Interferon-gamma, tumor necrosis factor-alpha, and transforming growth factor-beta inhibit cyclic AMP-induced Schwann cell differentiation.

Schwann cells differentiate in vivo in response to contact with axons, and cAMP simulates some of these aspects of differentiation in vitro, particularly morphologic changes and expression of certain phenotypic molecules. Unfractionated inflammatory cytokines inhibit cAMP-induced Schwann cell expression of galactolipids (Gal). We sought to identify which cytokines were responsible for this inhibition and to determine whether other phenotypic indicators of Schwann cell differentiation were also affected. Neonatal rat Schwann cells were incubated in vitro with 1 mM 8 Bromo cAMP (8 Br cAMP) with or without the addition of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-6, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or transforming growth factor-beta (TGF-beta). Cells were then examined for morphologic changes and for expression of surface Gal and low-affinity nerve growth factor receptor (NGFRp75), employing indirect immunofluorescence. 8 Br cAMP induced Schwann cell upregulation of Gal, downregulation of NGFRp75, and the cells became enlarged and somewhat amorphous and irregular in appearance. Cells treated with IFN-gamma or TNF-alpha alone were more bipolar and more evenly distributed on coverslips than were control cells, whereas TGF-beta alone induced elongated cells often in a swirling pattern. None of the cytokines alone induced upregulation of Gal or downregulation of NGFRp75. TNF-alpha, IFN-gamma, and TGF-beta inhibited the 8 Br cAMP-induced morphologic changes, as well as the upregulation of Gal and downregulation of NGFRp75. The other cytokines had no effects on Gal or NGFRp75 expression. Thus, these three cytokines, which are present in inflammatory lesions in the peripheral nervous system, are capable of inhibiting Schwann cell differentiation.

TNF-alpha and TGF-beta act synergistically to kill Schwann cells.

Interactions between cytokines and Schwann cells (SC) are important in development, repair, and disorders of the peripheral nervous system (PNS). Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) are two prominent cytokines which may be involved in these processes and their gene products are upregulated in some experimental neuropathies. This study focuses on the in vitro effects of these cytokines, both singly and in combination, on cultured SC. Expression of both Type I and Type II TNF-alpha receptors was demonstrated on the SC surface by immunocytochemistry. Treatment of SC with a combination of TNF-alpha plus TGF-beta causes significant detachment and cell death while treatment with each cytokine alone is not significantly cytotoxic. When compared with control cultures, SC treated with the combination of cytokines exhibit an increase in the number of cells with condensed nuclei and evidence of DNA fragmentation, characteristics consistent with cells undergoing programmed cell death. Thus, TNF-alpha plus TGF-beta induce SC loss of adhesion which is predominantly due to cell death. Apoptotic mechanisms are likely to contribute to some extent to this cell death. These findings provide in vitro evidence to support the hypothesis that cytokines can directly damage SC in PNS disorders.

Antibodies to glycolipids and cholera toxin B subunit do not initiate Ca++ signaling in rat Schwann cells.

Antibodies to glycolipids have been implicated in the pathogenesis of several immune-mediated PNS demyelinating diseases. This study focuses on antibodies to galactocerebroside (GalC) and sulfatide and on the B subunit of cholera toxin (CTB), which reacts with GM1 ganglioside, to examine whether these agents have any direct effects on Schwann cells (SC) as measured by Ca++ responses. While surface levels of GalC and sulfatide were markedly upregulated by 8 Br-cAMP treatment, as reported by others, very little expression of surface GM1 ganglioside was detected with or without 8 Br-cAMP treatment. Schwann cells, under either condition, showed no changes in intracellular Ca++ levels when exposed to purified monoclonal antibodies reacting with GalC or sulfatide. Thus upregulation of surface levels of GalC or sulfatide does not lead to antibody-induced Ca++ influx, in contrast to previous findings in mature oligodendrocytes (OLs) exposed to antibodies to GalC. Further, cross-linking with one of the antibodies (R-mAb) did not produce Ca++ responses. No Ca++ responses were elicited by CTB in Schwann cells either with or without 8 Br-cAMP treatment. Since surface binding of CTB was very low and sparsely punctate in Schwann cells +/- 8 Br-cAMP, we tested whether increasing levels of GM1 ganglioside on the surface would lead to induction of a Ca++ signaling pathway, as reported for fibroblasts. GM1 ganglioside on the surface of SC was markedly increased by exposing cells to exogenous GM1 ganglioside, but no Ca++ responses were observed in the treated cells. Thus undifferentiated or partially differentiated SC lack the glycoconjugate-mediated Ca++ signaling pathways found in mature OLs or fibroblasts.

The effect of cytoplasmic domain mutations on membrane anchoring and glycoprotein processing of herpes simplex virus type 1 glycoprotein C.

The predicted amino acid sequence of herpes simplex virus type 1 glycoprotein C (HSV-1 gC) shows that it has the features of a typical type 1 integral membrane protein: a cleavable N-terminal signal sequence, a glycosylated ectodomain, a single transmembrane domain, and a small, charged cytoplasmic domain. In an earlier investigation of the function of the gC cytoplasmic domain, it was shown that the gC synthesized by a gC mutant, dl2, which lacked the last three residues of the transmembrane domain and the entire cytoplasmic domain, was initially synthesized as a membrane bound glycoprotein, but was not stably anchored in the plasma membrane. In this study, we generated a panel of four HSV-1 gC mutants with novel cytoplasmic domains in order to further delineate the role of this domain in stable anchoring and to investigate the role of charged residues in this process. The cytoplasmic domain mutants produced significant quantities of a novel precursor (pgC-86K), approximately 6K smaller than the wild-type gC precursor. The quantity of pgC-86K correlated with the number of positive charges in the cytoplasmic domain. Although the nature of the novel form of the precursor is unclear, its correlation with cytoplasmic domain charge suggests that an important function of this domain is to influence gC processing. Significantly, restoration of the carboxy-terminal 3 amino acids of the gC transmembrane domain restored the wild-type anchoring phenotype to gC, indicating that the cytoplasmic domain is not required for membrane anchoring. Further characterization of dl2 gC confirmed that this glycoprotein is not released from the membrane by proteolysis. We suggest that the addition of three additional hydrophobic residues to the dl2 transmembrane domain increases its hydrophobicity enough to stabilize membrane anchoring of the glycoprotein.

Bicarbonate concentrations in the renal cortex.

Total CO2 (CO2+HCO3-) was measured in alkaline extracts of quickly frozen rat renal cortex. The mean concentration calculated for tissue water was 18.7 +/- SD 1.6 mEq/l (n = 10) when that measured in plasma water was 23.5 +/- 1.7. In other experiments (n = 6-10), the mean concentration in tissue water increased to 28.3 mEq/l after the animals were treated with benzolamide to inhibit carbonic anhydrase and to 38.1 mEq/l after they were infused with NaHCO3. On the other hand, the tissue concentration decreased to 14-15 mEq/l when they were hyperventilated or infused with HCl. Calculations based on published micropuncture data indicate that the usual intracellular concentration is 20-25 mEq/l or about twice that usually reported for skeletal muscle; and in contrast to muscle, the findings are suggestive of large variations in metabolic disorders.