Phospholipid metabolism of stimulated lymphocytes. Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A.

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [3H]arachidonate and [14C]oleate or [3H]arachidonate and [14C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3-7-fold) and phosphatidylethanolamine (2-3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.

Induction of an immune response to keyhole-limpet hemocyanin in surgical patients with anergy.

Groups of surgical patients, classified as reactive or anergic on the basis of delayed type hypersensitivity skin testing with five recall antigens, were immunized with keyhole-limpet hemocyanin (KLH) alone or KLH together with mediators derived from leukocytes of a KLH immune donor cultured with antigen. Patients with anergy injected with KLH alone did not generate an immune response as judged by a T cell proliferative reaction performed 14 days after immunization. In contrast, leukocytes of patients with anergy immunized with KLH together with the mediators reacted to KLH in vitro in similar numbers and with a magnitude comparable to that given by reactive, hospitalized patients without anergy immunized with KLH alone. These results confirm and extend our previous observations showing that anergy defined as a lack of cell-mediated immunity to recall antigens such as purified protein derivative extends to the generation of a systemic immune response to a neoantigen such as KLH and mediators that could restore a state of delayed hypersensitivity to purified protein derivative could also be instrumental in inducing cell-mediated immunity de novo when injected together with the antigen.

Endotoxin tolerance is associated with reduced secretion of tumor necrosis factor.

Bacterial endotoxin effects are partially mediated by tumor necrosis factor (TNF). It is known that sublethal doses of endotoxin induce transient refractoriness (tolerance) to some of its effects. We studied the role of TNF in endotoxin tolerance in rats. Weight loss, lethality, and TNF production were measured after an initial dose of endotoxin and after subsequent doses. Weight loss reached its peak 72 hours after the initial endotoxin challenge, followed by recovery even under continued administration of endotoxin. While tolerant, rats could survive a dose of endotoxin that was lethal for 100% of naive rats. The high serum levels of TNF, observed 90 minutes after the first dose of endotoxin, markedly diminished when rechallenged during tolerance. Recovery of responsiveness to these effects followed the refractory phase by 3 weeks. We concluded that endotoxin tolerance is associated with a reduced secretion of TNF.

Tumor necrosis factor alone does not explain the lethal effect of lipopolysaccharide.

Lethality and tumor necrosis factor production induced by different types of lipopolysaccharide were studied in naive (non-primed) rats during the late phase of endotoxin tolerance. The correlation with antilipopolysaccharide antibodies was also analyzed. No correlation was found between tumor necrosis factor levels and lipopolysaccharide-induced mortality in naive animals. Low-toxicity lipopolysaccharide preparations induced levels of tumor necrosis factor similar to those induced with more toxic types of lipopolysaccharide. Late tolerance was associated with progressively lower levels of lipopolysaccharide-induced tumor necrosis factor and increasing titers of antilipopolysaccharide antibodies after repeated injections of homologous lipopolysaccharide. During late endotonxin tolerance, a direct correlation between the lipopolysaccharide dose and peak tumor necrosis factor serum levels was found. We conclude that since tumor necrosis factor serum levels do not correlate with mortality, tumor necrosis factor alone cannot explain the lethal effect of lipopolysaccharide.

Blastogenic factor: its role in the mixed leukocyte culture reaction.

The MLC-CML reaction depends on cooperation between different subsets of T lymphocytes in which soluble mediators may play a critical role. Evidence is presented that cell-free supernatants from mixed cultures, containing blastogenic factor (BF) activity can, together with solubilized membrane preparations, induce a primary cell-mediated lympholysis (CML) reaction of the same order of magnitude as intact allogeneic stimulating cells. Furthermore, BF alone can induce secondary mixed leukocyte culture (MLC) and CML reactions very efficiently. The results described suggest that BF is produced in response to activation by LD determinants, probably by helper T cells, and that the target of BF is the cytotoxic lymphocyte. It would appear that regulation of CML activity may be mediated by BF, and exhaustion of the mediator may be responsible for the decline of cell proliferation and effector function in the MLC reaction.

Regulation of the mixed leukocyte culture reaction by suppressor cells.

Regulation of the mixed leukocyte culture (MLC) reaction by suppressor cells raised and tested in MLC has been evaluated. The experiments described suggest that suppressors are distinct from cytotoxic T lymphocytes (CTL), both in terms of conditions optimal for their activation, as well as through demonstration of efficient suppression in the total absence of cytotoxic activity. Suppressor cells inhibit the activation of the precursors of CTL, and may also prevent the production of the lymphokine blastogenic factor; they do not appear to interfere with the reaction of primed cells. It is suggested that suppressor cells may regulate the MLC reaction by preventing recruitment of fresh competent cells, and by limiting the duration of the reaction of cells already activated.

Lymphocyte activation by purified HLA-DR molecules fused into autochthonous "stimulating cells".

Affinity-purified Ia molecules derived from the Daudi cell line were reconstituted into vesicles with Sendai virus envelope glycoproteins. These vesicles inserted into human peripheral leukocytes could induce stimulation of autologous lymphocytes, as measured by thymidine uptake, 6 days later. It is suggested that this method could provide a means to study allostimulation at the molecular level.

