Modulation of immune response by bacterial lipopolysaccharide (LPS): multifocal effects of LPS-induced suppression of the primary antibody response to a T-dependent antigen.

Spleen cells from mice injected with 2 to 50 microgram bacterial lipopolysaccharide (LPS) have a reduced capacity to make an antibody response in vitro to trinitrophenylated sheep erythrocytes (TNP-SRBC) when tested 1 to 7 days later. Recovery is gradual, and these cells are full functional 2 weeks after in vivo LPS treatment. Unresponsiveness resides in the nonadherent splenic cell populations, and can be shown to have a suppressive cell component, which is irradiation sensitive and has somme characteristics of a thymus-derived lymphocyte (T cell). In addition, neither bone marrow-derived lymphocytes (B cells) nor T cells in the spleens of LPS-treated mice are functionally normal in their abilities to cooperate during an antibody response in vitro. LPS-B cells cooperated poorly with nylon wool-enriched T cells from normal mice but cooperated well with irradiated carrier-primed T cells or nylon wool-purified splenic T cells from carrier-primed mice. LPS-T cells have a reduced capacity to interact with normal B cells and appear to contain a suppressor cell component. These results indicate that the effects of exposure of immunocompetent cells to LPS are multifocal and can include suppression as well as stimulation of antibody formation.     

Effect of bacterial lipopolysaccharide on the in vitro secondary antibody response in mice. I. Description of the suppressive capacity of lipopolysaccharide.

Bacterial lipopolysaccharide (LPS) suppressed the in vitro secondary antibody response in mice to the protein antigens human gamma globulin (HGG) and turkey gamma globulin (TGG). This is the first report of LPS inhibiting a secondary antibody response. Consistent suppression was dependent on the time of LPS addition; LPS added at culture initiation was less effective than LPS added 12 to 48 hr later. The mitogenic moiety of LPS was the inhibitory principle, as shown by the lack of suppression of spleen cells from C3H/HeJ mice, and the inability of the polysaccharide component, but not the lipid component of LPS, to suppress A/J spleen cells. The mechanism of suppression by LPS was not due to large numbers of B cells proliferating in response to LPS, since removal of B cells not bearing specificity for the priming antigen did not reduce suppression by LPS. However, the possibility exists that LPS may act through B cells specific for the priming antigen.     

Potent adjuvant action of lipopolysaccharides possessing the O-specific polysaccharide moieties consisting of mannans in antibody response against protein antigen

It was previously reported that Klebsiella O3 lipopolysaccharide (LPS) exhibits extraordinarily strong adjuvant activity in augmenting antibody response against protein antigens in mice compared with other kinds of LPS, for example, LPS from Escherichia coli O55, O111, and O127 and Salmonella enteritidis. The present study was undertaken to clarify the relationship between the strong adjuvant activity in augmenting antibody response against deaggregated bovine gammaglobulin and the chemical structure of LPS. Among LPS from Klebsiella O1, O4, O5, and O7, only O5 LPS exhibited nearly the same degree of the strong adjuvant activity as did O3 LPS. The adjuvant activity of the other LPS was very weak in a degree similar to that of LPS from E. coli O55 and O127. Even when the natural forms of Klebsiella O3 LPS and O1 LPS were converted to various defined uniform salt forms, their adjuvant activity did not significantly differ from that of the respective natural forms. It is therefore unlikely that the difference in strength of the adjuvant activity between Klebsiella O3 LPS and O1 LPS is due to the difference in their salt forms. The common feature in the structures of Klebsiella O3 LPS and O5 LPS is their O-specific polysaccharide chains consisting of the mannose homopolysaccharides (mannans). LPS from E. coli O8 and O9, the O-specific polysaccharide chains of which consist of the mannans, also exhibited much stronger adjuvant activity than do LPS from E. coli O55 and O127, and the strength of the adjuvant activities of the former two was comparable to that of LPS from Klebsiella O3 and O5. On the other hand, LPS from Klebsiella O3 and O5 and E. coli O8 and O9 showed the ability to activate B lymphocytes polyclonally in vivo in a degree similar to that of the other kinds of LPS. From the present results it can be concluded that LPS possessing the O-specific polysaccharide moieties consisting of the mannans exhibit extraordinarily strong adjuvant activity in augmenting antibody response against protein antigen.     

