Lennart Lögdberg

An intriguing member of the lipocalin protein family: α1-microglobulin

The plasma protein alpha 1-microglobulin is a member of the lipocalin protein superfamily. In the last few years, the work on alpha 1-microglobulin has given unexpected and promising new results. Of particular interest are its molecular association with immunoglobulin A and with proteinase inhibitors, and its interactions with the immune system.

Purification of antibodies using protein L-binding framework structures in the light chain variable domain

Protein L from the bacterial species Peptostreptococcus magnus binds specifically to the variable domain of Ig light chains, without interfering with the antigen-binding site. In this work a genetically engineered fragment of protein L, including four of the repeated Ig-binding repeat units, was employed for the purification of Ig from various sources. Thus, IgG, IgM, and IgA were purified from human and mouse serum in a single step using protein L-Sepharose affinity chromatography. Moreover, human and mouse monoclonal IgG, IgM, and IgA, and human IgG Fab fragments, as well as a mouse/human chimeric recombinant antibody, could be purified from cultures of hybridoma cells or antibody-producing bacterial cells, with protein L-Sepharose. This was also the case with a humanized mouse antibody, in which mouse hypervariable antigen-binding regions had been introduced into a protein L-binding kappa subtype III human IgG. These experiments demonstrate that it is possible to engineer antibodies and antibody fragments (Fab, Fv) with protein L-binding framework regions, which can then be utilized in a protein L-based purification protocol.

Immunosuppressive Properties of α1-Microglobulin

α1‐Microglobulin (α1m), a serum glycoprotein (26,000 d). was found to impede the proliferative response of human lymphocytes to purified protein derivative (PPD) and tetanus toxoid. The data suggest that, α1m operates through an unstable suppressor mechanism, which no longer can function after 24 h of preculturing. This effect of α1m on antigen stimulation did not seem to be due to binding of α1m to PPD or cells, to altered kinetics of the PPD response, or to non‐specific cytotoxicity. In contrast, PPD‐induced leucocyte migration inhibition was not reversed by α1m. α1m did not cause significant inhibition in experiments in which lymphocytes were stimulated by the mitogens phytohaemagglutinin or concanavalin A. Finally, α1m had its own leucocyte migration inhibitory effect.

Rat β2-microglobulin isolation, properties and relation to β2-microglobulins from other species

Abstract Rat s -microglobulin was isolated from the urine of animals with sodium-chromate induced tubular damage. It was purified by sequential use of gel chromatography, ion exchange chromatography, zone electrophoresis, and finally repeated gel chromatography. The rat protein was similar to human β 2 -microglobulin in properties such as molecular weight and the presence of an apparently analogous disulfide loop. Electrophoretically, however, it migrated in the γ-globulin region, which was reflected in a higher pI-value (pH 7.4). A minor rat β 2 -microglobulin form with a somewhat lower pI (pH 6.8) was found. On both Ouchterlony immunodiffusion analyses and radioimmunoassays, rat β 2 -microglobulin was found to cross-react with purified β 2 -microglobulins from four other species: rabbit, guinea pig, mouse, and man. The gel chromatography profile of β 2 -microglobulin in rat serum was investigated by radioimmunoassay. β 2 -microglobulin was found in high mol. wt complexes and in free form.

Cross-reacting monoclonal anti-α1-microglobulin antibodies produced by multi-species immunization and using protein G for the screening assay

In order to generate monoclonal antibodies (MAb) directed against the low molecular weight glycoprotein alpha 1-microglobulin, a BALB/c mouse was immunized with a mixture of human, guinea pig, rat and rabbit alpha 1-microglobulin homologues (multi-species immunization) and boosted several times. On day 194, the mouse splenocytes were fused to SP2/0 myeloma cells. The resulting hybridomas were screened for anti-alpha 1-microglobulin activity against the alpha 1-microglobulin mixture or against the individual homologues. For this screening, protein G (the newly described IgG-binding streptococcal protein) was used in a solid-phase radioimmunoassay. The binding of protein G to immobilized antigen-antibody complexes was enhanced by pre-incubation with rabbit anti-mouse immunoglobulin G. The result was a panel of nine established hybridoma lines, all producing unique monoclonal antibodies, of IgG1 or IgG2a class, to alpha 1-microglobulin. The antibodies were not only reactive in solid-phase radioimmunoassay, but they could also immunoprecipitate 125I-labeled soluble alpha 1-microglobulin. Moreover, they reacted specifically with the alpha 1-microglobulin band in Western blots of urinary proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Such monoclonal antibodies are potentially valuable reagents for the further characterization of alpha 1-microglobulin.

Binding of β2‐Microglobulin by Heterologous Sera

Purified human, rat or guinea‐pig β2‐microglobulin (β2m) was mixed with sera from guinea‐pig, rat, mouse rabbit, horse, goat, cow, rhesus monkey or man. The mixtures were incubated at.37°C for various lengths of time. When the sera were separated by gel‐chromatography on Sephadex G‐200, β2m was traced not only in ‘free’ form but also in fractions with higher molecular weights. Evidence is presented suggesting that heterologous β2m binds to β2m‐containing molecules in sera by exchange with the homologous counterpart.

