Infection-derived lipids elicit an immune deficiency circuit in arthropods.

The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.

Identification of a Potent Microbial Lipid Antigen for Diverse NKT Cells.

Semi-invariant/type I NKT cells are a well-characterized CD1d-restricted T cell subset. The availability of potent Ags and tetramers for semi-invariant/type I NKT cells allowed this population to be extensively studied and revealed their central roles in infection, autoimmunity, and tumor immunity. In contrast, diverse/type II NKT (dNKT) cells are poorly understood because the lipid Ags that they recognize are largely unknown. We sought to identify dNKT cell lipid Ag(s) by interrogating a panel of dNKT mouse cell hybridomas with lipid extracts from the pathogen Listeria monocytogenes. We identified Listeria phosphatidylglycerol as a microbial Ag that was significantly more potent than a previously characterized dNKT cell Ag, mammalian phosphatidylglycerol. Further, although mammalian phosphatidylglycerol-loaded CD1d tetramers did not stain dNKT cells, the Listeria-derived phosphatidylglycerol-loaded tetramers did. The structure of Listeria phosphatidylglycerol was distinct from mammalian phosphatidylglycerol because it contained shorter, fully-saturated anteiso fatty acid lipid tails. CD1d-binding lipid-displacement studies revealed that the microbial phosphatidylglycerol Ag binds significantly better to CD1d than do counterparts with the same headgroup. These data reveal a highly potent microbial lipid Ag for a subset of dNKT cells and provide an explanation for its increased Ag potency compared with the mammalian counterpart.

Persistent high levels of IgM antiphospholipid antibodies in a patient with recurrent pregnancy losses and rheumatoid arthritis.

OBJECTIVE: Recurrent pregnancy losses (RPL) and unexplained infertility (UI) are often associated with the presence of antiphospholipid antibodies (APA). We report one case with RPL, UI, and persistent IgM APA without B-cell isotype switch. METHOD OF STUDY: (i) A case report of a woman with RPL and UI who eventually developed rheumatoid arthritis and B-cell phenotype study of the case and controls by flow cytometric analysis; (ii) a retrospective cohort study of 1067 subjects with APA test. RESULTS: A 44-year-old woman with a history of RPL and UI was revealed to have high levels of APA, primarily of IgM isotype: IgM autoantibodies were specific to cardiolipin, phosphatidylglycerol, phosphatidylserine, and phosphatidylinositol. The monitoring of the patient's serological characteristics for 28 months did not reveal the development of IgG APA. B-cell phenotype analysis revealed decreased switched and double negative memory B cells and increased non-switched memory B cells in comparison with normal controls and reported ranges. A retrospective analysis of APA test revealed that total five patients (5/1067, 0.47%) with similar persistent IgM-only pattern were detected. CONCLUSION: Persistent IgM APA without isotype switch may be a rare variant form of APA manifestation, which is associated with dysregulated B-cell subsets.

Recognition of microbial and mammalian phospholipid antigens by NKT cells with diverse TCRs.

CD1d-restricted natural killer T (NKT) cells include two major subgroups. The most widely studied are Vα14Jα18(+) invariant NKT (iNKT) cells that recognize the prototypical α-galactosylceramide antigen, whereas the other major group uses diverse T-cell receptor (TCR) α-and ß-chains, does not recognize α-galactosylceramide, and is referred to as diverse NKT (dNKT) cells. dNKT cells play important roles during infection and autoimmunity, but the antigens they recognize remain poorly understood. Here, we identified phosphatidylglycerol (PG), diphosphatidylglycerol (DPG, or cardiolipin), and phosphatidylinositol from Mycobacterium tuberculosis or Corynebacterium glutamicum as microbial antigens that stimulated various dNKT, but not iNKT, hybridomas. dNKT hybridomas showed distinct reactivities for diverse antigens. Stimulation of dNKT hybridomas by microbial PG was independent of Toll-like receptor-mediated signaling by antigen-presenting cells and required lipid uptake and/or processing. Furthermore, microbial PG bound to CD1d molecules and plate-bound PG/CD1d complexes stimulated dNKT hybridomas, indicating direct recognition by the dNKT cell TCR. Interestingly, despite structural differences in acyl chain composition between microbial and mammalian PG and DPG, lipids from both sources stimulated dNKT hybridomas, suggesting that presentation of microbial lipids and enhanced availability of stimulatory self-lipids may both contribute to dNKT cell activation during infection.

Antigen-specific enhancement of natural human IgG antibodies to phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol-4-phosphate, cholesterol, and lipid A by a liposomal vaccine containing lipid A.

