Immunomodulation by alpha 2-macroglobulin and alpha 2-macroglobulin-proteinase complexes: the effect on the human T lymphocyte response

Alpha 2-macroglobulin (alpha 2M), various alpha 2M-proteinase complexes and methylamine-treated alpha 2M were added to human lymphocyte cultures stimulated with the specific antigen purified protein derivative of tuberculin (PPD), pokeweed mitogen (PWM) and anti-CD3. alpha 2M-trypsin diminished all reactions in a dose-dependent way. In the PPD-induced stimulation of peripheral blood mononuclear cells, both change of configuration and remaining proteinase activity contributed to the suppressive activity. Separate exposure of adherent cells or the highly purified T lymphocytes to alpha 2M-trypsin complexes indicated that the effect was mediated through adherent cells. Addition of indomethacin did not modify the results. In the interleukin 2 (IL2)-dependent stimulation of purified T lymphocytes by anti-CD3 the effect of alpha 2M-proteinase complexes was probably due to the digestion of IL 2 through remaining proteinase activity. As alpha 2M-proteinase complexes are formed at sites of inflammation, the multiple immunomodulatory effects of alpha 2M-proteinase complexes might contribute to the dysregulation of the immune system in inflammatory diseases.     

Effect of multiple sclerosis alpha 2-macroglobulin on mitogen induction of lymphocyte proliferation

Electrophoretic studies on microheterogeneity of alpha 2-macroglobulin (alpha 2M) have revealed the presence of an abnormal acidic form of alpha 2M in multiple sclerosis (MS). In the present study we have characterized alpha 2M chemically and compared the inhibitory effect of MS and normal alpha 2M on the mitogen induction of lymphocyte proliferation. The antitrypsin activity and total reducing sugar content of MS alpha 2M were comparable with those of normal alpha 2M. However, the sialic acid content of MS alpha 2M was found to be significantly higher than that of normal alpha 2M. In agreement with previous studies, normal alpha 2M was found to inhibit the mitogen induction of lymphocyte proliferation in more than 90% cell cultures. However, MS alpha 2M was found to inhibit the mitogen-induced lymphocyte proliferation in less than 35 percent cell cultures. The effects of MS or normal alpha 2M on the lymphocyte cultures of either groups were found to be apparently the same. The possible explanations for the loss of the above-mentioned inhibitory effect of MS alpha 2M have been discussed.     

Effect of α2-macroglobulin on the lymphocyte response

We studied the effect of α₂-macroglobulin (α₂MG) on the proliferative response of lymphocytes to several different lectins. This response to concanavalin A (Con A) and phytohaemagglutinin (PHA) was markedly reduced by addition of α₂MG to the reaction mixture, while that to lipopolysaccharide (LPS) was reduced to a lesser extent. Preincubation of lymphocytes with α₂MG had little effect on the response of the lymphocytes to lectin, when the protein was washed out before addition of the lectin. The response of the lymphocytes to lectin was reduced when α₂MG was added within 12 hr after the addition of the lectin, while the reduction was not apparent when the protein was added 24 hr after the lectin.

¹²⁵I-α₂MG prepared by the chloramine T method which maintained a 30% trypsin-protein esterase (TPE) activity did not bind to the cells, while the labelled protein prepared using the lactoperoxidase method which had full TPE activity, did bind to the cells. The binding of α₂MG with Con A was also demonstrated. Thus, the inhibitory effect of the protein on the proliferative response of lymphocytes to lectin may be due to binding of the protein to lymphocytes and consequently blocks the binding of the lectin to the cells or due to interaction of the protein with the lectin so as to diminish the number of the lectin molecules acting on the cells.

     

Quantification of free alpha 2-macroglobulin and alpha 2-macroglobulin-protease complexes by a novel ELISA system based on streptococcal alpha 2-macroglobulin receptors.

