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The marginal zone (MZ) of the spleen is an important site for the capture of blood-borne pathogens and a gateway for lymphocytes entering the white pulp. We have recently reported that Leishmania donovani infection results in a remarkably selective loss of MZ macrophages (MZM) from the MZ. To understand the basis of this observation, we have investigated how MZM maintain their anatomical distribution in the steady state in uninfected mice. We now report that plt/plt mice, which lack functional CCL19 and CCL21, have significantly reduced numbers of MZM compared with normal C57BL/6 (B6) mice. Similarly, in B6.CD45.1-->plt/plt chimeras, donor-derived MZM were rare compared with the number observed in reciprocal plt/plt-->B6.CD45.1 chimeras. Moreover, we show that administration of pertussis toxin, an inhibitor of chemokine receptor signaling, to B6 mice results in exit of MZM from the MZ, that MZM can migrate in response to CCL19 and CCL21 in vitro, and that MZM colocalize with CD31+CCL21+ endothelial cells. Collectively, these data indicate that CCL21 and, to a lesser extent, CCL19 play significant roles in the distinctive localization of MZM within the splenic MZ. Deficiency of CCL19 and CCL21, as also previously observed in mice infected with L. donovani, may thus account for the selective loss of MZM seen during this infection.

Experimental murine infection with the parasites that cause human visceral leishmaniasis (VL) results in the establishment of infection in the liver, spleen, and bone marrow. In most strains of mice, parasites are eventually cleared from the liver, and hepatic resistance to infection results from a coordinated host response involving a broad range of effector and regulatory pathways targeted within defined tissue structures called granulomas. In contrast, parasites persist in the spleen and bone marrow by mechanisms that are less well understood. Parasite persistence is accompanied by the failure of granuloma formation and by a variety of pathologic changes, including splenomegaly, disruption of lymphoid tissue microarchitecture, and enhanced hematopoietic activity. Here, we review the salient features of these distinct tissue responses and highlight the varied roles that cytokines of the tumor necrosis factor family play in immunity to this infection. In addition, we also discuss recent studies aimed at understanding how splenomegaly affects the survival and function of memory cells specific for heterologous antigens, an issue of considerable importance for our understanding of the disease-associated increase in secondary infections characteristic of human VL.

Interaction between dendritic cells (DCs) and T cells is essential for the generation of cell-mediated immunity. Here we show that DCs from mice with chronic Leishmania donovani infection fail to migrate from the marginal zone to the periarteriolar region of the spleen. Stromal cells were fewer, which was associated with loss of CCL21 and CCL19 expression. The residual stromal cells and endothelium produced sufficient CCL21 to direct the migration of DCs transferred from naïve mice. However, DCs from infected mice had impaired migration both in naïve recipients and in vitro, in response to CCL21 and CCL19. Defective localization was attributable to tumor necrosis factor-alpha-dependent, interleukin 10-mediated inhibition of CCR7 expression. Effective immunotherapy was achieved with CCR7-expressing DCs, without the need to identify protective Leishmania antigens. Thus defective DC migration plays a major role in the pathogenesis of this disease and the immunosuppression is mediated, at least in part, through the spatial segregation of DCs and T cells.

Monocyte chemoattractant protein-1 (MCP-1) is a protective cytokine in murine endotoxaemia induced by lipopolysaccharide (LPS). In this study, LPS-induced pathophysiology in the human (h) MCP-1 transgenic mouse (Tgm) line was investigated. The hMCP-1 Tgm showed a marked increase in the mortality and weight loss following LPS administration. In the Tgm spleens, disappearance of marginal zone macrophages (MZMphi) and dendritic cells (DC) was induced by a smaller amount of LPS than that required for the disappearance in non-transgenic littermates. A significant number of apoptotic cells were seen in these areas. Furthermore, expressions of tumour necrosis factor-alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), and IL-6 mRNA were enhanced and sustained in the LPS-treated Tgm. Neutralization of TNF-alpha considerably depressed the LPS-sensitivity of Tgm. These findings demonstrate that the continuous and systemic presence of MCP-1 is no more protective toward endotoxaemia and suggest that the high sensitivity of the MZMphi and DC to LPS is attributed to the enhanced TNF-alpha production in the hMCP-1 Tgm.

