Lymphotoxin and the amazing technicolor circus of intestinal homeostasis.

Recently a new lymphocyte subset called innate lymphoid cells has emerged and it includes key producers of interleukin (IL)-17 and IL-22 in the mucosal environment. Using Citrobacter rodentium infection to deliver a pathogenic insult to the colon, two studies have revealed an underlying role for lymphotoxin-ß receptor signaling in the generation of IL-22 by these cells. This observation links a system well known for its ability to organize lymphoid microenvironments into a basic mucosal response.

Death of HT29 adenocarcinoma cells induced by TNF family receptor activation is caspase-independent and displays features of both apoptosis and necrosis.

The HT29 adenocarcinoma is a common model of epithelial cell differentiation and colorectal cancer and its death is an oft-analyzed response to TNF family receptor signaling. The death event itself remains poorly characterized and here we have examined the involvement of caspases using pan-caspase inhibitors. zVAD-fmk did not block death of HT29 cells in response to activation of the Fas, TRAIL, TNF, TWEAK and LTbeta receptors. The secondary induction of TNF or the other known bona fide death inducing ligands did not account for death following LTbeta receptor activation indicating that TNF family receptors can trigger a caspase-independent death pathway regardless of the presence of canonical death domains in the receptor. To provide a frame of reference, the phenotype of HT29 death was compared to four other TNF family receptor triggered death events; Fas induced Jurkat cell apoptosis, TNF/zVAD induced L929 fibroblast necrosis, TNF induced death of WEHI 164 fibroblastoid cells and TNF/zVAD induced U937 death. The death of HT29 and U937 cells under these conditions is an intermediate form with both necrotic and apoptotic features. The efficient coupling of TNF receptors to a caspase-independent death event in an epithelial cell suggests an alternative approach to cancer therapy.

Elimination of colonic patches with lymphotoxin beta receptor-Ig prevents Th2 cell-type colitis.

Past studies have shown that colonic patches, which are the gut-associated lymphoreticular tissues (GALT) in the colon, become much more pronounced in hapten-induced murine colitis, and this was associated with Th2-type T cell responses. To address the role of GALT in colonic inflammation, experimental colitis was induced in mice either lacking organized GALT or with altered GALT structures. Trinitrobenzene sulfonic acid was used to induce colitis in mice given lymphotoxin-beta receptor-Ig fusion protein (LTbetaR-Ig) in utero, a treatment that blocked the formation of both Peyer's and colonic patches. Mice deficient in colonic patches developed focal acute ulcers with Th1-type responses, whereas lesions in normal mice were of a diffuse mucosal type with both Th1- and Th2-type cytokine production. We next determined whether LTbetaR-Ig could be used to treat colitis in normal or Th2-dominant, IFN-gamma gene knockout (IFN-gamma(-/-)) mice. Four weekly treatments with LTbetaR-Ig resulted in deletion of Peyer's and colonic patches with significant decreases in numbers of dendritic cells. This pretreatment protected IFN-gamma(-/-) mice from trinitrobenzene sulfonic acid-induced colitis; however, in normal mice this weekly treatment was less protective. In these mice hypertrophy of colonic patches was seen after induction of colitis. We conclude that Th2-type colitis is dependent upon the presence of colonic patches. The effect of LTbetaR-Ig was mediated through prevention of colonic patch hypertrophy in the absence of IFN-gamma. Thus, LTbetaR-Ig may offer a possible treatment for the Th2-dominant form of colitis.

BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF.

B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.

Essential role of lymph nodes in contact hypersensitivity revealed in lymphotoxin-alpha-deficient mice.

