Development of follicular dendritic cells in lymph nodes of B-cell-depleted mice

Summary We have studied follicular dendritic cells (FDC) in lymph nodes of normal and thymus dysgeneic nude mice depleted of B-cells by chronic treatment with anti-IgM antibodies. We found that B cell depletion was accompanied by the absence of mature FDC as defined morphologically at the ultrastructural level. Only precursor FDC (p-FDC) could be demonstrated. Upon release of B-cell suppression, the repopulation of lymph nodes with B-cells was associated with the reappearance of fully differentiated FDC in primary follicles of nude mice and in secondary follicles of T-cell competent mice. We conclude that mature B-cells and/or B-cell products are required for the development of mature follicular dendritic cells in the mouse lymph node.     

Establishment of lymphotoxin beta receptor signaling-dependent cell lines with follicular dendritic cell phenotypes from mouse lymph nodes

Follicular dendritic cells (FDCs) have been shown to play a crucial role in the positive selection of high-affinity B cells that are generated by somatic hypermutation in germinal center (GC). Because of technical difficulties in preparing and maintaining pure FDCs, a role for FDCs in this complicated process has not been fully elucidated. In this study, we established a cell line designated as pFL that retained major FDC phenotypes from a three-dimensional culture of mouse lymph node cells. pFL cells proliferated slowly in response to an agonistic anti-lymphotoxin beta receptor mAb and TNF-alpha. A more rapidly growing clone, named FL-Y, with similar requirements for growth was isolated from a long-term culture of pFL. Analysis of surface markers in these two cell lines by immunostaining, flow cytometry, and DNA microarray revealed the expression of genes, including those of CD21, FcgammaRIIB, lymphotoxin beta receptor, ICAM-1, VCAM-1, IL-6, and C4, which have been shown to be characteristic of FDCs. In addition, B cell-activating factor was expressed in these two cell lines. At the pFL or FL-Y:B cell ratio of 1:100, the cell lines markedly sustained B cell survival and Ab production during 2 wk of culture, while most B cells collapsed within 1 wk in the absence of the FDC-like cells. Interestingly, expression of typical GC markers, Fas and GL-7, was notably augmented in B cells that were cocultured with Th cells on these two cell lines. Thus, pFL and FL-Y cells may be useful for providing insight into the functional role for FDCs in GC.     

Trypanosoma cruzi: maintenance of parasite-specific T cell responses in lymph nodes during the acute phase of the infection

Mice infected with 5 x 10(3) forms of Trypanosoma cruzi showed a transient, but severe impairment of in vitro spleen cell responses to parasite antigens and to Concanavalin A (Con A). In contrast, inguinal and periaortic lymph node (LN) cells displayed high parasite-specific proliferative responses and only a partial reduction of the Con A-induced proliferation during the acute and chronic phases of infection. Lymphocytes that underwent blastic transformation in T. cruzi-stimulated cell cultures were of the L3T4+ phenotype. Suppression of spleen cell responses occurred in the acute phase whether mice were infected with high (3 x 10(5] or low (5 x 10(3] doses of T. cruzi by intraperitoneal or subcutaneous route. Suppression of the T. cruzi-specific proliferative response of LN cells was only observed in mice infected with high subcutaneous inocula. This suppression, however, was restricted to the LNs draining the site of inoculation without affecting distant LNs. Supernatants from parasite-stimulated proliferating LN cells displayed low or undetectable T cell growth factor (TCGF) activity, in contrast with the high TCGF levels found in supernatants of the same cells stimulated with Con A. Low levels of TCGF were also detected in cultures of LN cells from mice immunized with T. cruzi extracts. Neither the T. cruzi antigen used for in vitro stimulation nor the LN cell supernatants from infected mice inhibited TCGF activity. These findings indicate that (1) parasite-specific responses are present in the LN compartment throughout the acute phase of T. cruzi infection in mice and (2) the proliferative response of L3T4+ LN cells from infected mice to T. cruzi antigens is not associated with a high TCGF secretory response.     