The development of secondary lymphoid organs is a highly regulated process, mediated by tumor necrosis factor (TNF) family cytokines. In contrast, the mechanisms controlling changes in lymphoid architecture that occur during infectious disease are poorly understood. Here we demonstrate that during infection with Leishmania donovani, the marginal zone of mice undergoes extensive remodeling, similar in extent to developmental abnormalities in mice lacking some TNF family cytokines. This process is selective, comprising a dramatic and rapid loss of marginal zone macrophages (MZMs). As a functional consequence, lymphocyte traffic into the white pulp is impaired during chronic leishmaniasis. Significantly, MZMs were preserved in L. donovani-infected B6.TNF-alpha(-/-) mice or mice that received anti-TNF-alpha antibodies, whereas studies in CD8(+) T-cell-deficient mice and in mice lacking functional CD95L, excluded a direct role for either cytotoxic T lymphocyte activity or CD95-mediated apoptosis in this process. Loss of MZMs was independent of parasite burden, yet could be partially prevented by chemotherapy, which in turn reduced endogenous TNF-alpha levels. This is the first report of an infectious agent causing selective and long-lasting changes to the marginal zone via TNF-alpha-mediated mechanisms, and illustrates that those cytokines involved in establishing lymphoid architecture during development, may also play a role in infection-induced lymphoid tissue remodeling.

Monocyte chemoattractant protein (MCP)-1 is a chemotactic cytokine for monocytes, memoryT cells and dendritic cells (DC). However, the precise role of MCP-1 in a variety of immunological responses remains unclear. In the present study, we analyzed contact hypersensitivity (CHS) using human MCP-1 transgenic mice (hMCP-1Tgm) that constitutively produce high levels of hMCP-1 in the sera. Following 2,4-dinitrofluorobenzene (DNFB) sensitization, enhancement of CHS was demonstrated in Tgm as compared with that in non-Tgm. Anti-hMCP-1 antibodies significantly inhibited the CHS in Tgm. A prominent accumulation of B7-1+I-Ad+ Langerhans' cells (LC) bearing haptens was detected in draining lymph nodes (DLN) of Tgm 24 h after DNFB or fluorescein isothiocyanate (FITC) sensitization. Similar results were obtained with BALB/c mice administrated recombinant (r) hMCP-1. Langerhans' cells (LC) in the epidermal sheets of Tgm increased in size and expressed high levels of I-Ad and B7-1 12 h after FITC application compared with those of non-Tgm. After 18 h, the number of LC in the epidermis was reduced in Tgm. It was also shown that the B7-1 expression on LC of BALB/c mice was augmented after culture with rhMCP-1. These findings demonstrate that MCP-1 not only accelerates LC migration from epidermis into the DLN after sensitization with haptens but also up-regulates the I-Ad and B7-1 expressions, which results in the enhanced T cell activation and CHS.

