Self-reactive antibodies (natural autoantibodies) in healthy individuals.

Antibodies that are present in the serum of healthy individuals in the absence of deliberate immunization with any antigen, are refered to as natural antibodies. A vast majority of natural antibodies react with one or more self antigens and are termed as natural autoantibodies. The importance of natural autoantibodies in immune regulation has long been neglected, since tolerance to self was thought to be primarily dependent on the deletion of autoreactive clones, rather than on peripheral suppressive mechanisms. Clonal deletion and energy cannot account, however, for the prevalence of natural autoreactivity among healthy individuals. It is now well established that autoreactive antibodies and B cells, and autoreactive T cells, are present in healthy individuals, and in virtually all vertebrate species. Autoreactive repertoires are predominantly selected early in ontogeny. Questions pertaining to the role of natural antibodies in the regulation of the immune response and maintenance of immune homeostasis and to the distinction between natural autoreactivity and pathological autoimmunity have not been adequately addressed. Here, we focus on the current knowledge on the physicochemical and functional properties of NAA in man, and the use of NAA for therapeutic intervention. reserved.

Functional diversity and clonal frequencies of reactivity in the available antibody repertoire.

The present experiments address functional antibody diversity and clonal distribution in murine available repertoires. IgM-containing supernatants were prepared by unbiased, polyclonal stimulation of resting splenic B cells from C57BL/6 mice, to ensure similar numbers of responding clones/culture and equivalent growth and maturation of all clones. The repertoires of clones and clonal mixtures were quantitatively assayed by limiting dilution analysis (LDA) on immunoblots of sodium dodecylsulfate polyacrylamide gel electrophoresis of homologous liver extracts, allowing to determine specific clonal frequencies towards the many hundred blotted antigens. The clonal frequency of reactivity of B cells with the extract was shown to be a bi-modal distribution of specific frequencies between 1/220 and 1/100,000. Cross-correlation analysis of reactivity to different bands in individual supernatants revealed low levels of cross-reactivity, suggesting that the blotted extract provides a very diverse set of antigens. Investigation of the affinity/concentration thresholds for detection of antigen-antibody interactions of our assay supports the notion that global repertoire analyses on immunoblots were highly discriminative and non-degenerate. Furthermore, reactivity patterns obtained with complex antibody mixtures correlated with the frequency of clonal reactivities as determined by LDA. The results demonstrate a large functional diversity of resting B lymphocytes, indicating a minimal repertoire size that is orders of magnitude higher than previous theoretical proposals suggested, and extensively heterogeneous in the size of clonal specificities.

Maternal IgG stimulates B lineage cell development in the progeny.

To examine the physiological role of maternal natural IgG antibodies on the development of B lineage cells of the progeny, we have bred homozygous muMT/muMT or heterozygous muMT/+ females to muMT/muMT or muMT/+ males, respectively. We could thus compare normal or B cell-deficient mice born from Ig-deprived (Ig-) or phenotypically normal mothers (Ig+). B cell-deficient progeny of heterozygous mothers contain no detectable serum IgA or IgM, but IgG concentrations that peak at 2 mg/ml by 7-21 days of age, decay after weaning with a half-life of 7 days, and remain detectable for 2 months after birth. At 7 days after birth, muMT/+ progeny born of Ig+ mothers contain two- to threefold higher numbers of bone marrow (BM) pre-B and B cells, and of splenic B cells, compared to mice of the same age born from Ig mothers. In contrast, the former progeny exhibit two to four times lower numbers of Ig-secreting plasma cells in spleen and thymus, and contain sixfold lower serum IgM concentrations. A similar maternal IgG-dependent stimulation of BM B cell precursors is also observed in muMT/muMT progeny. No significant differences were detected between the groups on day 3 after birth, suggesting the requirement for a minimal IgG concentration in the serum.

Targeted disruption of the V(H) 81X gene: influence on the B cell repertoire.