Induction of ornithine decarboxylase in macrophages by bacterial lipopolysaccharides (LPS) and mycobacterial cell wall material

Lipopolysaccharide from $\text{E. Coli}$ (LPS) and BCG cell walls (BCGcw) are recognized immunoadjuvants that directly stimulate some macrophage functions. The macrophage cell line J774.1 and peritoneal exudate cells (PEC) from mice can be stimulated by LPS or other adjuvants $\text{in vitro}$ to synthesize and release protein factor(s) that activate thymus-derived lymphocytes. We have utilized J774.1 cells and PEC to demonstrate that an increase in ornithine decarboxylase (ODC) activity is a marker of early biochemical changes in adjuvant-stimulated macrophages. BCGcw and LPS increased ODC within 2 hours in J774.1 cells as well as murine peritoneal exudate macrophages. Maximal increases in ODC were detected 4 hours after the addition of adjuvants to J774.1 cells. The marked increases (12–23 fold) in ODC observed with BCGcw (20 μg/ml) did not appear to involve an effect on cell proliferation which was suppressed by this adjuvant. Cycloheximide inhibited the induction of ODC by LPS and BCGcw in the macrophage cell line. Evidence that the induction of ODC may be promoted by an increase in cyclic AMP was provided by experiments demonstrating that prostaglandin E₁ (PGE₁) and 8-bromo-adenosine-3′:5′-monophosphate (8Br-cyclic AMP) can mimic the effects of LPS and BCGcw in J774.1 cells. These observations indicate that one of the early biochemical changes in macrophages promoted by adjuvants is an induction of ODC.     

Effect of bacterial lipopolysaccharide on the in vitro secondary antibody response in mice. II. Abrogation of the suppressive capacity of endotoxin

Previous experiments have shown that bacterial endotoxin (ET) can be highly inhibitory to the in vitro secondary IgG antibody response when added 1–2 days after antigen. This paper examines the capacity of ET and another adjuvant, poly(AU), to circumvent the suppressive capacity of ET. It was found that ET or poly(AU) given simultaneously with antigen prevented any subsequent inhibition by ET added later to the cultures. Poly(AU) was effective in amounts as low as 1 μg/ml and ET in amounts as low as 0.1 μg/ml. Poly(AU) in greater amounts (50–100 μg/ml) also suppressed antibody synthesis when added 1–2 days after antigen, similar to ET. As with ET suppression, both ET and poly(AU) when added simultaneously with antigen were capable of overcoming the suppressive capacity of poly(AU). The capacity of small amounts of poly(AU) and ET to circumvent suppression in vitro by ET may help to explain why suppression by ET given after antigen has not been routinely observed in vivo. Lymphoid cells in vivo are most likely constantly exposed to either nuclear material released upon natural cell turnover or to ET from bacteria habitating the gut, resulting in an abrogation of any subsequent suppression by ET.     

Selective elimination of a B cell subset having acceptor site(s) for T cell-replacing factor (TRF) with biotinylated antibody to the acceptor site(s) and avidin-ricin A-chain conjugate

