BLyS: member of the tumor necrosis factor family and B lymphocyte stimulator.

The tumor necrosis factor (TNF) superfamily of cytokines includes both soluble and membrane-bound proteins that regulate immune responses. A member of the human TNF family, BLyS (B lymphocyte stimulator), was identified that induced B cell proliferation and immunoglobulin secretion. BLyS expression on human monocytes could be up-regulated by interferon-gamma. Soluble BLyS functioned as a potent B cell growth factor in costimulation assays. Administration of soluble recombinant BLyS to mice disrupted splenic B and T cell zones and resulted in elevated serum immunoglobulin concentrations. The B cell tropism of BLyS is consistent with its receptor expression on B-lineage cells. The biological profile of BLyS suggests it is involved in monocyte-driven B cell activation.

Disseminated growth of murine plasmacytoma: similarities to multiple myeloma.

Murine plasma cell tumors share a number of common features with human multiple myeloma, suggesting their possible use as a model for this disease. However, one major difference between the two is the peritoneal localization of murine tumors as opposed to bone marrow residence of malignant plasma cells in early stages of multiple myeloma. We have thus examined the ability of murine plasmacytoma to produce disseminated growth similar to that seen in myeloma or other lymphoid neoplasias. Of four murine cell lines evaluated, all were demonstrated to effect highly metastatic disease involving multiple organs, although variation was observed between lines. A temporal analysis was accordingly performed with the S107 line to assess the pattern of cellular localization. Both light microscopy and PCR analysis revealed that engraftment of plasma cells occurs first in the bone marrow, followed by dissemination to other sites including the spleen, lung, and liver. Cells passaged in vivo through the bone marrow display an entirely different metastatic pattern with no homing preference to bone marrow or any other organ, suggesting the occurrence of a phenotypic change. Microscopic osteolytic lesions were observed adjacent to plasma cell tumor masses in the bone marrow, indicating early stages of bone disease. These findings demonstrate previously unrecognized similarities between the murine and human diseases and suggest the use of this in vivo model for experimental approaches to the treatment of human disease.

PSGR, a novel prostate-specific gene with homology to a G protein-coupled receptor, is overexpressed in prostate cancer.

PSGR, a new prostate tissue-specific gene with homology to the G protein-coupled odorant receptor gene family, has been identified. Here we report the characteristics of the predicted protein sequence of PSGR and its prostate tissue specificity and expression profile in human prostate cancer and matched normal tissues. Using multiple tissue Northern blots from over 50 different tissues, PSGR expression was restricted to human prostate tissues. Paired normal and tumor specimens from 52 primary prostate cancers, obtained by laser capture microdissection or manual microdissection, were analyzed for PSGR expression by semiquantitative and real-time PCR assays. The differential expression of PSGR between normal and tumor tissues was highly significant (P < 0.001), and 32 of 52 (62%) matched prostate specimens exhibited tumor-associated overexpression of PSGR. Of note, there was very little or no expression of PSGR in many normal specimens in comparison with the generally high expression of PSGR seen in matched tumor specimens. In situ hybridization assays showed restricted PSGR expression in the epithelial cells of the normal and tumor tissue sections. Restricted expression of PSGR in prostatic epithelial cells, overexpression of the PSGR in a significant percentage of prostate cancers, and the predicted protein sequence of PSGR with seven transmembrane domains provide a foundation for future studies evaluating the potential of PSGR as a prostate cancer gene expression marker and the utility of PSGR protein as a novel target for developing immunotherapeutic strategies for prostate cancer.

Chromosomal translocations deregulating c-myc are associated with normal immune responses.

Plasmacytomas induced in BALB/c mice by pristane consistently evidence chromosomal translocations involving the c-myc gene and one of the Ig loci. This observation has lead to the suggestion that c-myc deregulation is a critical event in the generation of such tumors. However, it is not clear whether c-myc translocation is related to pristane treatment or occurs in normal lymphocyte populations nor whether such translocations occur normally, and at similar frequencies, in strains genetically resistant to plasmacytoma development, such as DBA/2. In order to address these questions, a Long Distance PCR assay with single copy sensitivity was employed to assess the frequency of c-myc/IgA translocations in normal and immunized mice of both plasmacytoma resistant and susceptible lineages in the absence of pristane treatment. Our data demonstrate that spontaneous translocations occur in normal DBA/2 and BALB/c mice with no significant differences in frequency. A 3-5-fold increase in translocation frequency was observed in mice immunized with cholera toxin, a strong stimulator of IgA responses. We conclude that c-myc deregulation by chromosomal translocation is associated with normal physiological processes of B-cell differentiation and, as such, can not be the determining factor leading to malignancy.

Adsorption of non-membrane proteins on the surface of model phospholipid membranes.