Lymph nodes (LNs) are important sentinal organs, populated by circulating lymphocytes and antigen-bearing cells exiting the tissue beds. Although cellular and humoral immune responses are induced in LNs by antigenic challenge, it is not known if LNs are essential for acquired immunity. We examined immune responses in mice that lack LNs due to genetic deletion of lymphotoxin ligands or in utero blockade of membrane lymphotoxin. We report that LNs are absolutely required for generating contact hypersensitivity, a T cell-dependent cellular immune response induced by epicutaneous hapten. We show that the homing of epidermal Langerhans cells in response to hapten application is specifically directed to LNs, providing a cellular basis for this unique LN function. In contrast, the spleen cannot mediate contact hypersensitivity because antigen-bearing epidermal Langerhans cells do not access splenic white pulp. Finally, we formally demonstrate that LNs provide a unique environment essential for generating this acquired immune response by reversing the LN defect in lymphotoxin-alpha(-/)- mice, thereby restoring the capacity for contact hypersensitivity.

Antagonism at 5-HT(2A) receptors potentiates the effect of haloperidol in a conditioned avoidance response task in rats.

High affinity for serotonin-2A (5-HT(2A)) over dopamine (DA) D(2) receptors is a leading hypothesis for clozapine's favorable therapeutic profile. Recent preclinical studies also indicate that a sufficient antipsychotic effect might be obtained by a combined high 5-HT(2A)/low D(2) receptor blockade. Thus, addition of a 5-HT(2A) receptor antagonist to an ineffective dose of a D(2) receptor antagonist produces a robust antipsychotic-like effect in the conditioned avoidance response (CAR) test. Electrophysiological and biochemical studies also show that 5-HT(2A) receptor antagonists can confer an atypical (clozapine-like) profile on a D(2) receptor antagonist. Improved therapeutic efficacy by adjunctive 5-HT(2A) receptor antagonist treatment to a traditional D(2) receptor blocking regimen has been suggested. However, the ability of 5-HT(2A) receptor blockade to protect against, or ameliorate, parkinsonian symptoms still remains unclear. Using the CAR and the catalepsy (CAT) tests as indices for antipsychotic activity and extrapyramidal side effect (EPS) liability, respectively, the effects of the selective 5-HT(2A) receptor antagonist MDL 100,907 in combination with the DA D(2) receptor antagonists haloperidol or raclopride were studied in rats. Haloperidol (0.025 or 0.1 mg/kg sc, -30 min) produced a dose-dependent suppression of CAR. Pretreatment with MDL 100,907 (0.5, 1.0, or 1.5 mg/kg sc; -60 min) enhanced and prolonged the haloperidol-induced suppression of CAR without escape failures. MDL 100,907 (1 mg/kg sc, -60 min) had no effect on CAT when coadministered with ineffective doses of raclopride. Raclopride (1 mg/kg sc, -30 min) alone produced a submaximal cataleptic response that was significantly enhanced by pretreatment with MDL 100,907. The present results confirm and extend previous results by showing that 5-HT(2A) receptor blockade can enhance the antipsychotic-like effects of a very low dose of a commonly used traditional antipsychotic. 5-HT(2A) receptor blockade does not, however, prevent EPS (CAT). The therapeutic advantage of this combination might, therefore, operate within a fairly narrow window.

A soluble form of B cell maturation antigen, a receptor for the tumor necrosis factor family member APRIL, inhibits tumor cell growth.

A proliferation-inducing ligand (APRIL) is a ligand of the tumor necrosis factor (TNF) family that stimulates tumor cell growth in vitro and in vivo. Expression of APRIL is highly upregulated in many tumors including colon and prostate carcinomas. Here we identify B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI), two predicted members of the TNF receptor family, as receptors for APRIL. APRIL binds BCMA with higher affinity than TACI. A soluble form of BCMA, which inhibits the proliferative activity of APRIL in vitro, decreases tumor cell proliferation in nude mice. Growth of HT29 colon carcinoma cells is blocked when mice are treated once per week with the soluble receptor. These results suggest an important role for APRIL in tumorigenesis and point towards a novel anticancer strategy.

BAFF mediates survival of peripheral immature B lymphocytes.