An ELISA test system has been developed for the quantification of the two distinct forms of the proteinase inhibitor alpha 2-macroglobulin (alpha 2M): (i) free alpha 2M (functionally active), which is the electrophoretically slow form (alpha 2 MS), and (ii) the alpha 2 M-proteinase complex (functionally inactive), which is the electrophoretically fast form of alpha 2 M (alpha 2 MF). Discrimination between the two types of alpha 2 M was achieved using extracts of the two independent streptococcal strains, M1 and Sc1, which express receptors for alpha 2 MS and alpha 2 MF, respectively, in combination with a monoclonal antibody specific for alpha 2 M. The assay system described is easy and reliable and permits quantitation of alpha 2 MS and alpha 2 MF in complex biological samples such as plasma and cutaneous suction blister fluid.     

A study of the alpha2-macroglobulin homologues of various species

The antigenic relationships between the α₂-macroglobulin homologues of a large number of species (predominantly mammalian) have been studied by the gel diffusion precipitin technique using a specific antiserum to human α₂-macroglobulin raised in a rabbit.

This antiserum has been absorbed with eleven different mammalian sera and the ability of the absorbed samples to cross-react with all these sera has again been tested.

From these results an attempt has been made to calculate the minimum number of antigenic determinants on each α₂-macroglobulin homologue recognized by this rabbit antiserum.

     

Rheumatoid-like synovitis in rabbits by intra-articular administration of alpha 2-macroglobulin . trypsin complexes and alkylamine-treated alpha 2-macroglobulin

The present animal experiments revealed that repeated intra-articular administration of alpha 2-macroglobulin . trypsin complexes caused a rheumatoid-like synovitis in not preimmunized rabbits. In early stages of synovitis, acute to subacute pathomorphological alterations were observed. In later stages, the chronic infiltration was accomplished by early onset of fibroplasia in some experimental joints. Similar lesions were caused by administration of methylamine-treated alpha 2-macroglobulin, an inactive alpha 2-macroglobulin (alpha 2-M), very like that produced in the trapping of a proteinase. In contrast, administration of native plasma alpha 2-M was ineffective, whereas active trypsin produced moderate inflammation. It is felt that the present experimental model resembles immunopathological processes in human arthritides, where the occurrence of alpha 2-M . proteinase complexes was verified.     

Interactions between cytokines and alpha 2-macroglobulin.

Cytokines are often thought of as short range, short half-life molecules. This view may now be challenged by recent findings that indicate that alpha 2-macroglobulin and antibodies to cytokines may alter cytokine kinetics in vivo. alpha 2-Macroglobulin, one of the major proteins in serum, can bind a wide range of physiologically important molecules. As Keith James reviews here, the interaction between alpha 2-macroglobulin and a number of cytokines has recently come under scrutiny, revealing a new and potentially important mechanism for the modulation of cytokine activity, while in the following paper a modulatory role for anti-cytokine antibodies is considered.     

Serum alpha 2-macroglobulin levels in diabetes.

Serum alpha 2-macroglobulin levels have been determined in diabetic patients by quantitative radial immunodiffusion and compared with those observed in age- and sex-matched controls. In addition, the results in diabetics have been analysed with respect to such variables as the age and sex of the patient, the duration of disease, treatment, control, and the occurrence of retinopathy or nephropathy. The alpha 2-macroglobulin levels in diabetic patients were found to be significantly higher than in age- and sex-matched controls, thus confirming previous observations. However, these differences were most apparent in the more extreme age groups. Multiple regression analysis also revealed that the only variables contributing significantly to the regression apart from age and sex were control and retinopathy.     

The association of alpha2-macroglobulin with lymphocyte membranes in chronic lymphocytic leukaemia and other disorders.

The association of alpha2-macroglobulin (alpha2M) with the surface membranes of human peripheral blood lymphocyte preparations has been investigated by the direct immunofluorescent technique. The percentage (and total number) of lymphocytes with detectable alpha2M on their surface is significantly increased in chronic lymphocytic leukaemia. The incidence of alpha2M-positive cells in normal and pathological conditions closely parallels that observed with conjugated antiserum to the kamma and gamma light chains of human immunoglobulin. It bears no relationship however to the plasma alpha2M levels or to the age of the donor. Additional blocking studies with aggregated human IgG, soluble antigen-antibody complexes and the F(ab')2 moiety of the anti-alpha2M, and indirect immunofluorescent studies with the latter, indicate that the fluorescence observed is not due to interaction of the conjugated reagent with Fc or C3 receptors.     

