An acute phase protein α1-acid glycoprotein mitigates AKI and its progression to CKD through its anti-inflammatory action.

The molecular mechanism for acute kidney injury (AKI) and its progression to chronic kidney disease (CKD) continues to be unclear. In this study, we investigated the pathophysiological role of the acute phase protein α1-acid glycoprotein (AGP) in AKI and its progression to CKD using AGP KO mice. Plasma AGP levels in WT mice were increased by about 3.5-fold on day 1-2 after renal ischemia-reperfusion (IR), and these values then gradually decreased to the level before renal IR on day 7-14. On day 1 after renal IR, the AGP KO showed higher renal dysfunction, tubular injury and renal inflammation as compared with WT. On day 14, renal function, tubular injury and renal inflammation in WT had recovered, but the recovery was delayed, and renal fibrosis continued to progress in AGP KO. These results obtained from AGP KO were rescued by the administration of human-derived AGP (hAGP) simultaneously with renal IR. In vitro experiments using RAW264.7 cells showed hAGP treatment suppressed the LPS-induced macrophage inflammatory response. These data suggest that endogenously induced AGP in early renal IR functions as a renoprotective molecule via its anti-inflammatory action. Thus, AGP represents a potential target molecule for therapeutic development in AKI and its progression CKD.

The effect of polyclonal activators on rabbit B cell antibody production.

Stimulation with polyclonal activators is a tool to increase antibody secretion in B cells. The aim of the present study was to select the most effective common commercially available polyclonal activators of rabbit B cells. Specifically, type B oligodeoxynucleotides with unmethylated deoxycytidyl-deoxyguanosine dinucleotides (CpG-ODN), recombinant rabbit interleukin-2 (rrIL-2), lipopolysaccharide (LPS), pokeweed mitogen (PWM) and Resiquimod (R848) were tested on B cells isolated from blood and spleen by fluorescence-activated cell sorting. Based on the obtained data, stimulation with CpG-ODN induced the highest antigen-specific antibody levels detected by ELISA in supernatants when a single activator was used. In contrast, LPS, PWM and R848 showed a weak or no stimulatory effect. Stimulation with a mix of activators was more effective than CpG-ODN alone, which indicates a synergistic effect in the stimulation of antibody production.

Alpha-2-macroglobulin loaded microcapsules enhance human leukocyte functions and innate immune response.

Synthetic microstructures can be engineered to deliver bioactive compounds impacting on their pharmacokinetics and pharmacodynamics. Herein, we applied dextran-based layer-by-layer (LbL) microcapsules to deliver alpha-2-macroglobulin (α2MG), a protein with modulatory properties in inflammation. Extending recent observations made with dextran-microcapsules loaded with α2MG in experimental sepsis, we focused on the physical and chemical characteristics of these microstructures and determined their biology on rodent and human cells. We report an efficient encapsulation of α2MG into microcapsules, which enhanced i) human leukocyte recruitment to inflamed endothelium and ii) human macrophage phagocytosis: in both settings microcapsules were more effective than soluble α2MG or empty microcapsules (devoid of active protein). Translation of these findings revealed that intravenous administration of α2MG-microcapsules (but not empty microcapsules) promoted neutrophil migration into peritoneal exudates and augmented macrophage phagocytic functions, the latter response being associated with alteration of bioactive lipid mediators as assessed by mass spectrometry. The present study indicates that microencapsulation can be an effective strategy to harness the complex biology of α2MG with enhancing outcomes on fundamental processes of the innate immune response paving the way to potential future development in the control of sepsis.

Immunotherapy of tumors with α2-macroglobulin-antigen complexes pre-formed in vivo.

The cell surface receptor CD91/LRP-1 binds to immunogenic heat shock proteins (HSP) and α(2)M ligands to elicit T cell immune responses. In order to generate specific immune responses, the peptides chaperoned by HSPs or α(2)M are cross-presented on MHC molecules to T cells. While the immunogenic HSPs naturally chaperone peptides within cells and can be purified as an intact HSP-peptide complex, the peptides have had to be complexed artificially to α(2)M in previous studies. Here, we show that immunogenic α(2)M-peptide complexes can be isolated from the blood of tumor-bearing mice without further experimental manipulation in vitro demonstrating the natural association of tumor antigens with α(2)M. The naturally formed immunogenic α(2)M-peptide complexes are effective in prophylaxis and therapy of cancer in mouse models. We investigate the mechanisms of cross-presentation of associated peptides and co-stimulation by APCs that interact with α(2)M. These data have implications for vaccine design in immunotherapy of cancer and infectious disease.