A covalent conjugate of avidin with ricin subunit A-chain (avidin-RA) was prepared by using N-succinimidyl 3-(2-pyridyldithio)propionate as a coupling agent. Selective cytotoxic activity after the combined treatment of spleen cells with biotinylated antibody and avidin-RA was demonstrated by the fact that the responsiveness to LPS was selectively abrogated by pretreatment of the cells with biotinylated rabbit anti-mouse immunoglobulin (MIg) antibody, but not with biotinylated anti-Thy-1.2 antibody. Neither the biotinylated antibody alone nor avidin-RA alone was effective in decreasing the responses to mitogens. Moreover, a high anti-DNP PFC response elicited by DNP-KLH-primed BALB/c mouse spleen cells stimulated in vitro with DNP-KLH was mostly abrogated by the pretreatment of the cells with biotinylated anti-MIg antibody and avidin-RA. Again, neither the biotinylated antibody alone nor avidin-RA alone was effective in decreasing the anti-DNP PFC response. This cell-killing method with the use of biotinylated antibody and avidin-RA was applied and evaluated in experimental systems in which the helper action of T cells on B cells was mediated by T cell-replacing factor (TRF) or was performed by the direct interaction of T cells with B cells (cognate interaction). When DNP-KLH-primed splenic B cells, pretreated with biotinylated F(ab')2 fragment of DCF1 male anti-BALB/c-B IgG antibody against acceptor site(s) for TRF followed by treatment with avidin-RA, were stimulated with DNP-OVA in the presence of monoclonal TRF, the anti-DNP PFC response was significantly decreased, whereas the same treated B cells responded well to stimulation with DNP-PPD in the presence of Tbc-primed T cells (cognate interaction). These results indicate that B cells responsible for the cognate interaction and those having TRF acceptor site(s) belong to a distinct subpopulation of B cells, and that the cytocidal action of the noncovalent conjugate of the antibody and RA formed from the biotinylated antibody and avidin-RA via an avidin-biotin complex has immunologic selectivity, eliminating only the latter subset of B cells recognized by the antibody.     

Inhibition of the mitogenic response to lipopolysaccharide (LPS) in mouse spleen cells by polymyxin B.

The addition of low doses of the cationic polypeptide antibiotic, polymyxin B (PB), to cultures of mouse spleen cells inhibits lipopolysaccharide-(LPS) induced DNA synthesis but not that stimulated by PPD, PHA, or Con A. Inhibition is stoichiometric; the mitogenic response is suppressed by 50% at a weight ratio of PB:LPS of 0.055 to 1. Furthermore, PB-LPS complexes have a much reduced mitogenic capacity. These complexes inhibit the mitogenic response of spleen cells to unmodified LPS but not to PPD, Con A, or PHA. The inhibitory activity of PB is less effective when added after LPS is mixed with responding cells, achieving 50% inhibition when addition is made at 4 to 6 hr. Time course experiments indicate that partial inhibition is a reflection of a lower rate of DNA synthesis. Thus, PB inhibition of LPS mitogenesis apparently occurs as a result of formation of PB-LPS complexes with reduced mitogenic capacity. Specific inhibition by the complexes of mitogenesis induced by native LPS suggests that the inactive complex may bind to B cells but is unable to trigger them.     

Suppression of in vitro antibody response by ribosome-associated factor(s) from interferon-treated cells.

Initiation factor preparation (eIF-IF) from mouse L cells treated with virus-type interferon suppressed the in vitro plaque-forming cell (PFC) response to sheep red blood cells. The eIF-IF preparation had previously been shown to block formation of the ternary complex Met-tRNA_f-eIF-GTP. The formation of this complex is a necessary step in initiation of protein synthesis. Initiation factor preparation (eIF) from untreated L cells affected neither the PFC response nor the participation of eIF in the formation of ternary complex. The induced factor was shown not to be Interferon by antibody neutralization experiments with anti-interferon. The factor must be present in the PFC cultures during the early stages of antigen induction in order to suppress the immune response. Speculatively, eIF-IF may act at the level of the macrophage, perhaps entering the cell by pinocytosis. This may account for its inability to inhibit virus replication in L cells. The production of the inhibitory factor is blocked or partially blocked by actinomycin D. It is possible that this factor is a mediator of the immunosuppressive effects of virus-type interferon. This is the first report of biological activity on cells, which is associated with a ribosome-associated factor induced by interferon.     

Role of lipopolysaccharide susceptibility in the innate immune response to Salmonella typhimurium infection: LPS, a primary target for recognition of Gram-negative bacteria.

Lipopolysaccharide is an important recognition marker by virtue of which the innate immune system senses and reacts against Gram-negative bacteria invading the LPS susceptible host. This review deals with the factors affecting LPS susceptibility and with the role of the latter in the course and outcome of Salmonella typhimurium infection.     