Preliminary studies on the interaction of TNF alpha and IFN gamma with alpha 2-macroglobulin.

The binding of 125I-labelled recombinant human TNF alpha and IFN gamma to isolated human blood alpha 2-macroglobulin has been investigated using molecular sieving procedures and non-denaturing PA gel electrophoresis in combination with autoradiography. These studies revealed that both cytokines readily bind to the electrophoretically fast form of alpha 2M generated by methylamine or protease treatment of this protein. PAGE/SDS gel investigations indicated that TNF alpha bound non-covalently while the IFN gamma interaction was covalent in nature. Preliminary competition studies also indicate that cold TNF alpha and IL-2 are more effective than cold IFN gamma at inhibiting the binding of labelled IFN gamma to alpha 2M. Bioassays revealed that "native" alpha 2M or its derivatives at 2 mg/ml concentration did not impair the antiproliferative effects of TNF alpha and IFN gamma on susceptible bladder tumour cell lines. Furthermore they did not interfere in the induction of Class II antigen expression by IFN gamma on inducible cell lines or in a 2-site ELISA assay for TNF.     

Suppression of experimental allergic encephalomyelitis by alpha 2-macroglobulin.

Lewis rats sensitized with guinea-pig spinal cord in Freund's complete adjuvant developed an acute-phase protein response. This was characterized by a marked increase in plasma alpha 2-macroglobulin (alpha 2 M) levels which, however, declined towards normal values before the onset of clinical signs of experimental allergic encephalomyelitis (EAE). In contrast, levels of two other acute-phase proteins, fibrinogen and caeruloplasmin, remained variably elevated over the entire study period. Recovery from EAE coincided with an increase in alpha 2 M levels. Infusion of purified alpha 2 M effectively protected the rats against clinical EAE and this was associated with a restimulation of the acute-phase response. The protected rats were shown to be sensitized to myelin basic protein and to have comparable mononuclear infiltration of the central nervous system with the diseased animals. It is postulated that the infusion of alpha 2 M leads to the inhibition of the effector pathways of the delayed type hypersensitivity response.     

Effect of lonidamine on alpha2-macroglobulin,hemopexin and alpha1-antitrypsin in the rat testis and epididymis

In a recent study (Leone et al., 2000) we reported that lonidamine (LND), an antispermatogenic drug, affected the concentration of selected testicular and epididymal proteins in the rat. Thus, the effect of LND on alpha2-macroglobulin (alpha2-M) and on other two acute phase proteins (APP), hemopexin (HPX) and alpha1-antitrypsin (alpha1-AT) was examined here. LND was administered orally at the dose of 100 mg/kg, the animals were killed after 24 and 48 hr and the samples were analyzed by immunoblotting. The drug did not induce any significant change of alpha2-M in the serum or testis and of HPX and alpha1-AT in the serum, testis or epididymis. Thus, the antispermatogenic action of LND was not accompanied by a significant change of these inflammatory markers, even if it did cause a decrease of alpha1-inhibitor-3, a negative APP, as previously reported (Leone et al., 2000).     

Activated alpha 2-macroglobulin is a principal defensin-binding protein.