Mouse allograft inflammatory factor-1 (AIF-1) cDNA was cloned and the AIF-1-specific monoclonal antibodies were established to examine its tissue distribution. The mouse AIF-1 was highly conserved among all reported AIF-1 from a variety of species, from invertebrates to mammals, and the cloned cDNA was in good accordance with putative expressed regions of genomic sequences in the mouse major histocompatibility complex (MHC) class III region. The messages of mouse AIF-1 were abundantly expressed in the testis, moderately in the spleen and lymph nodes and slightly in the liver and thymus of normal BALB/c mice. Immunohistological examination revealed that differentiating germ cells in the testis and presumably macrophages in the red pulp of the spleen were positive for AIF-1. To analyse the function of the AIF-1, a macrophage cell line, RAW 264.7, was transfected with mouse AIF-1 cDNA. Upon stimulation with bacterial lipopolysaccharide, the transfectants that overexpressed AIF-1 showed marked morphological changes and produced significantly large amounts of interleukin (IL)-6, IL-10 and IL-12p40 but not IL-12p70 compared with control cells. No difference was noted in production of tumour necrosis factor-alpha, transforming growth factor-beta1 and IL-1alpha. These results suggest that AIF-1 plays an important role in cells of a monocyte/macrophage lineage upon stimulation with inflammatory stimuli by augmenting particular cytokine production.

Atherosclerosis involves inflammatory processes between vascular tissues and hematocytes with a hyperlipidemic background. To examine whether variations of hematocytes constitute one of the genetic components in atherosclerosis, irradiated apolipoprotein E (apoE)-deficient (apoE(-/-)) mice with hypercholesterolemia and preexisting atherosclerotic lesions were reconstituted with mixed bone marrow cells (BMC) from syngeneic and wild-type (apoE(+/+); atherosclerosis-resistant SJL or -susceptible B10.S) mice. Stable mixed allogeneic chimeras with small amounts of serum apoE were established without any detrimental complications. Compared with untreated apoE(-/-) mice or apoE(-/-) mice transplanted with syngeneic BMC alone, significant reduction of the cholesterol level and significant lesion regression were observed in the mixed chimeras. Furthermore, mixed chimeras given SJL BMC showed marked reductions in numbers of lesions compared with those reconstituted with B10.S BMC. Cholesterol levels in the former SJL chimeras, however, were significantly higher than those in the latter B10.S chimeras. These findings indicate that the resistance of SJL to atherosclerosis resides in the bone marrow-derived cells.

We have previously shown that the generation of an NK1.1+TCRalphabeta+ (NK-T) cell population is severely impaired in an alymphoplasia mutant (aly/aly) mouse strain and the defect resides in the thymic environment. In the present study, to elucidate the thymic stromal component(s) that affects the development of NK-T cells, radiation bone marrow chimeras were established with the aly/aly mouse as a donor and either the beta2 microglobulin knockout (beta2m-/-) or the CD1d1-/- mouse that also lacks the NK-T cell population as a recipient. A normal population of NK-T cells with a typical NK-T phenotype and functions was detected in both the thymus and the spleen of these chimeras. These findings indicated that a radiation-resistant CD1(-) component of the thymus supported generation of functional NK-T cells from aly/aly precursors. Furthermore, transfer of an intact medullary thymic epithelial cell line into aly/aly thymus significantly induced the generation of NK-T cells in the thymus. These findings suggest that CD1 molecules of bone marrow-derived cells and the medullary epithelial cells acted in concert in the generation of the NK-T cell population and that a function(s) of the medullary thymic epithelial cells other than direct presentation of CD1 molecules to the NK-T precursors is indispensable for the development of NK-T cells., (Copyright 2000 Academic Press.)

A human monocyte chemoattractant protein-1 (hMCP-1) transgenic mouse (Tgm) line which constitutively produces a large amount of hMCP-1 (7-13 ng/ml in the serum) was established. Although expression of the transgene was detected in various tissues, an accumulation of macrophages (Mphi) was seen in only lymphoid organs which might be attributed to the high concentration of hMCP-1 in these organs. A reduced phagocytosis by peritoneal Mphi in vivo and a delayed clearance of granulomas in the liver following zymosan administration were observed in these Tgm. However, peritoneal exudate cells (PEC) from Tgm exhibited normal in vitro phagocytic activity and nitric oxide (NO) production upon stimulation with IFN-gamma as compared with those from non-Tgm. In addition, high activities of src-family protein tyrosine kinases (PTK), Fgr and Hck, were also noted in the peritoneal resident cells from Tgm, whereas the level of mitogen-activated protein kinase (MAPK) activity was almost the same as that of non-Tgm. It was suggested that the low functional activities of Tgm Mphi seen in vivo were attributed to down-regulation of the unique transducing system of hMCP-1 signals under the influence of a high concentration of the hMCP-1. It seemed that the depressed functions were recovered when the peritoneal cells were released ex vivo from such a high hMCP-1 environment.

