Normal human serum contains a natural IgM antibody cytotoxic for human neuroblastoma cells.

Neuroblastoma (NB) is characterized by the second highest spontaneous regression of any human malignant disorder, a phenomenon that remains to be elucidated. In this study, a survey of 94 normal human adult sera revealed a considerable natural humoral cytotoxicity against human NB cell lines in approximately one-third of the tested sera of both genders. Specific cell killing by these sera was in the range of 40% to 95%. Serum cytotoxicity was dependent on an intact classical pathway of complement. By several lines of evidence, IgM antibodies were identified as the cytotoxic factor in the sera. Further analyses revealed that a 260-kDa protein was recognized by natural IgM of cytotoxic sera in Western blots of NB cell extracts. The antigen was expressed on the surface of seven human NB cell lines but not on human melanoma or other control tumor cell lines derived from kidney, pancreas, colon, bone, skeletal muscle, lymphatic system, and bone marrow. Furthermore, no reactivity was observed with normal human fibroblasts, melanocytes, and epidermal keratinocytes. The antigen was expressed in vivo as detected by immunohistochemistry in both the tumor of a NB patient and NB tumors established in nude rats from human NB cell lines. Most interestingly, the IgM anti-NB antibody was absent from the sera of 11 human NB patients with active disease. The anti-NB IgM also could not be detected in tumor tissue obtained from a NB patient. Collectively, our data suggest the existence of a natural humoral immunological tumor defense mechanism, which could account for the in vivo phenomenon of spontaneous NB tumor regression.

Anti-nuclear antibody production and immune-complex glomerulonephritis in BALB/c mice treated with pristane.

The pathogenesis of systemic lupus erythematosus is thought to be primarily under genetic control, with environmental factors playing a secondary role. However, it has been shown recently that intraperitoneal injection of pristane (2,6,10,14-tetramethylpentadecane) induces autoantibodies typical of lupus in BALB/c mice, a strain not usually considered to be genetically susceptible to the disease. In this study, the induction of autoimmune disease by pristane was investigated. BALB/c mice receiving pristane were tested for autoantibody production and histopathological evidence of glomerulonephritis. Six of 11 mice developed IgM anti-single-stranded DNA antibodies shortly after receiving pristane and 4 developed IgM anti-histone antibodies, but anti-double-stranded DNA antibodies were absent. IgG anti-DNA and anti-histone antibodies were absent. In contrast, the lupus-associated anti-nuclear ribonucleoprotein/Sm and anti-Su autoantibodies produced by these mice were predominantly IgG. In addition to autoantibodies, most of the mice developed significant proteinuria. Light microscopy of the kidney showed segmental or diffuse proliferative glomerulonephritis. Electron microscopy showed subepithelial and mesangial immune-complex deposits and epithelial foot process effacement. Immunofluorescence revealed striking glomerular deposition of IgM, IgG, and C3 with a mesangial or mesangiocapillary distribution. Thus, pristane induces immune-complex glomerulonephritis in association with autoantibodies typical of lupus in BALB/c mice. These data support the idea that lupus is produced by an interplay of genetic and environmental factors and that unlike the MRL or (NZB x W)F1 mouse models, in which genetic susceptibility factors are of primary importance, environmental factors are of considerable importance in the autoimmune disease of pristane-treated BALB/c mice.

Precursor B cells for autoantibody production in genomically Fas-intact autoimmune disease are not subject to Fas-mediated immune elimination

The Fas/Fas ligand (FasL) system participates in regulation of the immune system through the apoptotic process. However, the extent to which abnormalities in this system are involved in the loss of self-tolerance and development of autoimmune disease not associated with Fas/FasL mutations remains unknown. The present study addresses this issue in Fas/FasL-intact, systemic lupus erythematosus (SLE)-prone (NZB × NZW) (NZB/W) F1 mice. While splenic B cells from 2-month-old mice before overt SLE expressed Fas poorly, in vitro stimulation with an agonistic anti-CD40 mAb up-regulated their Fas expression, thus revealing the existence of two populations: one was Fashigh and highly susceptible to anti-Fas mAb-induced apoptosis, and the other was Faslow and apoptosis-resistant. The Faslow cells were included in the CD5+ B cell subpopulation and contained most of the cells that produced IgM anti-DNA antibodies. The isotype of anti-DNA antibodies switches from IgM to IgG in NZB/W F1 mice at ages beginning at about 6 months. These IgG anti-DNA antibodies were produced almost exclusively by a subpopulation of splenic B cells that spontaneously expressed low levels of Fas in vivo and were apoptosis-resistant. The findings indicate that precursor B cells for autoantibody production and presumably autoantibody-secreting cells in these mice are relatively resistant to Fas-mediated apoptosis, a finding supporting the concept that abnormalities of Fas-mediated apoptotic process are involved in the development of autoreactive B cells in Fas/FasL-intact autoimmune disease.

Restoration of surface IgM-mediated apoptosis in an anti-IgM-resistant variant of WEHI-231 lymphoma cells by HS1, a protein-tyrosine kinase substrate.