B cell maturation is a very selective process that requires finely tuned differentiation and survival signals. B cell activation factor from the TNF family (BAFF) is a TNF family member that binds to B cells and potentiates B cell receptor (BCR)-mediated proliferation. A role for BAFF in B cell survival was suggested by the observation of reduced peripheral B cell numbers in mice treated with reagents blocking BAFF, and high Bcl-2 levels detected in B cells from BAFF transgenic (Tg) mice. We tested in vitro the survival effect of BAFF on lymphocytes derived from primary and secondary lymphoid organs. BAFF induced survival of a subset of splenic immature B cells, referred to as transitional type 2 (T2) B cells. BAFF treatment allowed T2 B cells to survive and differentiate into mature B cells in response to signals through the BCR. The T2 and the marginal zone (MZ) B cell compartments were particularly enlarged in BAFF Tg mice. Immature transitional B cells are targets for negative selection, a feature thought to promote self-tolerance. These findings support a model in which excessive BAFF-mediated survival of peripheral immature B cells contributes to the emergence and maturation of autoreactive B cells, skewed towards the MZ compartment. This work provides new clues on mechanisms regulating B cell maturation and tolerance.

A chemokine-driven positive feedback loop organizes lymphoid follicles.

Lymphoid follicles are B-cell-rich compartments of lymphoid organs that function as sites of B-cell antigen encounter and differentiation. CXC chemokine receptor-5 (CXCR5) is required for B-cell migration to splenic follicles, but the requirements for homing to B-cell areas in lymph nodes remain to be defined. Here we show that lymph nodes contain two types of B-cell-rich compartment: follicles containing follicular dendritic cells, and areas lacking such cells. Using gene-targeted mice, we establish that B-lymphocyte chemoattractant (BLC/BCA1) and its receptor, CXCR5, are needed for B-cell homing to follicles in lymph nodes as well as in spleen. We also find that BLC is required for the development of most lymph nodes and Peyer's patches. In addition to mediating chemoattraction, BLC induces B cells to up-regulate membrane lymphotoxin alpha1beta2, a cytokine that promotes follicular dendritic cell development and BLC expression, establishing a positive feedback loop that is likely to be important in follicle development and homeostasis. In germinal centres the feedback loop is overridden, with B-cell lymphotoxin alpha1beta2 expression being induced by a mechanism independent of BLC.

BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population.

The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.

Reversal of virus-induced systemic shock and respiratory failure by blockade of the lymphotoxin pathway.

At present, little is known about the pathogenesis of acute virus-induced shock and pulmonary failure. A chief impediment in understanding the underlying disease mechanisms and developing treatment strategies has been the lack of a suitable animal model. This study describes a mouse model of virus-induced systemic shock and respiratory distress, and shows that blockade of the lymphotoxin beta receptor pathway reverses the disease.

A role for lymphotoxin beta receptor in host defense against Mycobacterium bovis BCG infection.

To investigate the role of membrane lymphotoxin (LT)alpha1 / beta2 and its LTbeta receptor (LTbetaR) in the protective immune response to Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection, we have used a soluble fusion molecule (LTbetaR-IgG1). LTbetaR-Ig treatment interferes with granuloma formation mainly in the spleen by inhibiting macrophage activation and nitric oxide synthase activity. In addition, a large accumulation of eosinophils was observed in the spleen of LTbetaR-Ig-treated infected mice. Decreased blood levels of IFN-gamma and increased IL-4 were also observed, suggesting that the LTbetaR pathway is important in BCG infection to favor a Th1 type of immune response. The treatment of transgenic mice expressing high blood levels of a soluble TNFR1-IgG3 fusion protein with LTbetaR-Ig resulted in a still higher sensitivity to BCG infection, and extensive necrosis in the spleen. In conclusion, these results suggest that the LTbetaR and the TNFR pathways are not redundant in the course of BCG infection and protective granuloma formation: the LTbetaR pathway appears to be important in spleen granuloma formation, whereas the TNFR pathway has a predominant role in other tissues.

Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations.