Administration of rat acute-phase protein α(2)-macroglobulin before total-body irradiation initiates cytoprotective mechanisms in the liver.

Previously, we showed that administration of the acute-phase protein α(2)-macroglobulin (α(2)M) to rats before total-body irradiation with 6.7 Gy (LD(50/30)) of X-rays provides the same level of radioprotection as amifostine. Here, we compare the cytoprotective effects of α(2)M and amifostine on rat liver. The potential of the liver to replenish cells destroyed by ionizing radiation was assessed by immunoblot analysis with antibody to proliferating cell nuclear antigen (PCNA). After irradiation, in unprotected rats PCNA decreased 6-fold from the basal level. In rats pretreated with either α(2)M or amifostine, PCNA was increased throughout a 4 week follow-up period, indicating that hepatocyte proliferation was unaffected. Since PCNA is an important component of the repair machinery, its increased expression was accompanied by significantly lower DNA damage in α(2)M- and amifostine-treated rats. At 2 weeks after irradiation, the Comet assay revealed a 15-fold increase in DNA damage in unprotected rats, while in α(2)M- and amifostine-treated rats we observed 3- and 4-fold rise in damage, respectively. The improved protection to DNA damage was supported by elevated activity of the antioxidant systems. Compared to untreated rats, pretreatments with α(2)M and amifostine led to similar increases in levels of the inflammatory cytokine IL-6 and the redox-sensitive transcription factor NFκB, promoting upregulation of MnSOD, the major component of the cell's antioxidant axis, and subsequent increases in Mn/CuZnSOD and catalase enzymatic activities. The results show that α(2)M induces protein factors whose interplay underlies radioprotection and support the idea that α(2)M is the central effector of natural radioprotection in the rat.

The rat acute-phase protein α2-macroglobulin plays a central role in amifostine-mediated radioprotection.

Previously we reported that elevated circulating concentrations of the acute-phase (AP) protein α(2)-macroglobulin (α(2)M), either as typically occurring in pregnant female rats or after administration to male rats, provides radioprotection, displayed as 100% survival of experimental animals exposed to total-body irradiation with 6.7 Gy (LD(50/30)) x-rays, that is as effective as that afforded by the synthetic radioprotector amifostine. The finding that amifostine administration induces a 45-fold increase in α(2)M in the circulation led us to hypothesise that α(2)M assumes an essential role in both natural and amifostine-mediated radioprotection in the rat. In the present work we examined the activation of cytoprotective mechanisms in rat hepatocytes after the exogenous administration of α(2)M and amifostine. Our results showed that the IL6/JAK/STAT3 hepatoprotective signal pathway, described in a variety of liver-injury models, upregulated the α(2)M gene in amifostine-pretreated animals. In both α(2)M- and amifostine-pretreated rats we observed the activation of the Akt signalling pathways that mediate cellular survival. At the cellular level this was reflected as a significant reduction of irradiation-induced DNA damage that allowed for the rapid and complete restoration of liver mass and ultimately at the level of the whole organism the complete restoration of body weight. We conclude that the selective upregulation of α(2)M plays a central role in amifostine-provided radioprotection.

The radioprotective effect of alpha2-macroglobulin: a morphological study of rat liver.

BACKGROUND: Administration of the acute-phase protein alpha2-macroglobulin (MG) prior to total-body irradiation of rats with a 6.7 Gy (LD50) dose of X-rays exerts a radioprotective effect. MATERIAL/METHODS: MG was administered 30 min before irradiation with a 6.7 Gy (LD50) dose of X-rays. Its radioprotective efficacy was compared with that of the synthetic agent amifostine (WR-2721), a sulfhydryl compound which is currently the most effective radioprotector in clinical use. After administration of either MG or amifostine, changes in body and liver weight were recorded and histological liver sections were examined during a four-week follow-up period. RESULTS: As observed in the experimental group administered amifostine, rats that received MG prior to irradiation exhibited 100% survival and restoration of the body and liver weight to the control values. The morphological damage seen in the liver after irradiation of untreated rats was absent in both the MG- and amifostine-pretreated rats. Also, hepatocytes and granulated cells had prominent nuclei and did not exhibit major changes in volume. Dilation of the central vein was not observed. CONCLUSIONS: Administration of MG before irradiation, similar to pretreatment with amifostine, provided complete survival of experimental rats and recovery of liver weight and preserved major histological parameters of the liver.