1. Two new methods are proposed for enhancement of the binding of hydrophilic proteins by liposomes. 2. An alkylating derivative of phosphatidic acid has been obtained by its reaction with N,N,N'-tris(2-chloroethyl)-N'-(p-formylphenyl)propylene-1,3-diamine. The alkylating activity of this derivative is very low due to the electron-acceptor effect of the formyl residue. Phosphatidylcholine liposomes which contain this alkylating derivative in the lipid bilayer may be obtained. The compound residing in the outer monolayer may be reduced by NaBH4. Upon reduction, the formyl residue is transformed into a hydroxymethyl residue. Therefore, the alkylating group of the compound is activated, and proteins may be attached covalently to the outer monolayer by alkylation with such chemically reactive liposomes. 3. Reaction of alkylating liposomes with myoglobin results in covalent binding of this hydrophilic protein. Complement-mediated leakage of such myoglobin-carrying liposomes may be induced by antibodies against myoglobin. 4. Modification of hydrophilic proteins with dansyl chloride results, even at small extents of modification, in a dramatic increase of the affinity of such proteins to phosphatidylcholine liposomes.

Interleukin-2-mediated modulation of plasma cell tumor growth in a model of multiple myeloma.

A number of studies have demonstrated that inoculation of certain types of cancer cells engineered for expression of the interleukin-2 (IL-2) gene results in reduced tumorigenicity and/or protection from subsequent challenge with a tumorigenic dose of wild-type cells. In the current studies, we have employed murine plasma cell tumors to examine IL-2-mediated tumor rejection as a possible model for therapeutic approaches to human myeloma or plasma cell leukemia. Two murine plasma cell tumor lines, S107 and X24, were infected with a retroviral vector expressing the human IL-2 gene, and the antitumor potential of IL-2-expressing infectants was characterized in syngeneic BALB/c and BALB/c nu/nu mice. Results demonstrate that tumorigenicity of both lines correlates inversely with the amount of IL-2 produced by the tumor cells. However, there are clear differences between the two lines in terms of reduced tumorigenicity and the ability to protect against co-injected parental tumor cells that appear unrelated to IL-2 levels. More importantly, intravenous immunization of animals with irradiated, IL-2 secreting cells from either line leads to significant protection from challenge with highly metastatic parental cells. These results suggest that such an approach may warrant consideration in the treatment of human plasma cell dyscrasias.

A new system for ATP-metric immunoanalysis.

Treatment of amino-group-containing antigens with adenosine-5'-trimetaphosphate results in their chemical modification by -pppA residues. An immunoanalytical system is proposed based upon competition of these ATP-labelled antigens with those of the sample for immobilized antibodies. Mild acidic treatment of complexes of ATP-labelled antigens with immobilized antibodies results in quantitative liberation of intact ATP. The latter may be determined by the ultrosenstive bioluminescent techniques based upon emission of light with firefly luciferase. The validity of the system has been studied with two clinically important antigens, thyroxine and myoglobin.

High serum levels of B-lymphocyte stimulator are associated with clinical-pathological features and outcome in classical Hodgkin lymphoma.

B-lymphocyte stimulator (BLyS) acts as survival factor for B lymphocytes. As Hodgkin and Reed-Sternberg (HRS) cells express receptors through which BLyS promotes their growth and chemotherapy resistance, we investgated whether this molecule was increased in sera from patients with classical Hodgkin lymphoma (cHL) and whether it correlates with clinical-pathological features and outcomes. Enzyme-linked immunosorbent assay was used to measure soluble BLyS (sBLyS) in sera from 87 patients and 33 donors; higher levels were detected in patients (mean +/- standard error 4493.9 +/- 264.9 pg/ml vs. 2687.0 +/- 200.9 pg/ml; P < 0.0001). Levels above the median value (4242.0 pg/ml) were associated with age > or = 45 years (P = 0.042), advanced stages of disease (P = 0.005), systemic symptoms (P = 0.014) and extranodal involvement (P = 0.009). Five-year failure-free survival (FFS) of patients with sBLyS below or equal to median levels was 88.6% as compared to 65.1% of those with levels above the median (P = 0.009). Statistical analyses confirmed the prognostic significance of sBLyS (P = 0.046). When patients were analysed according to variables associated with high levels, sBLyS showed an independent predictive power in terms of FFS. Our findings support the involvement of BLyS in cHL pathogenesis. The association between high serum levels and an inferior FFS indicates that sBLyS is a possible prognostic predictor with a potential significance as a therapeutic target.

Elevated serum B lymphocyte stimulator levels in patients with systemic immune-based rheumatic diseases.