We have generated a mutant mouse in which the most D-proximal V(H) gene (V(H)81X) has been disrupted by introducing a neomycin-resistance gene into the V(H)81X exon by means of gene targeting in embryonic stem cells. The mutant mice generated are unable to express the V(H)81X gene but appear to display a normal pattern of B cell differentiation as well as normal numbers of bone marrow and peripheral B cells from fetal life all through ontogeny. They mount normal immune responses to several different antigens tested. In contrast, the distribution of V(H) gene rearrangements in the V(H)7183 family is altered in homozygous mutant mice. Thus, the antibody repertoire of the targeted mice is modified, at least as far as the expression of V(H)7183 genes is concerned.

Studies on the T cell dependence of natural IgM and IgG antibody repertoires in adult mice.

The present experiments address the thymic dependence of IgM and IgG natural antibody repertoires in adult euthymic and athymic BALB/c mice, as well as in athymic animals reconstituted with a fixed number of syngeneic T cells. Within 3 weeks of the transfer of 10(7) syngeneic splenic T lymphocytes to athymic mice, the T cell compartment is essentially reconstituted in the peritoneal cavity (up to 80% of the numbers in euthymic animals), but is only 10-20% of controls in the spleen and lymph nodes. Early after transfer, there is an increase in the numbers of activated B cells and of immunoglobulin-secreting cells in the spleen, and within 1-2 weeks, the serum concentrations of IgG1 and IgG2a are fully reconstituted to control levels (30-40-fold increased). Multiparametric analyses of serum IgM and IgG repertoires revealed that euthymic and athymic mice share essentially all natural antibody reactivities towards syngeneic extracts of liver and muscle. When tested at the same immunoglobulin concentrations, however, nude sera consistently show higher values of reactivity in all detectable bands. The transfer of 10(7) splenic T cells into athymic mice results in a general decrease of serum IgM reactivities, some of which become undetectable, and in alterations of the serum IgG repertoire as early as 1 week, and for at least 4 weeks after transfer. T cell transfer, however, fails to restore the euthymic IgM and IgG repertoires within 4 weeks. The present observations demonstrate that, after limited T cell reconstitution of nude mice, there is a rapid and quantitatively important increase of serum IgG1 and IgG2a production; the serum IgM reactivity repertoire is qualitatively similar in euthymic and athymic animals, but is generally decreased by T cell activity; and the serum IgG repertoire, which is qualitatively similar in euthymic and athymic animals, is amplified by T cell activity and partially altered by T cell transfer into athymic animals. These results raise questions on the mechanisms of B cell activation and natural antibody repertoire selection in T cell-deficient adult individuals.

Global analysis of antibody repertoires. II. Evidence for specificity, self-selection and the immunological "homunculus" of antibodies in normal serum.

The serum IgM repertoires of C57BL/6, DBA/2 and BALB/c mouse strains were analyzed using a recently developed global and quantitative assay that measures antibody reactivities to a very large number of antigens. A characteristic repertoire could be assigned to each strain. The different repertoires could be successfully classified with multivariate statistics. Many common reactivities were also observed among the different strains, which allows the definition of a mouse-specific repertoire. Analysis of human sera support this notion. To investigate the impact of minor genetic differences on the serum IgM repertoire, the congenic strains B10.D2/oSn and B10.D2/nSn, which differ in the expression of the C5 component of complement, were analyzed. The two strains could be separated based on the reactivity profiles obtained. The analysis of the results reveals that many antigenic proteins are not recognized at all by natural antibodies, while others are disproportionately reactive, the resulting patterns giving rise to what could be the definition of an "immunological homunculus". The relevance of this type of analysis for clinical applications is discussed.

Protection by nedocromil sodium of active immunization-induced bronchopulmonary alterations in the guinea pig.

