Barnet M. Sultzer

Lipopolysaccharide nonresponder cells: The C3H/HeJ defect

Since its initial discovery as endotoxin resistant, the C3H/HeJ mouse has been extensively studied and used as a comparative model to help reveal the mechanism under genetic control which governs host responses to endotoxin. Most of the research has focused on the B lymphocyte and macrophage of this strain which fail to be activated by LPS. Recently, specific LPS binding proteins have been isolated on lymphocytes and other cells; however a receptor which transduces an activation signal has not been isolated as yet from responder cells which is missing or altered on C3H/HeJ nonresponder cells. Investigations into the signal transduction pathways used by C3H/HeJ B cells when they are activated by a protein mitogen have been found to be similar to those used by LPS responder cells when activated by LPS. Protein kinase C and tyrosine kinase, which phosphorylate signal proteins in cells have been found to be operative in C3H/HeJ and C3H/OuJ B cells. In both cases, DNA synthesis is shut off by either PKC or PTK blockade; however, PTK inhibition will also block activation of PKC stimulated DNA synthesis, indicating tyrosine kinase initiated phosphorylation may regulate the PKC signal pathway. Further analysis of the proteins that are phosphorylated in LPS responder and LPS nonresponder B cells is needed before conclusions can be drawn as to whether the defect in C3H/HeJ cells resides in the signal pathway leading to gene activation and proliferation. Nevertheless, the notion of a missing or defective signal receptor still remains as a working hypothesis to explain C3H/HeJ cell hyporesponsiveness to LPS. Isolation of the Lpsn gene and its product will provide the evidence needed for a clearer understanding of how LPS reacts with cells.

The activation of C3H/HeJ cells by certain types of lipopolysaccharides

Experimentation with the endotoxin non-responder C3H/HeJ mouse has provided considerable insight into the genetic control of the pathophysiological and immunobiological effects induced by lipopolysaccharide endotoxin (LPS); however, the mechanism underlying this deficiency has never been fully elucidated. At the cellular level much of the research has focused on the B-lymphocyte of the C3H/HeJ strain. Although it is known that C3H/HeJ B-cells can respond to protein mitogens (8, 13, 14), there is no conclusive evidence that typical protein-free LPS or lipid A can stimulate DNA synthesis or polyclonal antibody production by these cells equivalent to that seen with normal responder cells. However, recently several reports have appeared indicating that C3H/HeJ spleen cells in culture could be stimulated by certain forms of LPS to proliferate to some extent. By gel filtration chromatography, Vukajlovich and Morrison prepared homogeneous monomeric components of E. coli 055:B5 LPS (17). One of these fractions, which was protein-free, enriched in lipid A and contained a trace of 0-antigen, induced a significant but limited amount of 3H-thymidine incorporation by C3H/HeJ splenic lymphocytes as compared to responder C3HeB/FeJ cells (17). Vogel et al., (16) reported that a lipid A precursor obtained from a temperature sensitive mutant of S. typhimurium was active for C3H/HeJ B-cells and macrophages although the preparation contained a trace of protein. In contrast, the lipid X precursor of Raetz et al., (10) from E. coli failed to stimulate C3H/HeJ cells although it is a polyclonal B-cell activator (PBA) for LPSresponder cells. With the advent of synthetic lipid A, studies on its activity and various analogues were initiated with responder and non-responder cells. Kumazawa et al., (3) reported that one non-posphorylated derivative containing 3-hydroxytetradecanoic acid stimulated 3H-thymidine uptake 3 to 6-fold in C3H/HeJ spleen cells although the net cpms were half that obtained with C3H/HeN responder cells. Synthetic lipid A as well as all of the other derivatives which were monophosphorylated, diphosphorylated, or nonphosphorylated and contained various mixtures of myristic and hydromyristic acids were inactive on C3H/HeJ cells (3). Finally, Girard et al., (2) reported that the LPS from B. pertussis was active in stimulating C3H/HeJ splenic lymphocytes to proliferate but the activity was ascribed to the polysaccharide components rather than the lipid A.

Immunomodulation of C3H/HeJ Cells by Endotoxin Associated Protein and Lipopolysaccharide Endotoxin

Protein kinase C plays a vital role in the activation of C3H/HeJ B lymphocytes by endotoxin associated protein; however, it is unlikely that G proteins are involved in the early signals stimulated by EP. On the other hand, LPS suppresses C3H/HeJ B cell DNA synthesis induced by EP which may be the result of PKC down regulation. LPS inhibits C3H/HeJ B cells from progressing through the G1 phase of the cell cycle blocking RNA synthesis within the first 12 hr after the cells are stimulated. Finally, this inhibition extends to activation of the arachidonic acid metabolism in C3H/HeJ macrophages and T cell proliferation to a limited extent.

Polyclonal Lymphocyte Activation by M. tuberculosis and Its Products

The origin of the term polyclonal activation as applied to lymphocytes can be ascribed to the original finding of Nowell,1 who was the first to show that the proliferation of human peripheral blood lymphocytes in culture could be stimulated by a plant lectin phytohemagglutinin (PHA). With the development of an adequate culture system for mouse lymphocytes came an expansion of the use of this mitogen and the discovery of others, many of which are bacterial products. The value of these substances derived from the fact that lymphoid cells were stimulated non- specifically. Consequently, large numbers of cells were activated that permitted examination of the biochemical and immunologic events attendant to this process that was difficult at best to measure when dealing with a cell system based on specific antigenic recognition.