Inhibition of the in vitro 19S and 7S antibody response by immunoglobulin-binding factor (IBF) from alloantigen-activated T cells

A soluble factor secreted by alloantigen-activated mouse T cells which binds to the Fc fragment of IgG and inhibits complement activation by IgG (immunoglobulin-binding factor, IBF) suppressed the in vitro 19S and 7S antibody response by mouse spleen cells to T-dependent as well as T-independent antigens. IBF inhibited the 19S plaque response best when it was added late during PFC generation (between 48 and 72 hr). On the other hand, when it was left in cultures for up to 60 hr and then removed, antibody synthesis was not inhibited. However, its presence for only 2 hr starting after 72 hr of incubation was sufficient to inhibit PFC formation. The suppressive activity of IFB could be neutralized by adding aggregated mouse IgG prior to the critical stage around 72 hr. These data favour the view that IBF could be a suppressive T cell factor and point to the possibility that IBF may act on already triggered B cells during their final differentiation to active PFC.     

Shrimp invertebrate lysozyme i-lyz: gene structure, molecular model and response of c and i lysozymes to lipopolysaccharide (LPS)

The invertebrate lysozyme (i-lyz or destabilase) is present in shrimp. This protein may have a function as a peptidoglycan-breaking enzyme and as a peptidase. Shrimp is commonly infected with Vibrio sp., a Gram-negative bacteria, and it is known that the c-lyz (similar to chicken lysozyme) is active against these bacteria. To further understand the regulation of lysozymes, we determined the gene sequence and modeled the protein structure of i-lyz. In addition, the expression of i-lyz and c-lyz in response to lipopolysaccharide (LPS) was studied. The shrimp i-lyz gene is interrupted by two introns with canonical splice junctions. The expression of the shrimp i-lyz was transiently down-regulated after LPS injection followed by induction after 6 h in hepatopancreas. In contrast, c-lyz was up-regulated in hepatopancreas 4 h post-injection and slightly down-regulated in gills. The L. vannamei i-lyz does not contain the catalytic residues for muramidase (glycohydrolase) neither isopeptidase activities; however, it is known that the antibacterial activity does not solely rely on the enzymatic activity of the protein. The study of invertebrate lysozyme will increase our understanding of the regulatory process of the defense mechanisms.     

Phospholipids inhibit lipopolysaccharide (LPS)-induced cell activation: a role for LPS-binding protein

The inhibition of LPS-induced cell activation by specific antagonists is a long-known phenomenon; however, the underlying mechanisms are still poorly understood. It is commonly accepted that the membrane-bound receptors mCD14 and TLR4 are involved in the activation of mononuclear cells by LPS and that activation may be enhanced by soluble LPS-binding protein (LBP). Hexaacylated Escherichia coli lipid A has the highest cytokine-inducing capacity, whereas lipid A with four fatty acids (precursor IVa, synthetic compound 406) is endotoxically inactive, but expresses antagonistic activity against active LPS. Seeking to unravel basic molecular principles underlying antagonism, we investigated phospholipids with structural similarity to compound 406 with respect to their antagonistic activity. The tetraacylated diphosphatidylglycerol (cardiolipin, CL) exhibits high structural similarity to 406, and our experiments showed that CL strongly inhibited LPS-induced TNF-alpha release when added to the cells before stimulation or as a CL/LPS mixture. Also negatively charged and to a lesser degree zwitterionic diacyl phospholipids inhibited LPS-induced cytokine production. Using Abs against LBP, we could show that the activation of cells by LPS was dependent on the presence of cell-associated LBP, thus making LBP a possible target for the antagonistic action of phospholipids. In experiments investigating the LBP-mediated intercalation of LPS and phospholipids into phospholipid liposomes mimicking the macrophage membrane, we could show that preincubation of soluble LBP with phospholipids leads to a significant reduction of LPS intercalation. In summary, we show that LBP is a target for the inhibitory function of phospholipids.     