[The regulation of the activation of cytotoxic lymphocytes may be mediated by a lymphokine]. / Régulation de l'activation des cellules cytotoxiques par l'intermédiaire des lymphokines

MLC-CML reactivity declines after approximately the 7th day of culture. The experiments described were designed to probe the reason for this decline, envisaged as due to the signals (SD or LD determinants) required for CML activation. Either fresh stimulating cells or a lymphokine--blastogenic factor (BF)--were added daily to mixed cultures from the 5th to the 12th day of incubation. Whereas stimulating cells were without effect, added BF maintained both proliferative and cytotoxic activity of the cells in culture. These observations, together with previous results, were interpreted to suggest that helper cells may regulate the activation of cytotoxic lymphocytes by means of soluble mediators.

Dysfunction of humoral immunity in anergic surgical patients: absence of anti-tetanus IgG antibody production.

The experiments described have shown that, whereas the injection of tetanus toxoid (tet) into 8 of 12 control individuals resulted in the appearance of specific anti-tet-IgG antibodies in their plasma, immunization of 14 anergic patients did not elicit an antibody response. This observation was extended to an in vitro system, where cells from four control subjects were shown to secrete anti-tet-IgG antibodies in response to polyclonal activators whereas cells from eight anergic patients did not. It is suggested that this failure of humoral immunity could account for the high risk of bacterial infections in anergy.

Role of cytokines in the restoration of the delayed-type hypersensitivity reaction of anergic patients.

Soluble mediators from peripheral blood lymphocytes activated either by skin test antigens or by alloantigens restored the delayed type hypersensitivity (DTH) reaction in the majority of anergic surgical patients who are at increased risk for sepsis and mortality. Antigen had to be injected together with the mediators and the individual had to be reactive to the antigen for restoration. These results suggest that restoration of the DTH response depends on the ability of cytokines produced and acting in a non-specific manner to promote the response of the anergic patients' specific antigen-sensitive cells to antigen.

Protein fusions of beta-galactosidase to the ferrichrome-iron receptor of Escherichia coli K-12.

The fusion-generating phage lambda plac Mu1 was used to produce fusions of lacZ to fhuA, the gene encoding the ferrichrome-iron receptor (FhuA protein) in the outer membrane of Escherichia coli K-12. Fusions to the fhuA gene in a delta (lac) strain were selected by their resistance to bacteriophage phi 80 vir. Ten independent (fhuA'-'lacZ) fusions were all Lac+ and were resistant to the lethal agents which require the FhuA protein as receptor, i.e., phi 80 vir, T5, T1, UC-1, and colicin M; none could utilize ferrichrome as the sole iron source. Specialized transducing phages were obtained by illegitimate excision from the chromosome of each of the fusion-bearing strains, and EcoRI fragments which encoded the fusions were subcloned into the high-copy plasmid pMLB524. Physical mapping of the fusion-containing plasmids confirmed the presence of three restriction sites which were also located on the chromosomal DNA of sequences near the fhuA gene. The direction of transcription of the fhuA gene was deduced from the direction of transcription of the (fhuA'-'lacZ) gene fusion. Identification of the chimeric proteins was made by both radiolabeling cells and immunoprecipitating the LacZ-containing proteins with antibody to beta-galactosidase and by preparing whole cell extracts from Lac+ cells containing the cloned gene fusions. Two sizes of (FhuA'-'LacZ) proteins were detected, 121 kDa and 124 kDa. The DNA sequences at the unique fusion joints were determined. The sequence information allowed us to identify three distinct fusion joints which were grouped as follows, type I fusions, 5'-ACT GCT CAG CCA A-3'; type IIa fusions, 5'-GCG GTT GAA CCG A-3'; and type IIb fusions: 5'-ACC GCT GCA CCT G-3'. To orient these fhuA fusion joints, the complete nucleotide sequence of the fhuA gene was determined from a 2,902-base-pair fragment of DNA. A single open reading frame was found which translated into a 747-amino acid polypeptide. The signal sequence of 33 amino acids was followed by a mature protein with a molecular weight of 78,992. Alignment of the amino acid sequence of the FhuA protein with the amino acid sequences presented for two other tonB-dependent receptor proteins in the outer membrane of E. coli showed an area of local homology at the amino terminus of all three proteins.

Lymphocyte function in anergic patients.

The lymphocyte function of anergic surgical patients who are at increased risk for sepsis and mortality was studied. In vitro lymphocyte responses appear to be normal in most instances, in that over 80% of patients showed a normal response in a standardized mixed leucocyte culture reaction. Similarly, 56% of the lymphocytes from anergic patients showed a positive in vitro proliferative response with PPD. The ability of in vitro-activated lymphocytes to elicit a skin reaction was determined by culturing the cells of anergic patients with PPD and then reinjecting the lymphocytes or their supernatants intradermally into the original donor. When there was a positive proliferative response to PPD in vitro, the reinjected cells or supernatant elicited a positive skin reaction in 79% of the anergic patients. In contrast, a skin reaction was obtained in less than 20% of the instances when there was no in vitro proliferation to PPD or when the cells were cultured without antigen. These results suggest that one of the acquired immune defects in these anergic patients is an in vivo block of lymphocyte activation.