Mitogenic Effect of α1‐Microglobulin on Mouse Lymphocytes

Human α1‐m microglobulin (α1‐m), a low molecular weight plasma protein, was found to exert mitogenic effects on mouse lymphocytes from lymph nodes and spleen. The stimulatory effects appeared lo be strain‐restricted: α1‐m induced a varying degree of proliferation of lymphocytes from three strains, whereas one strain responded poorly. Experiments with lymphocyte subpopulations showed only weak stimulatory effects of α1‐m on purified T and B lymphocytes cultivated alone. The addition of mitomycin‐treated cells of the other subpopulation could not restore the proliferative responses in either T or B lymphocytes. Strong stimulations were recorded only when both T and B lymphocytes were present, indicating that the T and B lymphocytes cooperate to achieve the proliferation However, FACS studies on cultured splenocytes indicated (hat the proliferating cells are predominantly B lymphocytes. These data extend our earlier findings of a mitogenic effect of α1‐m on guinea pig lymphocytes. Furthermore. results were obtained indicating the presence of a receptor on mononuclear cells. Iodine‐labelled α1‐m was bound lo mononuclear cells prepared from spleens, and the binding could be blocked by an excess of non‐labelled α1‐m. Scatchard plotting of the data gave an equilibrium constant of 0.7 × 105/M for the binding between α 1‐m and the receptor. Together with the documented inhibitory activity of α1‐m on antigen‐driven proliferation of lymphocytes, these results suggest an immunoregulatory role for α1‐m.

Alpha 1-microglobulin is mitogenic to human peripheral blood lymphocytes. Regulation by both enhancing and suppressive serum factors

Human alpha 1-microglobulin (alpha 1-m), a 26 kilodalton serum glycoprotein, was found to exert mitogenic effects on human peripheral blood lymphocytes (PBL) in serum-free medium. Purified T cells, but not B cells, responded with proliferation to alpha 1-m, but only in the presence of monocytes. The mitogenic activity could be partially neutralized by a mouse monoclonal antibody against alpha 1-m. The mitogenicity was species-specific, since alpha 1-m homologues from rats, guinea pigs and rabbits had no effect on human PBL. In a previous study, no effect of alpha 1-m was seen on PBL in the presence of 20% serum, and, therefore, we studied the influence of different concentrations of serum on the alpha 1-m-induced mitogenicity. Thus, human serum enhanced the mitogenic effects of alpha 1-m on human PBL at 1% concentration (v/v) and suppressed the effects at 10%. The suppressing effect of serum at 10%, but not the enhancing effect at 1%, seemed to be conserved among several species. To test the effect of serum proteins of different molecular sizes, human autologous serum was separated by gel chromatography on Sephadex G-200 into four fractions. Fractions 1 and 2 (roughly containing proteins larger than 100 kilodaltons) suppressed the mitogenic effects of alpha 1-m, while fractions 3 and 4 enhanced the stimulation by alpha 1-m, at 0.5% and concentrations above. It is concluded that the mitogenic effect of alpha 1-m on lymphocytes is regulated by several serum factors, both enhancing and suppressive, that does not have any proliferative effect of their own. It can be speculated that the balance between enhancing and suppressing co-factors in the blood determines the degree of the stimulation of lymphocytes by alpha 1-m. This is compatible with an immunomodulatory role for alpha 1-m, in spite of its relatively constant plasma levels in health and disease.

Experimental autoimmune encephalomyelitis in hybrids between Lewis and Brown Norway rats.

The susceptibility to experimental autoimmune encephalomyelitis (EAE) in Lewis (Lew) and Brown Norway (BN) rats was studied in breeding experiments, evaluating EAE from clinical signs of the disease. The Lew strain is highly susceptible, the BN strain is resistant to EAE. F1 hybrids between the strains show an intermediate susceptibility as described by earlier authors. Back-cross experiments verify that susceptibility is inherited in a complex way, at least according to a two-gene model previously suggested. Analysis of the F1 hybrids showed a bi-modal distribution of clinical scores, one group of rats which appear to have the same degree of susceptibility as the Lew strain, and another group with very low susceptibility. Study of F2 rats produced by F1 rats with high or low susceptibility showed that this property was probably not inherited, arguing against a residual heterozygosity in the parental strains. As an alternative hypothetical explanation, the possibility of allogeneic exclusion of genes regulating suppression of EAE is discussed.

On the Heterologous Interaction between β2‐Microglobulin and the Heavy Chain of Rat Major Histocompatibility Complex Class 1 Antigens

The heterologous interaction between β2‐microglobulin (β2m) and rat major histocompatibility complex (MHC) (RT1) antigens was measured in a two‐step binding assay consisting of binding of radiolabelled β2‐m to RT1 antigens and immunoprecipitation of β2m‐RT1 antigen complexes with RT1 antisera. The effects of varying the concentrations of the three reactants involved were studied. The molecular events taking place in the two steps were analysed by gel chromatography. The β2m‐RT1 antigen complex had the apparent size of albumin and reacted completely with specific alloantisera. RT1 antigens prepared from Wistar/Furth (RT1u) and Brown Norway (RT1n), respectively, both effectively bound heterologous β2m. The times for association and dissociation, respectively, at 37°C, were of the same order, but dissociation was slightly slower. Association was markedly temperature‐dependent and was considerably slower at low temperatures. All these processes were slower for RT1u than for RT1u antigens. The association constant for the interaction between RT1u antigens and 125I‐human β2m was estimated by Scatchard analysis to be about 109 M‐1. Contribution to the heterologous interaction by products from various rat MHC subloci (A, B, and C) was investigated by the introduction of sublocus‐specific antisera in step 2. The reaction apparently involved neither class 2 antigens (sublocus B) nor the presumed rat Qa homologue (sublocus C). Classical class 1 antigens (suhlocus A) clearly contributed to the binding. However, a monoclonal antibody against products from rat MHC class 1 genes only precipitated less than half of the RTI antigen‐complexed β2m. Thus, at least two RT1u class 1 alloantigen molecules seem to participate in the reaction. This, in turn, indicates that the rat genome may contain multiple class 1 genes, an is the case for most other mammals investigated.