Natural IgG antibodies (NA) to lipids are ubiquitously distributed in sera of healthy humans and are believed to serve beneficial functions. Although NA to lipids generally exhibit germ line or near germ line binding specificities, the antibodies commonly increase transiently in the acute phases of most, if not all, infectious diseases and may serve as a first line of defense. In order to determine whether similar anti-lipid antibodies can be induced by a vaccine in humans, we examined stored sera obtained from volunteers who had previously received a candidate vaccine to Plasmodium falciparum. The vaccine had consisted of liposomes that contained both the recombinant protein antigen and also contained monophosphoryl lipid A (MPLA) as an adjuvant. All of the pre-immune sera contained NA to one or more of the liposomal lipids in the vaccine: dimyristol phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), cholesterol, and MPLA. After initial immunization, followed by a boost, increased levels of IgG antibodies to all of the liposomal lipids, especially DMPG and MPLA, were observed by ELISA. Antibodies to phosphatidylinositol-4-phosphate (PIP) above the normal pre-immune NA to PIP were also observed. Although PIP was not present in the immunizing liposomes, based on the adsorption of anti-PIP antibodies by DMPG the anti-PIP antibodies were thought to represent cross-reacting anti-DMPG antibodies. The immune response was apparently antigen-specific in that NA to unrelated lipids, other than PIP, that were not present in the liposomes, galactosyl ceramide and ganglioside GM1, were not increased by the immunization. We conclude that antibodies to DMPC, DMPG, PIP, cholesterol, and MPLA can be induced in humans by immunization with liposomes containing MPLA.

Pulmonary surfactant phosphatidylglycerol inhibits Mycoplasma pneumoniae-stimulated eicosanoid production from human and mouse macrophages.

Mycoplasma pneumoniae is a human pathogen causing respiratory infections that are also associated with serious exacerbations of chronic lung diseases. Membranes and lipoproteins from M. pneumoniae induced a 4-fold increase in arachidonic acid (AA) release from RAW264.7 and a 2-fold increase in AA release from primary human alveolar macrophages. The bacterial lipoprotein mimic and TLR2/1 agonist Pam3Cys and the TLR2/6 agonist MALP-2 produced effects similar to those elicited by M. pneumoniae in macrophages by inducing the phosphorylation of p38(MAPK) and p44/42(ERK1/2) MAP kinases and cyclooxygenase-2 (COX-2) expression. M. pneumoniae induced the generation of prostaglandins PGD(2) and PGE(2) from RAW264.7 cells and thromboxane B(2) (TXB(2)) from human alveolar macrophages. Anti-TLR2 antibody completely abolished M. pneumoniae-induced AA release and TNFα secretion from RAW264.7 cells and human alveolar macrophages. Disruption of the phosphorylation of p44/42(ERK1/2) or inactivation of cytosolic phospholipase A(2)α (cPLA(2)α) completely inhibited M. pneumoniae-induced AA release from macrophages. The minor pulmonary surfactant phospholipid, palmitoyl-oleoyl-phosphatidylglycerol (POPG), antagonized the proinflammatory actions of M. pneumoniae, Pam3Cys, and MALP-2 by reducing the production of AA metabolites from macrophages. The effect of POPG was specific, insofar as saturated PG, and saturated and unsaturated phosphatidylcholines did not have significant effect on M. pneumoniae-induced AA release. Collectively, these data demonstrate that M. pneumoniae stimulates the production of eicosanoids from macrophages through TLR2, and POPG suppresses this pathogen-induced response.

Diagnostic performance of phospholipid-specific assays for the evaluation of antiphospholipid syndrome.

The diagnostic performance of commercially available nonstandard antiphospholipid (aPL) assays for the evaluation of antiphospholipid syndrome (APS) is unknown. In 62 patients with APS, 88 with recurrent pregnancy loss, 50 healthy blood donors, and 24 women with one or more successful pregnancies, we measured antiphosphatidic acid (aPA), antiphosphatidyl-choline (aPC), antiphosphatidylethanolamine (aPE), antiphosphatidylglycerol (aPG), antiphosphatidylinositol (aPI), and antiphosphatidyl-serine (aPS) IgG and IgM antibodies from 2 manufacturers. We computed the areas under the curve (AUC), sensitivities, specificities, positive and negative predictive values, and 95% confidence intervals to assess diagnostic performance. The AUC analyses of the IgM assays demonstrated significant differences (P < .01) for all markers except aPC, whereas the IgG markers showed comparable performance for most assays with the exception of aPE (P < .01) and aPS (P = .02) antibodies. Overall, the combined sensitivity of the aPL assays differed significantly between manufacturers and did not improve the diagnostic yield for APS.