Defensins are highly abundant and variably cationic peptides that possess antimicrobial, cytotoxic, and chemoattractant properties and equip mammalian phagocytes for participation in host defense and inflammatory processes. We studied the binding of the human defensin HNP-1 by proteins in plasma and serum and identified activated (F-form) alpha 2-macroglobulin (alpha 2M) as a principal binding protein for HNP-1. In contrast, native (S-form) alpha 2M bound little HNP-1. The binding of HNP-1 by F-form alpha 2M was resistant to salt and boiling in 2% sodium dodecyl sulfate but was ablated by dithiothreitol. Pretreatment of methylamine-activated serum or plasma by iodoacetamide substantially decreased the binding of HNP-1 to alpha 2M, suggesting that thiol groups in activated alpha 2M play a role in defensin binding, possibly by covalently trapping defensins via thiol-disulfide exchange. Western blots of conventionally collected sera showed endogenous defensins complexed with the F-form of alpha 2M, indicating that the generation of defensin-alpha 2M complexes was not limited to the in vitro model of methylamine-activated serum or plasma and radiolabeled HNP-1. Previous studies indicated that native alpha 2M can be converted to its F-form by many proteases, including those released by neutrophils and platelets, and that the F-form is recognized and internalized by specific receptors on macrophages and hepatocytes. Our findings suggest that the alpha 2M system may function as a scavenger of defensins and other peptide mediators in inflamed tissues and may constitute an important mechanism for the regulation and containment of inflammation.     

Regulation of macrophage agglutination factor production by alpha 2-macroglobulin.

Alpha-2-macroglobulin (alpha 2M) is a major mammalian plasma proteinase inhibitor and a well-established suppressor of T cell blastogenesis. Its role in modulating T cell-mediated lymphokine production is less well documented. We have isolated and characterized guinea-pig plasma alpha 2M in order to study its role in regulating the elicitation of macrophage agglutination factor (MAggF), a T cell-dependent inflammatory lymphokine closely related to fibronectin. Alpha-2-macroglobulin was purified by a combination of gel chromatography and immunoadsorption to remove contaminating IgM. Purified alpha 2M showed a double band on polyacrylamide gel electrophoresis. It had a molecular weight of 714,000 which fell to 190,000 on reduction, a pI of 4.8 and bound up to 1.3 moles of trypsin or elastase per mole. The elicitiation of MAggF from 25 X 10(6) purified protein derivative (PPD)-stimulated guinea-pig lymph node cells was inhibited 99% by 2-3 micrograms of biologically active alpha 2M only if the proteinase inhibitor was added to the lymph node cells at the same time as antigen. No inhibition of MAggF production was observed when active alpha 2M was added at the end of culture or when biologically inactive alpha 2M was added to the cultures at any time. MAggF was not elicited from normal cells by PPD, nor did alpha 2M have any effect on these cultures. Purified alpha 2M had no direct effect on MAggF activity in culture supernatants. We suggest that alpha 2M may be involved in modulating antigen-elicited lymphokine production in vivo.     

Binding of native alpha 2-macroglobulin to human group G streptococci.

Binding of human alpha 2-macroglobulin (alpha 2M) to group G streptococci and to their immunoglobulin G (IgG)-binding proteins (protein G) was investigated. Native alpha 2M bound specifically to strain G-148 with an apparent dissociation constant of (2.2 +/- 1.5) x 10(-9) M. Proteinase-complexed alpha 2M did not compete for the binding sites, and 125I-labelled proteinase-complexed alpha 2M did not bind to the bacteria. Binding of native alpha 2M to the cells was not affected by IgG or protein G consisting of only IgG-binding domains. 125I-labelled recombinant protein G did not bind to native or proteinase-complexed alpha 2M. However, a lysate of G-148 cells inhibited binding of alpha 2M to the bacteria, and immobilized wild-type protein G bound alpha 2M directly from fresh human plasma. In 13 group G streptococcal isolates, IgG-binding proteins were immunologically identified as protein G. In 11 isolates, these molecules reacted also with alpha 2M and human serum albumin (HSA). Western blots (immunoblots) of two wild-type protein G variants revealed identical bands reactive with goat IgG, HSA, and native alpha 2M. Digestion of wild-type protein G with clostripain destroyed in both variants the binding sites for alpha 2M but not for albumin and IgG. N-terminal fragments of protein G (lacking the IgG-binding region) bound both alpha 2M and HSA, whereas a similar HSA-binding peptide lacking the first 80 amino acids did not react with alpha 2M. Our findings are consistent with a specific binding site for native alpha 2M in the N-terminal region of protein G and suggest that binding of alpha 2M via IgG-binding proteins may be a general feature of human group G streptococci.