The association of Sertoli-stromal cell tumor with testicular feminization syndrome (TFS) has been elaborated in the past studies. Here, we described immunohistochemical studies on Sertoli cell tumor of the gonad in a TFS patient and compare with 2 other cases of spontaneous ovarian Sertoli-stromal tumor. [Case 1] The case was a 73 year-old Japanese patient (46XY karyotype), who had had primary amenorrhea. High level of testosterone was noted in laboratory investigation (1900 ng/ml). No ambiguous morphology of external genitalia was present, but atrophy of vagina was noted. The patient was diagnosed as TFS. A left gonadal tumor was identified histologically showing well differentiated Sertoli cell tumor. The tumor cells were positive for anti-vimentin antibody but negative for anti-keratin, EMA and p53 antibodies by immunohistochemistry. The right gonad was an immature testis. [Case 2] The case was a 33 year-old female with ovarian Sertoli-Leydig cell tumor. Immunohistochemically, positive reaction for anti-keratin and p53 antibodies were observed. [Case 3] The case was a 17 year-old female with moderately differentiated Sertoli cell tumor of the ovary. The tumor cells were positive for anti-keratin, EMA and p53 antibodies by immunohistochemistry. Difference in immunohistochemical reactions between Sertoli cell tumor in TFS and Sertoli-stromal cell tumors of the ovaries was probably due to variation in the degree of gonadal development.

MCAF/MCP-1 is a chemotactic cytokine which belongs to beta- or C-C chemokine subfamily. MCAF/MCP-1 activates not only monocytes but also other types of leukocytes. However, precise roles of MCAF/MCP-1 in vivo remain unclear. To elucidate these issues, we established a human MCAF/MCP-1 transgenic mouse (Tgm). The Tgm produced constitutively a high concentration of MCAF/MCP-1 (7-13 ng/ml) in the serum under the influence of beta-actin promoter in the vector of transgene. The expression of transgene was recognized in multiple organs, especially in lymphoid organs and an accumulation of macrophages was seen in these lymphoid organs. However, peritoneal macrophages of the Tgm exhibited reduced phagocytosis toward latex beads which had been injected intraperitoneally. It was also demonstrated that these Tgm had prolongation of liver granuloma reaction against the stimulation of zymosan. In contrast, activities of Fgr and Hck, macrophage-associated src-family protein tyrosine kinases, were increased in the peritoneal cells of Tgm. When peritoneal macrophages of Tgm were developed in a non-Tgm circumstance after bone marrow transplantation from Tgm into AKR, these macrophages exhibited increased expression of Mac-1. Moreover, when co-cultured with IFN-gamma, these macrophages produced a large amount of nitric oxide as compared to non-Tgm cells cultured in the same condition as Tgm cells. These findings suggested that the dysfunction of macrophages seen in human MCAF/MCP-1 Tgm in vivo was attributed to down-regulation and/or desensitization of receptors for MCAF/MCP-1 caused by the high concentration of MCAF/MCP-1.