Pulmonary surfactant proteins and lipids as modulators of inflammation and innate immunity.

OBJECTIVES: The pulmonary surfactant system of the human lung consists of unique lipids and proteins that contribute to the biophysical and innate immune properties of the organ. Surfactant protein A (SP-A) is an oligomeric protein consisting of 18 protomers with collagen and lectin-like domains that recognizes glycoconjugates, lipids and protein determinants on both host cells and invading microorganisms. The authors examined the interaction of SP-A with Mycoplasma pneumoniae and the influence of the protein upon the innate immune response to the bacteria. METHODOLOGY: The authors quantified SP-A interaction with bacteria using ELISA, and identified the major surface ligand by thin layer chromatography, HPLC and mass spectrometry. The inflammatory response of human and rat macrophages was measured by quantifying tumour necrosis factor-alpha secretion using ELISA, and nitric oxide production. RESULTS: SP-A bound the bacteria with high affinity and enhanced the inflammatory response of human and rat macrophages to the organism and its membranes. Analysis of the interaction of SP-A with the bacteria revealed that the major ligand was a phospholipid. The lipid ligand was purified by a combination of thin layer and HPLC, and identified by mass spectrometry. The mass spectrometry demonstrated that the SP-A reactive lipid consisted of several disaturated molecular species of phosphatidylglycerol (PtdGro). Additional experiments were performed to determine if disaturated PtdGro was capable of interfering with the action of SP-A as an inhibitor of bacterial lipopolysaccharide-induced inflammatory mediator production by macrophages. The disaturated PtdGro failed to alter the anti-inflammatory action of SP-A but unexpectedly these same studies revealed that unsaturated PtdGro can modify the host response to lipopolysaccharide. CONCLUSIONS: These findings reveal that both the lipids and proteins of pulmonary surfactant play a role in regulating the host response to invading microorganisms.

Assessing the variation in antiphospholipid antibody (APA) assays: comparison of results from 10 centers.

OBJECTIVE: This study was undertaken to determine the variability of antiphospholipid antibody (APA) assay results of cardiolipin and other frequently tested phospholipids. Study design Ten centers performing APA assays submitted samples that were positive in their assay. Twenty samples were identified to cover a broad range of APA results. Samples were distributed to the 10 participating centers for evaluation of immunoglobulin G (IgG), IgM, and IgA antibodies to phospholipids. RESULTS: Of 20 patients, 9 (45%) were identified as positive by all 10 centers and 2 of 20 patients (10%) were identified as positive by 8 or 9 centers. However, 9 of 20 samples (45%) returned with mixed results. The average percent of positive samples was 29%, but results from the 10 centers ranged from a low number of positives (13%) to a high number of (37%). CONCLUSION: When considering a clinical diagnosis of APA syndrome, laboratory concordance of results from 20 patient samples among the 10 centers was only 55%. However, when considering a single phospholipid of a single immunoglobulin isotype, agreement of test results was 83.8%. Isolated positive APA results should be correlated with the clinical history and confirmed by repeat testing.

Immunogenicity of immunoliposomes: reactivity against species-specific IgG and liposomal phospholipids.

Immunoliposomes with surface-linked avidin-biotinylated mouse IgG2a were prepared from dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylglycerol (DMPG) and biotinylated dipalmitoylphosphatidylethanolamine (DPPE-biotin) in the molar ratio 10:1:0.1 with or without 5 mol% poly(ethylene glycol) dipalmitate (PEG-(C18)2). The ability of IgG2a immunoliposomes to elicit anti-IgG2a antibodies in mice was compared with alum and N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP). IgG2a 5 microgram/mouse) did not elicit an IgG1 antibody response after 4 s.c. injections. Alum-adsorbed IgG2a elicited 2.1 +/- microgram IgG1 antibody/ml serum, whereas MDP elicited 24.3 +/- microgram/ml serum. IgG2a immunoliposomes elicited 12.4 +/- 3.7 microgram IgG1 antibody/ml serum, while immunoliposomes containing lipophilic PEG-(C18)2 elicited 21.4 +/- 5.1 microgram IgG1 antibody/ml serum. Elicited antibodies were specific for IgG2a, with no cross-reactivity with IgG2b. Anti-DPPC or anti-DMPG IgG antibody levels did not change during immunization. Anti-DPPE IgG antibody levels were slightly but significantly elevated during immunization, and there was a significant increase in the level of anti-DPPE-biotin antibodies. These results demonstrate that immunoliposomes prepared with species-specific antibody are immunogenic and induce significant levels of isotypespecific antibody upon repeated injection.

Phospholipid antibodies in alcoholic liver disease.