Cigarette smoke exposure impairs dendritic cell maturation and T cell proliferation in thoracic lymph nodes of mice

Respiratory tract dendritic cells (DCs) are juxtaposed to directly sample inhaled environmental particles. Processing and presentation of these airborne Ags could result in either the development of immunity or tolerance. The purpose of this study was to determine the consequences of cigarette smoke exposure on DC function in mice. We demonstrate that while cigarette smoke exposure decreased the number of DCs in the lungs, Ag-induced DC migration to the regional thoracic lymph nodes was unaffected. However, cigarette smoking suppressed DC maturation within the lymph nodes as demonstrated by reduced cell surface expression of MHC class II and the costimulatory molecules CD80 and CD86. Consequently, DCs from cigarette smoke-exposed animals had a diminished capacity to induce IL-2 production by T cells that was associated with diminished Ag-specific T cell proliferation in vivo. Smoke-induced defects in DC function leading to impaired CD4(+) T cell function could inhibit tumor surveillance and predispose patients with chronic obstructive pulmonary disease to infections and exacerbations.     

Suppression of the bacterial antigen-specific T cell response and the dendritic cell migration to the lymph nodes by osteopontin

Osteopontin (OPN) has been reported to enhance the interferon (IFN)-gamma-producing Th1-type T cell response through the induction of interleukin (IL)-12 and the suppression of IL-10. We therefore investigated whether OPN could enhance Th1 induction by vaccination against bacterial antigen in vivo. Unexpectedly, the co-inoculation of OPN suppressed the induction of IFN-gamma-producing CD4(+) T cells and T cell proliferative response after the subcutaneous heat-killed Listeria monocytogenes(HKLM) immunization. These results suggest that OPN down-regulates T cell priming. Since dendritic cells (DC) play a pivotal role in T cell priming, we next analyzed the effects of OPN on DC. The addition of OPN into the culture of either bone marrow-derived immature DC or an immature DC line JAWSII showed no effects on the expression of MHC class II, CD80, and CD86 molecules before and after HKLM stimulation. Consistently, in vitro OPN-treated DC showed a normal antigen-presenting function to an established Listeria-specific Th1-type T cells. However, when the DC were transferred into the footpad with HKLM and OPN, the migration of the transferred DC into the regional LN was suppressed in comparison to the DC transferred with HKLM alone. Furthermore, the addition of OPN into the culture of the DC line and HKLM severely suppressed the HKLM-induced expression of CCR7 chemokine receptor which is an important factor in the migration of DC into LN. All the results suggest the existence of an OPN-mediated negative feedback mechanism in the T cell immune response through the regulation of DC migration.     

Origin of follicular dendritic cell in the chicken spleen

The ellipsoid-associated cell (EAC) is a blood-borne phagocytic cell, residing in the antigen trapping zone of the chicken spleen. Binding and endocytosis of βGalactosidase (βGal) are independent from the Fc and complement receptors, because sulfated polysaccharides, in a concentration manner, inhibit the bacterial antigen uptake. The βGal-positive cells migrate to the periarterial lymphatic sheath (PALS), the preexisting germinal centers (GC), and form clusters with B- and T-cells. βGal, E5G12 double positive cells on the surface of the ellipsoid and in the PALS, GC and clusters prove that the EACs carry the enzyme. The EAC and the follicular dendritic cell (FDC) express, 68.2 and E5G12 and, 74.3 and E5G12, antigens, respectively. During migration the cessation of 68.2 and expression of 74.3 indicate the differentiation of EAC to FDC. By day 14 the clusters had disappeared, and in several GC the presence of double positive cells (74.3 and βGal; E5G12 and βGal) showed that the clusters had developed to GC. The presence of βGal⁺ cells in the PALS, where interdigitating dendritic cells (IDC) cooperate with the T-cells, suggests that in the spleen alternate routes exist for the EAC differentiation to FDC: EAC to FDC: βGal-loaded cells in the preexisting GC; and EAC through IDC to FDC: βGal⁺ EAC in the PALS and clusters. The EAC-FDC axis works exclusively inside the spleen; therefore; this system may be operated in pneumococcus infection.This work was supported by OTKA Grant number: T-042558.     

