Tetracyclines inhibit activated B cell function.

Tetracyclines have recently been shown to exert a number of pleiotropic anti-inflammatory and immunomodulatory activities, independent of their antibiotic properties. These include the ability to inhibit metalloproteinases (MP), a class of enzymes involved in crucial cellular functions such as the shedding of soluble mediators and their receptors from the cell surface, as well as interaction with, and remodeling of, the extracellular matrix. Here we report that doxycycline at therapeutic concentrations (1--5 microg/ml) significantly suppresses Ig secretion and class switching by in vitro activated murine B cells. Suppression of Ig secretion correlates with a decrease in levels of mRNA for the terminal B cell differentiation-associated genes Blimp-1 and mad-4, as well as to a reduction in expression of the plasma cell markers Syndecan-1 and J chain. Inhibition of class switching occurs at the recombination stage and is also induced by other MP inhibitors, including tetracycline analogs lacking antibiotic activity and the chemically unrelated hydroxamate KB8301. These novel, direct effects of MP inhibitors on B lymphocytes suggest an intrinsic role for MP in B cell activation and likely explain some of the observed in vivo immunomodulatory properties of tetracyclines. Moreover, these findings have significant implications for tetracycline therapy in Ig-mediated autoimmune or allergic diseases and raise questions about the use of doxycycline-inducible transgenic systems for the study of B cell function.

Normal isotype switching in B cells lacking the I mu exon splice donor site: evidence for multiple I mu-like germline transcripts.

Ig class switch recombination (CSR) in activated B cells is preceded by the generation of "switch" transcripts from the heavy chain constant region (CH) genes targeted for rearrangement. Switch transcripts include a sterile "I" exon spliced onto the first CH exon. Targeted mutations disrupting the expression or splicing of I exons severely hamper CSR to all tested CH loci, except mu. However, all mu switch transcript mutations tested so far have left the I mu exon splice donor site intact. To test the possibility that the residual CSR activity in I mu mutants could be due to splicing of a truncated I mu exon, we generated new mutants specifically lacking the I mu splice donor site. Surprisingly, normal CSR was observed in the I mu splice donor mutants even in the absence of detectable spliced I mu transcripts. In a search for potential alternative sources of switch-like transcripts in the mu locus, we identified two novel exons which map just upstream of the Smu region and splice onto the C mu 1 exon. Their expression is detectable from early B cell developmental stages, and, at least in hybridomas, it does not require the Emu enhancer. These studies highlight a unique structure for the mu locus I exon region, with multiple nested switch transcript-like exons mapping upstream of Smu. We propose that all of these transcripts directly contribute to mu class switching activity.

The Use of Spectral Analysis of Limiting Dilution for Investigating the Immune Response.

In this study we present a method for characterizing a spectrum of cells engaged in the humoral response by two parameters: the frequency and potential activity of antigen specific precursor cells. Spleen cells from mice immunized with ovalbumin were placed in microcultures in a limiting dilution assay. The values derived from positive cultures in an activity/cell number plot were compatible with Poisson distributions for distinct families of precursor cells displaying either positive or negative (suppressor-like) activities. Proposed limiting dilution spectral analysis combines the potentialities of the standard limiting dilution analysis with those provided by histograms of distribution according to the activity of positive cultures. This method opens new opportunities in detecting and characterizing distinct groups of limiting precursor cells.

Comparative study of immunomodulatory properties of muramyl peptides on immune system cells of young and old mice.

The immunomodulatory effects of two synthetic muramyl peptides (MP): muramyl dipeptide and glucosaminyl- muramyl dipeptide have been compared. It was shown, that MP effects on immune response are a consequence of the alteration in T lymphocyte regulators balance. MP action on old mice immune response and lymphocyte function was stimulating only: increasing of T helper precursors frequency and IL-1 production by macrophages. In the latter both MPs acted as correctors, recovering the decreased IL-1 production by old mice macrophages to young control level.

Muramyl dipeptide-induced changes in murine splenocyte responses to concanavalin A.

The effect of muramyl dipeptide (MDP) on Con A-stimulated activation of murine spleen cells was studied. MDP was found to enhance or suppress the proliferative response of splenocytes when different concentrations of Con A were used. MDP was shown to change the IL-2 content in culture supernatants of stimulated cells and to influence IL-2-dependent proliferation of Con A-blasts. A high degree of correlation was found between the proliferation of Con A-blasts and the expression of IL-2 receptors on Con A-blasts. This correlation, however, disappeared in the presence of MDP. The effects of MDP were shown to depend on the level of initial cell activity or rather on conditions leading to a given initial activity of cells.

Study of immunomodulatory properties of N-acetylmuramyl-L-alanyl-D-isoglutamine and N-acetylglucosaminyl-(beta 1----4)-N-acetylmuramyl-L-alanyl-D-isoglutamine.

The immunomodulatory activities of two synthetic muramylpeptides (MP), a muramyl dipeptide and a glucosaminyl-muramyl dipeptide, have been compared and have been found to exhibit many common features in their effects. In addition, the differential effects of low and high concentrations of MP on the primary humoral immune response in vitro were examined in detail. At high concentrations MP augmented the frequency of induced T-suppressor cells, while at low concentrations the primary immune response was stimulated by enhancement of the antigen-presenting function of accessory cells and by increasing the frequency of induced T-helper cells.

Target cells for immunomodulatory action of muramyl dipeptide.

Identification of the target cells for the immunomodulatory action of muramyl dipeptide (MDP) was addressed by investigation of various B-cell and T-cell lines. The lines used were: IM-9, a human lymphoblastoid B-cell line that spontaneously produces IgG; EL-4, a murine T-cell line that produces interleukin-2 (IL-2) on stimulation with phorbol myristate acetate; and CTLL-2, an IL-2-dependent murine T-cell line. MDP was shown to modulate such T-cell and B-cell functions as cell proliferation and secretion of IL-2 and IgG, respectively, in vitro. The effect of MDP in vitro was determined by both MDP dose and the control level of cell activity. The evidence obtained supports the possibility of the direct action of MDP on T and B lymphocytes.