The effect of nedocromil sodium on PAF-acether- and antigen-induced bronchoconstriction (BC) and mediator release in lungs from actively sensitized guinea pigs and on the eosinophil content of bronchoalveolar lavage (BAL) was investigated. Guinea pigs were actively sensitized by two subcutaneous injections of 10 micrograms ovalbumin in 1 mg AI(OH)3 at 2-wk intervals. One week after the second (booster) injection, the lungs were removed, perfused in the absence or presence of indomethacin, and challenged at 10-min intervals with PAF-acether (1 and 100 ng) and with ovalbumin (1 micrograms). No inhibition of PAF-acether- and antigen-induced BC or mediator release from sensitized lungs was observed when nedocromil sodium (10 microM) was added directly to the buffer solution. By contrast, when guinea pigs were treated for 1 wk before the experiment with nedocromil sodium (30 mg/kg per day), BC and the release of leukotrienelike material (but not of thromboxane B2) to 1 ng PAF-acether were reduced by around 50% (p less than 0.05) and 62% (p less than 0.05), respectively. No inhibition was observed for 100 ng PAF-acether and 1 micrograms ovalbumin. Furthermore, nedocromil sodium markedly impaired histamine secretion induced by both PAF-acether and antigen administration. Nedocromil sodium did not affect the titers of circulating ovalbumin-specific immunoglobulin G, as detected by an enzyme-linked immunosorbent assay. The 1-wk treatment with nedocromil sodium also reduced markedly the proportion of eosinophils in the BAL of sensitized guinea pigs, whereas it was ineffective when injected once subcutaneously at the dose of 30 mg/kg 2 h before the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

A new method to evaluate extravascular albumin and blood cell accumulation in the lung.

A method was developed to evaluate blood volume, accumulation of extravascular albumin (ALBev), and platelet (PL) or polymorphonuclear neutrophil (PMN) sequestration in lungs after challenge with inflammatory agents. Erythrocytes (RBC), albumin, and PL or PMN, labeled with 99mTc, 131I, and 111In,-respectively, were injected intravenously into anesthetized and ventilated guinea pigs. The different parameters were calculated from in vivo lung and blood radioactivity values. When N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) was injected intravenously at 10 micrograms.kg-1, lung RBC content dropped by 14.7 +/- 1.8% (SE; n = 10), indicating a reduced lung blood volume, ALBev rose to 15.0 +/- 3.2% of the initial albumin vascular content, and the circulating PMN were sequestered by 9.2 +/- 1.7%. A transient PL sequestration was also observed 1 min after the injection of fMLP (13.1 +/- 2.0%, n = 7). During the infusion of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine, the lung PL content rose dose dependently from 10.1 +/- 2.2% of the circulating pool with 3 ng.kg-1.min-1 to 54.9 +/- 20.1% with 44 ng.kg-1.min-1, the lung RBC content decreased by greater than 10%, and the ALBev increased beyond 16%. Our method allows the study of the correlations between cell entrapment and the variations of the albumin exchanges in the lung and may lead to a better understanding of the correlations between cell activation and edema.

Reduced responsiveness of bronchoalveolar cells from sensitised guinea-pigs to the cyclic AMP--stimulating effect of prostaglandin E2 and beta-adrenoceptor agonists.

Prostaglandin E2 and the beta-adrenoceptor agonist salbutamol were less effective in increasing the intracellular cAMP content of bronchoalveolar cells and adherent alveolar macrophages from ovalbumin-sensitised as compared to control guinea-pigs. This refractoriness to the cyclic AMP-stimulating effects of PGE2 and salbutamol induced by the sensitisation procedure may be relevant to the altered bronchopulmonary responsiveness in asthma.

Interference by the novel PAF-acether antagonist WEB 2086 with the bronchopulmonary responses to PAF-acether and to active and passive anaphylactic shock in guinea-pigs.