Correlation of viral replication activity, the clinical phase of the disease and immunologic deterioration in HIV infection

The authors have studied the relationship among HIV replicative activity, expressed by HIV isolation from peripheral blood lymphocytes and p24 antigenemia detection, and the evolution of infection, in terms of stage of disease and T4 cells absolute number, in a group of 120 infected subjects. Data presented indicate that the expression of viral gene products is related both to clinical and immunological deterioration and suggest that viral replication plays a central role in the progression of the disease.     

Development, maturation and subsequent activation of follicular dendritic cells (FDC): immunohistochemical observation of human fetal and adult lymph nodes

To elucidate the processes involved in development and activation of human follicular dendritic cells (FDC), immunohistochemistry was performed on paraffin sections of fetal lymph nodes (FLN) obtained from archived autopsy material, and of adult reactive lymph nodes (ARLNs) excised for diagnostic purpose, using a panel of antibodies. Our study showed that tiny clusters of CNA.42⁺ KiM4p⁺ cells, surrounded by some B-lymphocytes, initially arose in the cortical area of underdeveloped FLN around the 20th gestational week. No co-expression of CD21 and CD35 was found. In the relatively developed FLN of the same gestational age, small eddies of immature FDC, which expressed CD21, CD35, and nerve growth factor receptor (NGFR), as well as CNA.42 and KiM4p, were observed within ill-defined aggregations of B-lymphocytes. As gestation progressed, more B-lymphocytes assembled in a compact manner and formed primary lymphoid follicles containing an extending web of mature FDC, which expressed CNA.42, KiM4p, CD21, CD35, NGFR, and sometimes CD23 and X-11. In well-developed secondary follicles of ARLNs, activated FDC expressed additional molecules such as CD55, CD106, and S100α. Our observations identified the processes of phenotypic alteration of human FDC and established practical indicators determining their developmental stage and functional phase.     

Paracoccidioides brasilinsis-induced migration of dendritic cells and subsequent T-cell activation in the lung-draining lymph nodes

Paracoccidioidomycosis is a mycotic disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb), that starts with inhalation of the fungus; thus, lung cells such as DC are part of the first line of defense against this microorganism. Migration of DC to the lymph nodes is the first step in initiating T cell responses. The mechanisms involved in resistance to Pb infection are poorly understood, but it is likely that DC play a pivotal role in the induction of effector T cells that control Pb infection. In this study, we showed that after Pb Infection, an important modification of lung DC receptor expression occurred. We observed an increased expression of CCR7 and CD103 on lung DC after infection, as well as MHC-II. After Pb infection, bone marrow-derived DC as well lung DC, migrate to lymph nodes. Migration of lung DC could represent an important mechanism of pathogenesis during PCM infection. In resume our data showed that Pb induced DC migration. Furthermore, we demonstrated that bone marrow-derived DC stimulated by Pb migrate to the lymph nodes and activate a T helper (Th) response. To the best of our knowledge, this is the first reported data showing that Pb induces migration of DC and activate a T helper (Th) response.     

Dichotomy between CD1a+ and CD83+ dendritic cells in lymph nodes during SIV infection of macaques

The prevalence and differentiation of dendritic cells (DC) in lymphoid tissue of simian immunodeficiency virus (SIV)-infected cynomolgus monkeys was studied during disease progression. Lymph node biopsies were consecutively obtained from clinical rapid and slow progressors until the development of disease consistent with simian acquired immunodeficiency syndrome (sAIDS) occurred. Quantitative evaluation of CD1a+ DC and the expression of DC antigens related to maturation (CD83, DC-LAMP and S100b) were performed at the single cell level by in situ image analysis. Despite a persistent prevalence of CD1a+ DC in lymphoid tissue during disease progression, there was a subsequent drop of mature CD83+, DC-LAMP+ and S100b+ DC, correlating with the decline of CD4+ T cells in blood. Thus, disease progression to sAIDS was associated with impaired maturation of DC, and lack of CD83, DC-LAMP and S100b expression.     