Alcoholic liver injury has been reported to be directed preferentially against the proteins of the cell membrane, sparing the phospholipids. However, antiphospholipid antibodies against certain cell membrane phospholipids are known to be associated with a variety of diseases. We undertook this investigation to determine whether antiphospholipid antibodies were present in the serum of patients with alcoholic liver disease. We investigated seventy long-term alcoholic patients (> 80 gm ethanol/day for > 1 yr) and 8 normal nonalcoholic controls by means of enzyme-linked immunosorbent assay to determine whether serum antibodies were generated against the following membrane phospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol (cardiolipin) and phosphatidic acid. Group 1 comprised alcoholic patients with normal liver function (n = 13), group 2 comprised alcoholic patients with abnormal liver function (n = 16), group 3 comprised patients with alcoholic hepatitis or cirrhosis (n = 41) and group 4 comprised nonalcoholic controls (n = 8). The antibody prevalence was 15% in group 1, 31% in group 2, 81% in group 3 and 0% in group 4. In group 3, 20 of 41 patients had antibodies against several cell membrane phospholipids (i.e., phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and phosphatidylinositol). The antiphosphatidylethanolamine isotype was IgA or IgM in 25 of 41 of these patients. Both IgA (p < 0.01) and IgM (p < 0.008) antiphosphatidylethanolamine correlated significantly with disease severity. Antiphospholipid antibodies in alcoholic patients seem to reflect disease progression and correlate significantly with disease severity.

Multiples of the median: an alternative method for reporting antiphospholipid antibodies in women with recurrent pregnancy loss.

OBJECTIVE: To determine whether using multiples of the median (MoM) is a useful method of standardization for reporting antiphospholipid antibody results. METHODS: Each antibody was measured by enzyme-linked immunosorbent assay using the Harris calibration set no. 2, with values recorded as phospholipid units and as MoM. The levels of antiphospholipid antibodies were compared in 100 nonpregnant women diagnosed with recurrent pregnancy loss (three or more spontaneous, consecutive losses) and 100 nonpregnant parous women with no history of reproductive problems (controls). Results were analyzed based on phospholipid units for immunoglobulin (Ig) G and IgM and were considered positive if phospholipid units were at least 20 or if MoM was at least 2.5. All lower values were considered negative. RESULTS: When results were calculated using at least 2.5 MoM as positive, none of the controls had positive levels. Overall, IgG anticardiolipin identified the largest number of women with elevated antiphospholipid antibodies. Immunoglobulin G antiphosphatidyl glycerol was more "selective" when elevated since none of the controls were positive by either analysis; however, only half of the subjects determined to be positive using anticardiolipin were identified. CONCLUSION: The MoM method of reporting results may be more useful than phospholipid units for IgG or IgM.

Immunomodulatory effects of pulmonary surfactant on natural killer cell and antibody-dependent cytotoxicity.

Alveolar natural killer (NK) cells are functionally weak compared to their blood and interstitial counterparts. Having previously demonstrated that pulmonary surfactant suppresses lymphocyte responses to a variety of stimuli we sought in this study to determine if surfactant exerts a similar suppressive effect on cytotoxic function. Lipids were purified from the bronchoalveolar lavage (BAL) fluid from normal human volunteers. Human peripheral blood lymphocytes (n = 10 subjects) were cultured overnight in the presence or absence of purified BAL lipids (0.2 mg/ml), or pure preparations of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) (0.4 mg/ml). Standard NK and antibody-dependent cytotoxicity (ADCC) assays were performed using K562 and Chang target cells. The pooled BAL lipids significantly suppressed both NK (P less than 0.01) and ADCC (P = 0.01) activity in a dose-dependent manner. Whereas pure PC did not exert a significant effect, PG significantly suppressed (P less than 0.01) and PE significantly enhanced (P less than 0.01) both cytotoxic functions. There was no change in the expression of leu 7 or 11b antigens by lymphocytes after culture in BAL lipids. These results suggest that under normal circumstances pulmonary surfactant may suppress alveolar cytotoxic responses but that imbalances in the phospholipid profile might affect this immunoregulatory property.

Antibody-independent activation of C1. I. Differences in the mechanism of C1 activation by nonimmune activators and by immune complexes: C1r-independent activation of C1s by cardiolipin vesicles.

