proliferating connective tissue and hemosiderin deposition with calcification are not sufficient, and their location and histogenesis are also important considerations in this definition.

Visualization of cholecystokinin receptors on a subset of human monocytes and in rat spleen

Direct radioreceptor binding experiments and Scatchard analysis reveal CCK receptors on elutriator purified human peripheral blood monocytes, but not on purified human T cells. The monocyte receptors have a single class of high (0.1 nM) affinity binding sites. A structure-function analysis of monocyte binding by different CCK analogs correlates well with previously demonstrated chemotactic responses in monocytes and receptors in brain tissue. Biochemical cross-linking indicates that the monocyte CCK recognition molecule is comparable in molecular size to that in brain membranes. Utilizing a novel fluoresceinated Texas Red-CCK conjugate we have visualized that up to 20% of human peripheral monocytes bear receptors for CCK. A discrete and anatomically significant distribution of CCK receptors in rat spleen is shown by film autoradiography of tissue sections. A more detailed microscopic analysis identifies a dendritic population of monocyte-derived cells within the periarteriolar lymphocyte sheath (PALS) of the white pulp as the CCK receptor-bearing cell in spleen. The anatomical localization of receptor-bearing cells within the PALS region suggests a role for CCK in the antigen processing and sensitization phases of the immune response via regulatory effects of this peptide on a specific, local macrophage-related cell population.

The Periarteriolar Lymphocyte Sheath in Immunodeficiency T- or B-Lymphocyte Area?

In 153 patients with biopsy-proven primary systemic amyloidosis (AL), serum creatinine level correlated with median survival (creatinine less than or equal to 115 mumol/L [1.3 mg/dL], 2.56 months; creatinine greater than 115 mumol/L [1.3 mg/dL], 14.9 months; P = 0.007). The 24-hour urine total protein excretion was not correlated with survival. Patients who excreted monoclonal lambda light chains in the urine had an inferior survival (12 months) compared with those with kappa (30 months) or no monoclonal protein (35 months) in the urine (P = 0.01). The presence of lambda monoclonal protein was not associated with a higher median creatinine level. A monoclonal protein was detected in the urine in 76% of patients; when patients with more than 1 g of protein in the urine were studied, however, a monoclonal protein was detectable in 86%. The overall kappa-lambda ratio was 1:2.56, but as the urinary protein loss increased, the kappa-lambda ratio shifted to 1:4.7 for those with more than 3 g of protein in the urine.

Direct intrafollicular differentiation of memory B cells into plasma cells

Antibody-forming (plasma) cells and memory B cells are both generated during a humoral immune response in the spleen. Antibody-forming cells develop in the outer parts of the periarteriolar lymphocyte sheaths (PALS) while memory B cells develop in the lymphoid follicles. As soon as the first antibodies appear in the circulation, immune complexes are formed, the bulk of which are ingested by cells of the mononuclear phagocyte system. A small proportion, however, is immobilized on the processes of follicular dendritic cells (FDCs). These immune complexes are thought to play a key role in the generation of memory B cells in the follicles. In this article Nico van Rooijen postulates that, in the continuing presence of soluble antigen, the newly generated memory B cells in the follicles may continue to differentiate into antibody-forming cells. Experimentally such conditions are fulfilled when adjuvants that slowly release antigen are used and when antigens used for immunization can replicate in the body. The postulated synergistic action of immobilized immune complexes and soluble antigen in the follicles may represent an efficient mechanism for the rapid production of large numbers of plasma cells.