Cyclooxygenase-2-mediated prostaglandin E2 production in mesenteric lymph nodes and in cultured macrophages and dendritic cells after infection with Salmonella

Although numerous studies have demonstrated the ability of intestinal epithelial cells to produce PGs after infection with wild-type strains of Salmonella, few studies have focused on Salmonella-induced prostanoids in mucosal lymphoid tissues. This is surprising in view of the profound effects PGs can have on the host response. To begin to address PG production at mucosal sites, mice were orally inoculated with Salmonella, and at varying times postinfection cyclooxygenase-2 (COX-2) mRNA expression and PGE(2) synthesis were investigated. COX-2 mRNA expression was highly inducible in the mesenteric lymph nodes, whereas COX-1 mRNA levels were constitutive. PGE(2) production also increased significantly in the mesenteric lymph nodes following exposure to viable Salmonella, but not after exposure to killed bacteria. This increased PGE(2) response could be blocked by treatment of mice with the selective COX-2 inhibitor, celecoxib. Treatment of mice with celecoxib during salmonellosis resulted in increased viable bacteria in the mesenteric lymph nodes by day 3 postinfection. However, celecoxib treatment prolonged the survival of lethally infected animals. In vitro studies demonstrated Salmonella-induced up-regulation of COX-2 mRNA expression and PGE(2) secretion by both macrophages and dendritic cells, which could also be blocked in the presence of celecoxib. Interestingly, exposure of these cultured APCs to viable Salmonella was a much greater stimulus for induction of PGE(2) synthesis than exposure to Salmonella-derived LPS. The present study demonstrates induction of PGE(2) synthesis in mesenteric lymph nodes, macrophages, and dendritic cells after infection with wild-type salmonella.     

Characterization of the systemic loss of dendritic cells in murine lymph nodes during polymicrobial sepsis

Dendritic cells (DCs) play a key role in critical illness and are depleted in spleens from septic patients and mice. To date, few studies have characterized the systemic effect of sepsis on DC populations in lymphoid tissues. We analyzed the phenotype of DCs and Th cells present in the local (mesenteric) and distant (inguinal and popliteal) lymph nodes of mice with induced polymicrobial sepsis (cecal ligation and puncture). Flow cytometry and immunohistochemical staining demonstrated that there was a significant local (mesenteric nodes) and partial systemic (inguinal, but not popliteal nodes) loss of DCs from lymph nodes in septic mice, and that this process was associated with increased apoptosis. This sepsis-induced loss of DCs occurred after CD3(+)CD4(+) T cell activation and loss in the lymph nodes, and the loss of DCs was not preceded by any sustained increase in their maturation status. In addition, there was no preferential loss of either mature/activated (MHCII(high)/CD86(high)) or immature (MHCII(low)/CD86(low)) DCs during sepsis. However, there was a preferential loss of CD8(+) DCs in the local and distant lymph nodes. The loss of DCs in lymphoid tissue, particularly CD8(+) lymphoid-derived DCs, may contribute to the alterations in acquired immune status that frequently accompany sepsis.     

Characterization of dendritic cells,isolated from normal and stimulated lymph nodes of the rat

Summary Non-lymphoid dendritic cells were isolated from normal and paratyphoid vaccine-stimulated lymph nodes draining the rat skin. They were studied using enzymecytochemical, immunocytochemical and electron-microscopical methods. These cells had an irregular outline and an eccentrically situated nucleus. All showed acid phosphatase activity in a central area and expressed Ia antigen on the plasma membrane. Birbeck granules were exclusively present in dendritic cells isolated from lymph nodes in the induction phase of the immune response. This observation concurs with the presence of Birbeck granules in interdigitating cells in situ during the same period of the immune response. It is concluded that the dendritic cells are the in-vitro equivalents of the non-actively phagocytizing population of interdigitating cells.     

A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge.