The cause of many autoimmune and inflammatory diseases is unresolved, although dysregulated production of tumor necrosis factor (TNF) family members appears to be important in many cases. BAFF, a new member of the TNF family, binds to B cells and costimulates their growth in vitro. Mice transgenic for BAFF have vastly increased numbers of mature B and effector T cells, and develop autoimmune-like manifestations such as the presence of high levels of rheumatoid factors, circulating immune complexes, anti-DNA autoantibodies, and immunoglobulin deposition in the kidneys. This phenotype is reminiscent of certain human autoimmune disorders and suggests that dysregulation of BAFF expression may be a critical element in the chain of events leading to autoimmunity.

The requirement of membrane lymphotoxin for the presence of dendritic cells in lymphoid tissues.

Although several cytokines, including tumor necrosis factor (TNF), can promote the growth of dendritic cells (DCs) in vitro, the cytokines that naturally regulate DC development and function in vivo have not been well defined. Here, we report that membrane lymphotoxin (LT), instead of TNF, regulates the migration of DCs in the spleen. LTalpha(-/-) mice, lacking membrane LTalpha/beta and LTalpha(3), show markedly reduced numbers of DCs in the spleen. Unlike wild-type mice and TNF(-/-) mice that have densely clustered DCs in the T cell zone and around the marginal zone, splenic DCs in LTalpha(-/-) mice are randomly distributed. The reduced number of DCs in lymphoid tissues of LTalpha(-/-) mice is associated with an increased number of DCs in nonlymphoid tissues. The number of splenic DCs in LTalpha(-/-) mice is restored when additional LT-expressing cells are provided. Blocking membrane LTalpha/beta in wild-type mice markedly diminishes the accumulation of DCs in lymphoid tissues. These data suggest that membrane LT is an essential ligand for the presence of DCs in the spleen. Mice deficient in TNF receptor, which is the receptor for both soluble LTalpha(3) and TNF-alpha(3) trimers, have normal numbers of DCs. However, LTbetaR(-/-) mice show reduced numbers of DCs, similar to the mice lacking membrane LT alpha/beta. Taken together, these results support the notion that the signaling via LTbetaR by membrane LTalpha/beta is required for the presence of DCs in lymphoid tissues.

Brain choline acetyltransferase and mental function in Alzheimer disease.

OBJECTIVE: To determine whether higher brain levels of choline acetyltransferase (ChAT) are associated with improved neuropsychological function in patients with Alzheimer disease (AD). DESIGN: Case series with single-blind post hoc analysis of biopsy specimens. SETTING: Urban hospital and medical school. PATIENTS: A consecutive sample of 8 patients with AD undergoing brain biopsy and surgical implantation of intraventricular pumps for administration of potential chemotherapeutic agents. INTERVENTIONS: Brain biopsy, surgical implantation of intraventricular pumps, and, in 1 patient, ventriculoperitoneal shunt placement. MAIN OUTCOME MEASURES: All patients underwent neuropsychological testing no more than 2 weeks before surgical biopsy. Levels of ChAT were determined in fresh brain tissue from biopsy samples. RESULTS: Significant positive correlations were found between ChAT levels and 2 neuropsychological test scores, Mini-Mental State Examination and the Logical Memory subtest of the Wechsler Memory Scale. CONCLUSION: Degeneration of the cholinergic system in vivo correlates with decreasing cognitive function in patients with AD.

BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth.

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family, designated BAFF (for B cell activating factor belonging to the TNF family), which is expressed by T cells and dendritic cells. Human BAFF was mapped to chromosome 13q32-34. Membrane-bound BAFF was processed and secreted through the action of a protease whose specificity matches that of the furin family of proprotein convertases. The expression of BAFF receptor appeared to be restricted to B cells. Both membrane-bound and soluble BAFF induced proliferation of anti-immunoglobulin M-stimulated peripheral blood B lymphocytes. Moreover, increased amounts of immunoglobulins were found in supernatants of germinal center-like B cells costimulated with BAFF. These results suggest that BAFF plays an important role as costimulator of B cell proliferation and function.

Signaling through the lymphotoxin-beta receptor stimulates HIV-1 replication alone and in cooperation with soluble or membrane-bound TNF-alpha.