The development of T cells within the thymus is largely dependent on intact cortical and medullary epithelial cells. However, it has been reported that positive selection of natural killer antigen 1.1+ (NK1.1+) T cell antigen receptor (TCR)-alpha/beta+ thymocytes recently identified among CD4+8- and CD4-8- subpopulations is attributable to major histocompatibility complex class Ib ligands expressed on bone marrow (BM)-derived components in the thymus. In the present study, we investigated generation of NK1.1+ TCR-alpha/beta+ cells in the thymus of the aly/aly mouse which lacks lymph nodes and Peyer's patches and shows abnormalities of thymic and splenic structure. We found that the proportion of the NK1.1+ TCR-alpha/beta+ thymocytes was extremely low in these mice as compared with aly/+ and normal C57BL/6 mice. Thymic reconstitution by BM cells from aly/+ mice that possess a normal population of NK1.1+ TCR-alpha/beta+ cell population did not restore the NK1.1+ TCR-alpha/beta+ cell population in the thymus of lethally irradiated aly/aly mouse. When deoxyguanosine-treated fetal thymi from (B6 x B10.G)F1 mice were transplanted to aly/aly mice that had been thymectomized and reconstituted with BM cells of aly/aly mice, normal proportions of the NK1.1+ TCR-alpha/beta+ thymocytes were present in the thymus grafts. These findings demonstrate that the development of NK1.1+ TCR-alpha/beta+ thymocytes is accomplished under the influence not only of BM-derived components, but also of irradiation-resistant or deoxyguanosine-resistant components and an intact microenvironment of the thymus.

The catalytic activity of src-family protein tyrosine kinases (src-PTK) is suppressed when a C-terminal tyrosine is phosphorylated by an intracellular PTK, C-terminal Src kinase (Csk). In the present report, to study the regulatory functions of the Csk in cells of monocyte/macrophage lineage, we transfected a eukaryotic expression vector containing rat csk cDNA in a macrophage cell line, J774A.1, and examined alterations of the response to lipopolysaccharide (LPS) in the transfectants which overexpressed Csk. Csk overexpression resulted primarily in a down-regulation of Fgr activity, an src-PTK expressed in J774A.1, and hyperphosphorylation of several cellular proteins of 35, 57, 66, 97 and 120-130 kDa. Furthermore, in these Csk transfectants, production of interleukin (IL)-1alpha, IL-6, tumor necrosis factor-alpha, and nitric oxide (NO) following LPS stimulation were reduced compared with those in parental J774A.1 or J774A.1 transfected with the vector alone. The extent of reduction paralleled the amounts of Csk proteins expressed in the Csk-transfected J774A.1. The reduced NO production in these cells was associated with low levels of mRNA of inducible NO synthetase. On the other hand, an enhancement of prostaglandin E2 production was observed in the Csk-transfected J774A.1 cells upon stimulation with LPS, which appeared to result from the high level of prostaglandin-H synthetase in the transfectants. The present findings indicate that overexpression of Csk has differential effects on the regulation of production of chemical mediators and monokines, probably via modulation of signal transduction downstream of LPS-mediated signals.

The c-fgr gene product (Fgr) is a member of the src-family of protein tyrosine kinases. We have established a monoclonal antibody (2H2) which recognizes the unique N-terminal domain of the murine Fgr. In the present study, using immunohistochemical analysis and immune complex kinase assay with the 2H2, we investigated expression of Fgr in various cell populations and tissues in a murine system. In resting conditions, Fgr expression was confined to subsets of a monocyte/macrophage lineage. Thus, Fgr+ cells were detected in paracortical areas and medullas of lymph nodes, but seen only in marginal zones of the spleen and the medulla of the thymus. No Fgr+ macrophage was detected in other tissues, Peyer's patches, brain, heart, lung, liver, pancreas, kidney and peritoneal cavity. However, immune complex kinase assay revealed that, upon stimulation, T and B cells as well as peritoneal macrophages expressed significant levels of Fgr molecules. Transformed cell lines of lymphoid origin, EL-4 and LK35.2, which are T and B lineage lymphomas, respectively, also expressed Fgr molecules. Thus, various cells of hematopoietic origin appeared to possess a potentiality to express Fgr following activation or transformation. The present findings may help elucidate the functional significance of Fgr in immunologically committed cells in either activated or non-activated conditions.