Control of lipopolysaccharide (LPS) binding and LPS-induced tumor necrosis factor secretion in human peripheral blood monocytes.

We used flow cytometry to determine how LPS-binding protein (LBP) effects the binding of fluorescein-labeled LPS to human monocytes via receptor-dependent mechanisms. The addition of human, rabbit, mouse, or FCS strikingly increased the binding of LPS to monocytes compared with controls incubated in serum-free medium. This binding was totally prevented by preincubation of monocytes with MY4, an anti-CD14 mAb, or by enzymatic removal of CD14 from monocytes. Depletion of LBP from rabbit serum with anti-LBP antibodies also produced a similar suppression. Solutions of albumin did not support the enhanced binding observed in serum but the addition of purified rabbit LBP to albumin solutions resulted in binding similar to that observed in serum-containing medium. When type-specific anti-LPS mAb was added to human serum, LPS binding to monocytes occurred but was only partly inhibited by anti-CD14 mAb, suggesting that receptors other than CD14 (presumably Fc or complement receptors) were involved. Serum increased by 100- to 1000-fold the sensitivity of monocytes to the triggering by LPS resulting in TNF secretion. TNF secretion was inhibited by anti-CD14 mAb up to 100 ng/ml of LPS and by anti-LPS mAb up to 1 to 10 ng/ml. The inhibition of TNF secretion by anti-LPS mAb appeared to be the result of directing LPS to monocyte receptors other than CD14. In contrast, in medium containing normal as well as acute serum and in the absence of anti-LPS antibodies, the binding of LPS to monocytes and the triggering of TNF secretion appeared to be mediated mainly by interactions between CD14 and LBP-LPS complexes.     

Surgical castration of pigs affects the behavioural response to a low-dose lipopolysaccharide (LPS) challenge after weaning

The objective of the present study was to evaluate the effect of an acute stressor in the early postnatal life of pigs, surgical castration, on post-weaning behaviour, and on the behavioural, endocrine and immune responses elicited by a low-dose lipopolysaccharide (LPS) challenge after weaning. At 5-days-of-age, 64 male piglets were randomly assigned to undergo surgical castration or were left untreated (treatment). Pigs were weaned at 28 days-of-age. Behaviour post-weaning and mixing was assessed during a 1-h period, during which agonistic interactions were recorded. One day post-weaning, pigs were injected intraperitoneally with a single dose of 0 or 5 μg/kg of BW of LPS from Escherichia coli (challenge). Sickness behaviour was studied by scan sampling every 5 min for 45 min at 0, 1, 2, 3, 4, 6 and 8 h after the challenge. Blood samples were taken at 0, 2, 12 or 24 h after injection and were analysed for plasma concentrations of tumor necrosis factor-alpha (TNF-), interleukin-1beta (IL-1β), C-reactive protein (CRP), serum amyloid A (SAA) and cortisol. Results showed that non-castrated pigs were more aggressive than castrated pigs immediately after weaning (P < 0.05). Administration of LPS provoked behaviours characteristic of sickness including a reduction in general activity, as well as decreased eating and exploratory behaviours (P < 0.05). These altered behaviours occurred predominantly 3-h post injection (P < 0.05). Significant treatment by challenge interactions showed that castration reduced the occurrence of sickness behaviours induced by LPS, such as depressed general activity (P < 0.01), anorexia (P < 0.01) and reduced exploratory behaviours (P < 0.05). LPS administration increased TNF- levels (P < 0.05), with peak concentrations 2 h after injection (P < 0.01). CRP levels of LPS-treated pigs were higher than saline-treated animals at 12 h (P < 0.05). LPS administration tended to increase plasma SAA levels (P < 0.1), but did not increase cortisol levels (P > 0.1). However, castration did not affect the response of pro-inflammatory cytokines, acute phase proteins and cortisol to the challenge. These results show that surgical castration reduces aggressiveness at weaning and affects specific sickness behaviours but not the endocrine and immune responses elicited by low-dose endotoxin challenge in weaned pigs.