A candidate live-virus vaccine strain of Venezuelan equine encephalitis virus (VEE) was configured as a replication-competent vector for in vivo expression of heterologous immunogens. Three features of VEE recommend it for use as a vaccine vector. (i) Most human and animal populations are not already immune to VEE, so preexisting immunity to the vector would not limit expression of the heterologous antigen. (ii) VEE replicates first in local lymphoid tissue, a site favoring the induction of an effective immune response. (iii) Parenteral immunization of rodents and humans with live, attenuated VEE vaccines protects against mucosal challenge, suggesting that VEE vaccine vectors might be used successfully to protect against mucosal pathogens. Upon subcutaneous (s.c.) inoculation into the footpad of mice, a VEE vector containing the complete influenza virus hemagglutinin (HA) gene expressed HA in the draining lymph node and induced anti-HA immunoglobulin G (IgG) and IgA serum antibodies, the levels of which could be increased by s.c. booster inoculation. When immunized mice were challenged intranasally with a virulent strain of influenza virus, replication of challenge virus in their lungs was restricted, and they were completely protected from signs of disease. Significant reduction of influenza virus replication in the nasal epithelia of HA vector-immunized mice suggested an effective immunity at the mucosal surface. VEE vaccine vectors represent an alternative vaccination strategy when killed or subunit vaccines are ineffective or when the use of a live attenuated vaccine might be unsafe.     

Oncostatin M enhances CCL21 expression by microvascular endothelial cells and increases the efficiency of dendritic cell trafficking to lymph nodes

CCL21, a lymphatic endothelial cell (LEC)-derived chemokine, and its receptor CCR7 regulate dendritic cell (DC) trafficking to lymph nodes (LN), but it is unclear how CCL21 expression is regulated. Oncostatin M (OSM) is an IL-6-like cytokine synthesized by activated DC and other leukocytes. In vitro, OSM (but not TNF-alpha) stimulated CCL21 mRNA and protein expression by human dermal microvascular EC (DMEC) in an ERK1/2-dependent fashion. Conditioned medium from OSM-treated DMEC stimulated CCL21-dependent chemotaxis of mouse bone marrow-derived DC (BMDC). Cultured BMDC expressed OSM, which was increased with the addition of LPS. Topical application of the contact-sensitizing hapten, trinitrochlorobenzene, resulted in enhanced OSM expression in the skin, whereas cutaneous injection of TNF-alpha did not. Injection of OSM into the footpad increased CCL21 mRNA expression in the draining LN by approximately 10-fold and in mouse skin by approximately 4-fold without increasing CCR7 mRNA. In vitro, OSM increased the permeability of DMEC and lung microvascular EC monolayers to FITC-dextran beads, and, in vivo, it enhanced accumulation of Evans blue dye in draining LN by approximately 3-fold (p = 0.0291). Of note, OSM increased trafficking of BMDC injected in footpads to draining LN by 2-fold (p = 0.016). In summary, OSM up-regulates CCL21 expression in skin and draining regional LN. We propose that OSM is a regulator of CCL21 expression and endothelial permeability in skin, contributing to efficient migration of DC to regional LN.     

All-trans retinoic acid enhances murine dendritic cell migration to draining lymph nodes via the balance of matrix metalloproteinases and their inhibitors

Cancers escape immune surveillance through the manipulation of the host's immune system. Sequestration of dendritic cells (DCs) within tumor tissues and the subsequent inhibition of their migration is one of the several mechanisms by which tumors induce immunosuppression. In view of recent findings depicting the improvement of tumor immune responses in cancer patients following all-trans retinoic acid (ATRA) treatment, we sought to identify the effects of ATRA on DC mobility in the context of tumor immunotherapy. Our results demonstrate that ATRA, added to differentiating murine bone marrow progenitor cells, enhances the invasive capacity of the resulting DCs. Immature DCs injected intratumorally in mice show increased accumulation in draining lymph nodes, but not in nondraining lymph nodes and spleens, when differentiated in the presence of ATRA. The in vitro migration of mature DCs through the basement membrane matrix toward the lymphoid chemokines CCL19 and CCL21 is enhanced in these cells, albeit not in the presence of a matrix metalloproteinase (MMP) inhibitor. An increase in MMP production with a simultaneous decrease in the production of their inhibitors (tissue inhibitors of matrix metalloproteinase or TIMPs) is provoked by ATRA. This affects the MMP/TIMP balance in DCs, in particular that of MMP-9 and TIMP-1, favoring protease activity and thus allowing for enhanced DC mobilization. In conclusion, this study demonstrates that ATRA is capable of improving DC trafficking in a tumor milieu and, in view of the encouraging results obtained in the clinic, further supports the notion that ATRA might be a valuable chemical adjuvant to current immunotherapeutic strategies for cancer.