No abstract

Lipopolysaccharides (LPS) 1 from gram-negative bacteria are powerful mitogens for B cells in vitro (1). In contrast to antigens LPS stimulates a broad spectrum of B cells regardless of their specificity for antigen (1,2). The mitogenie part of the LPS molecule has been found to be the lipid A component (3).Lipid A stimulation in vitro of cells from nonimmunized adult mice resulted in induction of IgM synthesis only (2). We wish to report results which show that IgG synthesis may be induced, provided lipid A is given to neonatal mice in vivo. In agreement with results obtained in vitro (1) we could not demonstrate an in vivo IgG stimulation in adult mice. In order to measure newly synthesized IgG in the newborn, a sensitive hemolytic assay was developed, which permits the determination of IgG of paternal allotype in concentrations of a few micrograms per milliliter. Materials and MethodsAnimals.--Offspring from reciprocal matings between BALB/c/AnNIcr (allotype-a) and C57BL/6NIcr (aUotype-b), which were heterozygous for the IgG allotypic marker, were used.Lipid A and Salmondla.--Lipid A was obtained by acid hydrolysis (4) of phenol-waterextracted LPS (5). 1 nag lipid A was dissolved in 1 ml distilled water by addition of 0.5/zl triethylamine (6). Salmonella anatum was cultivated in nutrient broth, inactivated by 1% phenol, washed in distilled water, and lyophilized. For injection, preparations were diluted in phosphate-buffered saline (PBS). This study has been done by Cornelia Kolb in partial fulfillment of the requirements for a Ph.D. thesis at the University of Konstanz. Treatment of Mice.---On1 Abbreviations used in this paper: An, natural unit of antibody; anti-a, anti-b, antiallotype antisera; BSA, bovinc serum albumin; DNP-KLH, dinitrophenyl-kcyhole limpet hemocyanin; EDTA/PBS, 0.012 M ethylenc-diaminc-tctraacetate in PBS; LPS, lipopolysaccharide; M + H, Mayer's barbital buffer supplemented with 0.1% human serum albu~fin; NKM/PBS, solution of 0.13 M NaCl, 0.005 M KCI, and 0.07 M MgCl2 in PBS 1:1; PBS, phosphate-buffered saline; SRBC, sheep rcd blood cells.

Lipopolysaccharide (LPS) 1 from gram-negative bacteria, or the lipid A component isolated from LPS, have a potent stimulatory effect on B lymphocytes. In vitro, they induce both cell proliferation and IgM secretion (reviewed in references 1-3). In vivo, we have demonstrated a pronounced effect of lipid A administered to mice during the first few days of life: serum IgG rose rapidly to levels 10-to 50-fold higher than in untreated controls of the same age (4). By contrast, adult animals did not show a clear response to LPS or lipid A; the ability to respond to lipid A with IgG production is lost between 14 and 20 days of age (unpublished results). The dramatic response of new-born mice to lipid A is of interest in terms of the ontogenetic development of the Ig-producing limb of the immune system. We therefore studied the early Ig production in congenitally thymusless Cnude") mice and their response to lipid A and thymus grafts. The experiments to be presented were also prompted by reports showing that the limiting factor governing the capacity of young mice to give a humoral immune response could be the presence or maturity of thymus-dependent ~'helper" cells (5,6).A number of studies in vitro are concerned with the question whether or not T cells are participants in the stimulation of B cells by LPS or lipid A (7-9). The interpretation of the results is complicated by the finding that the nude mouse does possess some 0-positive cells (10), and that, in fact, LPS itself can induce precursor cells to express T-cell-specific antigens (11).In the design of the present experiments [as with those reported previously (4)] we had to take cognizance of the fact, that young animals bear high serum levels of maternal IgG. To measure selectively the autochthonous production of IgG by the young, we had to use allotypically heterozygous offspring, and assay only the Ig bearing the paternal allotype.The experiments show that lipid A can induce accelerated IgG production in young nudes, albeit not to the same degree as in normal young mice. IgG production was also dramatically stimulated by nearly congeneic thymus grafts; and the effects of lipid A and thymus grafts were additive and possibly cooperative. Materials and MethodsMice. The following mice were bred in our laboratory: BALB/c-AnNIcr; BALB/c-nu, a backcross-line developed in Konstanz by B. M. Kindred, which carries the nude (athymic) factor on the * Supported by the ¢'Sonderforschungsboreich 138" of the Deutsche ForschungsgemeinschaR.

Several cloned T cell lines were isolated from primed mixed lymphocyte cultures immunized against minor histocompatibility antigens. These lines were selected with irradiated stimulator cells as antigen and require restimulation at intervals to keep growing. They are responsive, as measured by proliferation, to interleukin 2 (T cell growth factor) but cannot be grown in it continuously. These T cell lines have either the H-2d or H-2k haplotype. They all show exquisite H-2 restriction and minor histocompatibility antigen specificity. We did not observe any alloreactivity on 8 different H-2 haplotypes. For the H-2k T cell lines, the restriction element could be mapped to either the K or D end of the H-2 complex. No I-A-restricted cell line was found. It is of interest that all these T cell lines need the presence of T cells in the irradiated stimulator cell population. This suggests a more complex interaction between irradiated stimulators and responder T cells than just H-2K- or D-restricted antigen interaction. This recognition, though necessary, does not seem sufficient to induce the T cell clones to proliferate.

No abstract