OBJECTIVE: To determine whether serum levels of B lymphocyte stimulator (BLyS) are elevated in patients with systemic immune-based rheumatic diseases and correlate with serum Ig levels and/or autoantibody titers. METHODS: Sera from 185 patients with various systemic immune-based rheumatic diseases (95 with systemic lupus erythematosus [SLE], 67 with rheumatoid arthritis [RA], 23 with other diagnoses) were assayed for BLyS and Ig. In 7 patients who required arthrocentesis of a swollen knee, coincident serum and synovial fluid samples were assayed for BLyS. Medical charts were retrospectively reviewed for elevated autoantibody titers and proteinuria within a 1-month period before or after collection of sera for BLyS and Ig determination. Sera concurrently collected from 48 normal healthy subjects served as controls. RESULTS: Serum BLyS levels were elevated in 38 of 185 patients (21%) and correlated significantly with serum IgG levels. Serum BLyS levels did not correlate with the patients' age, sex, race, or medications, but correlated positively with anti-double-stranded DNA antibody titers among SLE patients and with rheumatoid factor titers among seropositive RA patients. In contrast, serum BLyS levels correlated inversely with nephrotic-range proteinuria among SLE patients. In every case tested, BLyS levels in clinically inflamed synovial fluids were greater than those in simultaneously obtained sera. CONCLUSION: BLyS may be an important factor in driving polyclonal hypergammaglobulinemia and elevated autoantibody titers in patients with systemic immune-based rheumatic diseases. Local production of BLyS in the joints may contribute to joint pathology. Patients with elevated serum BLyS levels may be ideal candidates for therapeutic targeting of BLyS.

Inverse association between circulating APRIL levels and serological and clinical disease activity in patients with systemic lupus erythematosus.

OBJECTIVE: To assess longitudinal expression of a proliferation-inducing ligand (APRIL) in patients with systemic lupus erythematosus (SLE) and its correlation with B lymphocyte stimulator (BLyS) expression, serum anti-dsDNA titres, and clinical disease activity. METHODS: Sixty eight patients with SLE were longitudinally followed up for a median of 369 days. At each visit the physician assessed disease activity by SLEDAI, and blood was collected for determination of serum APRIL and BLyS levels and of blood APRIL and BLyS mRNA levels. Fifteen normal control subjects underwent similar laboratory evaluation. RESULTS: Dysregulation of APRIL was not as great as that of BLyS. Changes in serum levels of APRIL and BLyS over time were usually discordant, whereas blood levels of APRIL and BLyS mRNA strongly paralleled each other. Serum APRIL levels modestly, but significantly, inversely correlated with serum anti-dsDNA titres in anti-dsDNA positive patients analysed in aggregate. Moreover, serum APRIL levels modestly, but significantly, inversely correlated with clinical disease activity in all patients analysed in aggregate. CONCLUSION: Serum levels of APRIL and BLyS are differentially regulated. APRIL may serve as a down modulator of serological and/or clinical autoimmunity in patients with SLE. This may have important ramifications for BLyS targeted treatment, and it remains to be determined whether agents which neutralise only BLyS will be preferable to agents which neutralise both BLyS and APRIL.

Cutting edge: a role for B lymphocyte stimulator in systemic lupus erythematosus.

Increased levels of B lymphocyte stimulator (BLyS) are associated with systemic autoimmunity in animal models of spontaneous autoimmune disease, and transgenic animals expressing BLyS develop typical autoimmune disease. Here, we demonstrate significant elevations of BLyS in the patients with systemic lupus erythematosus (SLE). The BLyS isolated from the sera of SLE patients had the same m.w. as the natural soluble form and was able to stimulate B cell activation in vitro. Increased BLyS in SLE patients was partially associated with higher levels of anti-dsDNA Ab of the IgG, IgM, and IgA classes, but not associated with the disease activity. Our results suggest that BLyS may be a useful marker for early activation of an autoimmune diathesis and likely plays a critical role in triggering activation of self-Ag-driven autoimmune B cells in human SLE. BLyS may provide an effective therapeutic target in systemic autoimmunity.

Synthesis and release of B-lymphocyte stimulator from myeloid cells.

B-lymphocyte stimulator (BLyS) is a recently identified novel member of the tumor necrosis factor ligand superfamily shown to exist in a membrane-bound and soluble form. BLyS was found to be specifically expressed on cells of myeloid lineage and to selectively stimulate B-lymphocyte proliferation and immunoglobulin production. The expression of a cytokine involved in potentiation of humoral immune responses, such as BLyS, is expected to be strictly controlled. The goal of the present study was to examine regulation of BLyS levels in monocytic cells in response to cytokines and during their differentiation to macrophages and dendritic cells. The presence of BLyS on the cell surface and in the culture medium of both normal blood monocytes and on tumor cells of myelomonocytic origin was demonstrated. BLyS gene expression and levels of membrane-associated and soluble BLyS were found to be regulated by cytokines, in particular interferon (IFN)-gamma and to a lesser extent interleukin-10 (IL-10). The expression of BLyS on monocyte membranes was retained following differentiation into macrophages, but detection on the surface of monocyte-derived dendritic cells required stimulation with IFN-gamma. Both IFN-gamma and IL-10 enhanced the release of soluble BLyS that was active in B-cell proliferation assays. Cells transfected with BLyS complementary DNA mutated in a predicted cleavage site failed to release BLyS into the culture medium, thereby suggesting that soluble BLyS was derived from the membrane form. These results provide further support for an important role for BLyS expressed in myeloid cells in B-cell expansion and antibody responses.