C1 activation is controlled by the regulatory protein C1-inhibitor (C1-INH). In contrast to immune-complex-induced activation, which is insensitive to C1-INH, antibody-independent activation of C1 is modulated by C1-INH. The mechanisms regulating nonimmune activation were studied with two phospholipids varying in their capacity to activate C1 in the presence of C1-INH: cardiolipin (CL) and phosphatidylglycerol (PG). Whereas C1-INH consistently suppressed activation by PG vesicles, a dose-dependent increase in C1 activation was measured with CL vesicles above 40 mole %. A similar dose-response binding of C1s requiring C1q, but not C1r, was detected only on CL vesicles, but neither on PG vesicles nor on immune complexes. This binding was Ca2+-dependent, suggesting that dimeric C1s is involved and was inhibited by spermine. The C1q-bound C1s was specifically cleaved at 37 degrees C into its active 58 kDa and 28 kDa chains, in the absence of C1r. On the addition of anti-CL antibodies, the C1q-mediated cleavage of C1s by CL vesicles was specifically inhibited. The cleavage of C1r on CL vesicles was also determined. When macromolecular C1 was offered in the presence of C1-INH, C1r cleavage was detected; however, the presence of C1s was a critical factor for C1r activation, because it was required on CL vesicles, but not on immune complexes. These results show that nonimmune activation of C1 presents specific features which distinguish it from immune complex-induced activation. These characteristics varied with the capacity of antibody-independent activators to activate C1 in the presence of C1-INH.

Murine monoclonal anti-DNA autoantibodies bind to endogenous bacteria.

Several bacterial species (including Streptococcus faecalis, Bacillus cereus, Staphylococcus aureus, and Escherichia coli) were tested for their ability to react with monoclonal anti-DNA antibodies that were derived from MRL-lpr/lpr mice. S. faecalis reacted with 8/15 of such antibodies. The binding was unaffected by DNase, but it was competitively inhibited by DNA. F(ab')2 fragments of the monoclonal antibodies reacted with the bacteria, but Fc fragments did not. Phospholipids extracted from the bacterial cells were able to bind to three representative anti-DNA antibodies that also bound to whole bacteria. The results suggest that bacterial phospholipids might provide an immunogenic stimulus for the production of antibodies that cross-react with DNA. We propose that some anti-DNA auto-antibodies and anti-bacterial antibodies evolve from a restricted group of antibodies with high avidity for the phosphodiester groups that occur in DNA and bacterial cells walls.

Binding of antibodies onto the thylakoid membrane. I. Maximal antibody binding and adsorption of antibodies to lipids.

The binding of antibodies onto the lamellar system of Antirrhinum majus was determined in dependence on the serum addition. The unspecific adsorption of serum proteins was taken into account or eliminated. The binding of antibodies as a function of the amount of serum added is seem from a saturation curve. From an antiserum obtained by hyperimmunization with stroma-freed chloroplasts, the chloroplasts bind maximally 1 gram antibodies per gram stroma-freed chloroplasts. From an antiserum to the proteins of the thylakoid membrane prepared in the same way an equal amount of antibodies is adsorbed. It is assumed that with this amount the surfaces of the lamellar system accessible to antibodies is completely covered by antibodies. For an antiserum to monogalactosyl diglyceride a maximal antibody binding of 0.16 g, for sulphoquinovosyl diglyceride 0.12 g and for phosphatidyl glycerol 0.13 of antibodies per gram stroma-freed chloroplasts are obtained. The significance of these results with respect to the molecular surface structure of the thylakoid membrane is discussed.

Immunological studies on the localization of phosphatidylglycerol in the membranes of Mycoplasma hominis.

Phosphatidylglycerol is the main component (87%) of the membrane phospholipids of Mycoplasma hominis. It is immunologically active. Antibodies directed against phosphatidylglycerol were detected in rabbits intravenously immunised with native M. hominis or isolated M. hominis membranes. The intravenous method of immunisation was chosen in order to select for a response to surface antigenic determinants. Anti-phosphatidylglycerol antibodies were induced in rabbits by intravenously injecting the flocculated complexes of methylated bovine serum albumin and a phosphatidylglycerol/phosphatidylcholine/cholesterol mixture. These antibodies were specifically bound to intact M. hominis, as shown by complement fixation and Coombs tests. Native M. hominis were not agglutinated by anti-phosphatidylglycerol antibodies; but after partial digestion of the membrane proteins with Pronase, the mycoplasmas were heavily agglutinated by the anti-phosphatidylglycerol antibodies. The same amount of anti-phosphatidylglycerol antibodies was bound to intact M. hominis, containing 600 mug of phosphatidylglycerol as to 6 mug of phosphatidylglycerol in the optimal configurational arrangement of a mixed phosphatidylglycerol/phosphatidylcholine/cholesterol micelle. It is concluded that the major part of the phosphatidylglycerol in native M. hominis membranes is masked, probably by membrane proteins, and is not accessible to the anti-phosphatidylglycerol antibodies.