The interaction between the triazolothienodiazepine WEB 2086 and the in vitro and in vivo bronchopulmonary effects of PAF-acether and active/passive anaphylaxis in the guinea-pig was studied. WEB 2086 (1-100 nM) inhibited PAF-acether (10-100 ng)-induced bronchoconstriction and TXB2 release from isolated and perfused guinea-pig lungs without affecting the response to 100 micrograms arachidonic acid. In addition, 1-10 microM WEB 2086 significantly reduced antigen-induced TXB2 and histamine release from lungs from actively and passively sensitized guinea-pigs. In the presence of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), mepyramine, methysergide, indomethacin and atropine, WEB 2086 (20-50 microM) inhibited by 30-40% the residual contraction of lung parenchyma strips from guinea-pigs actively sensitized by 0.1-10 micrograms antigen. In vivo, WEB 2086 (0.1-1 mg/kg) reversed or abolished the bronchoconstriction, hypotension, thrombocytopenia and leukopenia evoked by perfusion of PAF-acether (3 or 44 ng/kg per min). At 3 mg/kg, WEB 2086 also markedly decreased the bronchoconstriction and leukopenia induced by 100 micrograms/kg antigen in mepyramine (5 micrograms/kg)-treated passively sensitized guinea-pigs. In contrast, WEB 2086 was ineffective against active anaphylaxis in vivo. These results demonstrate that WEB 2086 antagonizes the bronchopulmonary effects due to PAF-acether and to anaphylactic shock in the guinea-pig.

Pharmacological modulation of the effects of N-formyl-L-methionyl-L-leucyl-L-phenylalanine in guinea-pigs: involvement of the arachidonic acid cascade.

The intravenous administration of the chemotactic and secretagogue peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 0.3-30 micrograms kg-1) to the guinea-pig induces bronchoconstriction and dose-dependent leukopenia accompanied by mild thrombocytopenia. No electron microscopic evidence of platelet aggregation in lungs or significant accumulation of 111In-labelled platelets in the thoracic region at the height of bronchoconstriction was noted. Bronchoconstriction and leukopenia induced by FMLP were not affected by prostacyclin, by platelet depletion, by the platelet-activating factor (Paf-acether) antagonist BN 52021 or by the histamine H1-antagonist mepyramine. Bronchoconstriction, but not leukopenia, was inhibited by aspirin, whereas the peptido-leukotriene antagonist compound FPL 55712 and the cyclo-oxygenase lipoxygenase inhibitor indomethacin reduced bronchoconstriction to a limited extent only. The mixed cyclo-oxygenase/lipoxygenase inhibitor compound BW 755C was very effective in blocking bronchoconstriction by the highest dose of FMLP used, but failed to interfere with leukopenia. FMLP-induced dose-dependent contraction of parenchymal lung strips was accompanied by the formation of immuno-reactive thromboxane B2 in amounts markedly less than those formed from exogenous arachidonic acid at concentrations equieffective in inducing contractions. FMLP-induced contractions of the guinea-pig lung strip were not modified by mepyramine nor by FPL 55712. They were reduced by indomethacin and aspirin and an even greater reduction was obtained with aspirin used in combination with FPL 55712. BW 755C suppressed the effects of all the concentrations of FMLP tested, whereas tert-butyloxy-carbonyl-L-methionyl-L-leucyl-L-phenylalanine, a chemical analogue of FMLP, displaced the concentration-response curve to the right, without reducing the maximal contraction obtained. The present results indicate that: (a) bronchoconstriction by FMLP is not due to platelet activation, to cyclo-oxygenase-dependent mechanisms or to peptido-leukotriene formation. The inhibitory effect of aspirin and BW 755C involves a property other than cyclo-oxygenase inhibition, which is not shared by indomethacin. (b) The contractile effects of FMLP on parenchymal lung strips follow an interaction with specific receptor sites, as shown by the effectiveness of tert-butyloxy-carbonyl-L-methionyl-L-leucyl-L-phenylalanine, and involves the combined effects of cyclo-oxygenase and lipoxygenase metabolites.