The level of ongoing HIV-1 replication within an individual is critical to HIV-1 pathogenesis. Among host immune factors, the cytokine TNF-alpha has previously been shown to increase HIV-1 replication in various monocyte and T cell model systems. Here, we demonstrate that signaling through the TNF receptor family member, the lymphotoxin-beta (LT-beta) receptor (LT-betaR), also regulates HIV-1 replication. Furthermore, HIV-1 replication is cooperatively stimulated when the distinct LT-betaR and TNF receptor systems are simultaneously engaged by their specific ligands. Moreover, in a physiological coculture cellular assay system, we show that membrane-bound TNF-alpha and LT-alpha1beta2 act virtually identically to their soluble forms in the regulation of HIV-1 replication. Thus, cosignaling via the LT-beta and TNF-alpha receptors is probably involved in the modulation of HIV-1 replication and the subsequent determination of HIV-1 viral burden in monocytes. Intriguingly, surface expression of LT-alpha1beta2 is up-regulated on a T cell line acutely infected with HIV-1, suggesting a positive feedback loop between HIV-1 infection, LT-alpha1beta2 expression, and HIV-1 replication. Given the critical role that LT-alpha1beta2 plays in lymphoid architecture, we speculate that LT-alpha1beta2 may be involved in HIV-associated abnormalities of the lymphoid organs.

Lymphotoxin alpha/beta and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen.

Mice deficient in the cytokines tumor necrosis factor (TNF) or lymphotoxin (LT) alpha/beta lack polarized B cell follicles in the spleen. Deficiency in CXC chemokine receptor 5 (CXCR5), a receptor for B lymphocyte chemoattractant (BLC), also causes loss of splenic follicles. Here we report that BLC expression by follicular stromal cells is defective in TNF-, TNF receptor 1 (TNFR1)-, LTalpha- and LTbeta-deficient mice. Treatment of adult mice with antagonists of LTalpha1beta2 also leads to decreased BLC expression. These findings indicate that LTalpha1beta2 and TNF have a role upstream of BLC/CXCR5 in the process of follicle formation. In addition to disrupted follicles, LT-deficient animals have disorganized T zones. Expression of the T cell attractant, secondary lymphoid tissue chemokine (SLC), by T zone stromal cells is found to be markedly depressed in LTalpha-, and LTbeta-deficient mice. Expression of the SLC-related chemokine, Epstein Barr virus-induced molecule 1 ligand chemokine (ELC), is also reduced. Exploring the basis for the reduced SLC expression led to identification of further disruptions in T zone stromal cells. Together these findings indicate that LTalpha1beta2 and TNF are required for the development and function of B and T zone stromal cells that make chemokines necessary for lymphocyte compartmentalization in the spleen.

Both the lymphotoxin and tumor necrosis factor pathways are involved in experimental murine models of colitis.

BACKGROUND & AIMS: Membrane lymphotoxin (LT) alpha/beta, a member of the tumor necrosis factor (TNF) family of immune regulatory molecules, is involved both in the development of secondary lymphoid tissues and the maintenance of organized lymphoid tissues in the adult. Defects observed in the mucosal immune system in animals with a genetically disrupted LTalpha/beta pathway coupled with the expression of LTalpha/beta in activated T cells motivated an examination of the importance of this pathway in experimental colitis. METHODS: Soluble LTbeta receptor (LTbetaR) immunoglobulin fusion protein was used to inhibit the LTalpha/beta/light axis in two independent rodent models of colitis: CD45RBhi CD4(+)-reconstituted SCID mice and bone marrow-transplanted tg26 mice (BM --> tg26). RESULTS: Treatment with LTbetaR immunoglobulin attenuated the development of both the clinical and histological manifestations of the disease in these two murine models of colitis. Given the success of TNF inhibitors in the treatment of human Crohn's disease, the effects of LTbetaR immunoglobulin have been compared with antibody to TNF in the BM --> tg26 model, and both treatments were equally efficacious. CONCLUSIONS: The LT pathway plays a role in the development of colitis as important as that of the TNF system and, therefore, represents a potential novel intervention point for the treatment of inflammatory bowel disease.