The HS1 protein is one of the major substrates of non-receptor-type protein-tyrosine kinases and is phosphorylated immediately after crosslinking of the surface IgM on B cells. The mouse B-lymphoma cell line WEHI-231 is known to undergo apoptosis upon crosslinking of surface IgM by anti-IgM antibodies. Variants of WEHI-231 that were resistant to anti-IgM-induced apoptosis expressed dramatically reduced levels of HS1 protein. Expression of the human HS1 protein from an expression vector introduced into one of the variant cell lines restored the sensitivity of the cells to apoptosis induced by surface IgM crosslinking. These results suggest that HS1 protein plays a crucial role in the B-cell antigen receptor-mediated signal transduction pathway that leads to apoptosis.

A role for intracellular pH in membrane IgM-mediated cell death of human B lymphomas

We show that anti-IgM-induced cell death in a human B lymphoma cell line, B104, is associated with early intracellular acidification and cell shrinkage. In contrast, another human B cell lymphoma line, Daudi, less susceptible to B cell antigen receptor-mediated cell death, responded to anti-IgM with an early increase in intracellular pH (pHi). The anti-IgM-induced changes of pHi were associated with different levels of activation of the Na+/H+ exchanger isoform 1 (NHE1) as judged by its phosphorylation status. Prevention of anti-IgM-induced cell death in B104 cells by the calcineurin phosphatase inhibitor, cyclosporin A, abrogated both intracellular acidification and cell shrinkage and was associated with an increase in the phosphorylation level of NHE1 within the first 60 min of stimulation. This indicates a key role for calcineurin in regulating pHi and cell viability. The potential role of pHi in cell viability was confirmed in Daudi cells treated with an Na+/H+ exchanger inhibitor 5-(N,N-hexamethylene)amiloride. These observations indicate that the outcome of the anti-IgM treatment depends on NHE1-controlled pHi. We suggest that inactivation of the NHE1 in anti-IgM-stimulated cells results in intracellular acidification and subsequently triggers or amplifies cell death.

Tumor dormancy and cell signaling: anti-mu-induced apoptosis in human B-lymphoma cells is not caused by an APO-1-APO-1 ligand interaction.

Signal transduction initiated by crosslinking of antigen-specific receptors on T- and B-lymphoma cells induces apoptosis. In T-lymphoma cells, such crosslinking results in upregulation of the APO-1 ligand, which then interacts with induced or constitutively expressed APO-1, thereby triggering apoptosis. Here we show that crosslinking the membrane immunoglobulin on human lymphoma cells (Daudi) (that constitutively express APO-1) does not induce synthesis of APO-1 ligand. Further, a noncytotoxic fragment of anti-APO-1 antibody that blocks T-cell-receptor-mediated apoptosis in T-lymphoma cells does not block anti-mu-induced apoptosis. Hence, in B-lymphoma cells, apoptosis induced by signaling via membrane IgM is not mediated by the APO-1 ligand.

Invariance and restriction toward a limited set of self-antigens characterize neonatal IgM antibody repertoires and prevail in autoreactive repertoires of healthy adults.

Analysis of the reactivity of IgM with self-antigens in tissues by a quantitative immunoblotting technique showed striking invariance among newborns in the human and in the mouse. The self-reactive repertoire of IgM of adults was also markedly conserved; it comprised most anti-self reactivities that prevailed among neonates. Multivariate analysis confirmed the homogeneity of IgM repertoires of neonates toward self- and non-self-antigens. Multivariate analysis discriminated between newborn and adult repertoires for reactivity with two of five sources of self-proteins and with non-self-antigens. Our observations support the concept that naturally activated B lymphocytes are selected early in development and throughout life for reactivity with a restricted set of self-antigens.

B-cell activation by crosslinking of surface IgM or ligation of CD40 involves alternative signal pathways and results in different B-cell phenotypes.

Treatment of small resting B cells with soluble F(ab')2 fragments of anti-IgM, an analogue of T-independent type 2 antigens, induced activation characterized by proliferation and the expression of surface CD5. In contrast, B cells induced to proliferate in response to thymus-dependent inductive signals provided by either fixed activated T-helper 2 cells or soluble CD40 ligand-CD8 (CD40L) recombinant protein displayed elevated levels of CD23 (Fc epsilon II receptor) and no surface CD5. Treatment with anti-IgM and CD40L induced higher levels of proliferation and generated a single population of B cells coexpressing minimal amounts of CD5 and only a slight elevation of CD23. Anti-IgM- but not CD40L-mediated activation was highly sensitive to inhibition by cyclosporin A and FK520. Sp-cAMPS, an analogue of cAMP, augmented CD40L and suppressed surface IgM-mediated activation. Taken together these results are interpreted to mean that there is a single population of small resting B cells that can respond to either T-independent type 2 (surface IgM)- or T-dependent (CD40)-mediated activation. In response to different intracellular signals these cells are induced to enter alternative differentiation pathways.