Enhancement of tendon-bone healing of anterior cruciate ligament grafts by blockage of matrix metalloproteinases.

BACKGROUND: The use of soft-tissue grafts for anterior cruciate ligament reconstruction delays the healing process. This delay may be due to biochemical and/or biomechanical insults. We hypothesized that the blocking effect of alpha2-macroglobulin on synovial matrix metalloproteinase activity may enhance the healing of tendon graft in a bone tunnel. METHODS: The study was performed on twenty-eight healthy, skeletally mature New Zealand White rabbits. Each rabbit underwent bilateral anterior cruciate ligament reconstruction with use of the ipsilateral semitendinosus tendon. Alpha-2-macroglobulin (alpha2-macroglobulin) was injected into the knee joint in one limb, and the contralateral limb served as a control. The rabbits were killed two weeks (fourteen rabbits) or five weeks (fourteen rabbits) after the operative procedures. The presence of matrix metalloproteinases in synovial fluid, and the blocking effect of alpha2-macroglobulin on them, were determined with enzymatic assays. Healing between the tendon and the bone tunnel was assessed morphologically by determining the presence of fibrovascular tissue and collagen fibers. Healing also was assessed quantitatively by measuring the ultimate load to failure of the reconstructed complex. RESULTS: There was an increase in matrix metalloproteinases in the control group; in contrast, there was a decrease in the study group (p < 0.05). In the control specimens, the fibrovascular tissue at the bone-tendon interface had developed into dense connective tissue with poor vascularization. In the treated specimens, the bone tunnel had more areas of denser connective-tissue ingrowth. The interface tissue was more mature and contained numerous perpendicular collagen bundles (Sharpey fibers). The ultimate load to failure was significantly greater in the alpha2-macroglobulin-treated specimens than in the untreated controls at both two and five weeks. CONCLUSIONS: The present study demonstrated that alpha2-macroglobulin blockade of matrix metalloproteinases can enhance bone-tendon healing. This effect of alpha2-macroglobulin could occur through its effect solely on collagenase or on a subset of matrix metalloproteinases that are present at the healing interface.

The effect of alpha-2 macroglobulin on the healing of ruptured anterior cruciate ligament in rabbits.

To investigate the effect of modification of biological environmental conditions, one of the factors influencing the healing of anterior cruciate ligament rupture, we performed experimental anterior cruciate ligament ruptures on New Zealand rabbits. After experimental rupture, intra-articular alpha-2 macroglobulin was injected into the knees of the rabbits in the experiment group to prevent structural changes resulting from the enzymatic reactions in the ruptured anterior cruciate ligament. At the end of 10th day of the experiment, we observed that the anterior cruciate ligaments in the experiment group had retained their prerupture brightness and volume when compared with the control group in which intraarticular alpha-2 macroglobulin had not been injected. We also noted that the anterior cruciate ligaments in the experiment group had not retracted or swollen, the incision sites were regular and clean, and they did not show any signs of degeneration. In the histological examination, the anterior cruciate ligaments in the control groups showed disruption of the collagen network and a significant diminution in number of fibroblasts and fibrocytes (p <.001). At the end of this study, we concluded that the necessary conditions for the healing and repair of ruptured anterior cruciate ligament could exist if the enzymatic and biological environments were under control.

Covalent complexes of antigen and alpha(2)-macroglobulin: evidence for dramatically-increased immunogenicity.

A safe, effective, more potent adjuvant than currently available would be beneficial in developing new therapeutics and diagnostic reagents. We report here a technique for the rapid, efficient incorporation of non-proteolytic antigens into alpha(2)-macroglobulin (alpha(2)M; tradename, SynerVax), allowing us to covalently couple much larger subunit antigens to alpha(2)M than previously possible. Our goal was to determine if incorporation of HB, the monomeric form of Hepatitis B virus (HBV) surface antigen (HBsAg), into alpha(2)M would result in increased immune reactivity. Earlier attempts to immunize animals using HB did not generate significant levels of antibodies. Using HB complexes prepared with alpha(2)M we now report dramatically-increased immunogenicity of HB in BALB/c mice. Combining these soluble complexes with a depot-generating agent (alum), titers>1:1,000,000 are obtained with a single injection. This novel adjuvant technology should provide a valuable tool for the development of either prophylactic and therapeutic vaccines, or monoclonal antibodies against hitherto poorly-immunogenic subunit antigens.

Adjuvanticity of alpha 2-macroglobulin, an independent ligand for the heat shock protein receptor CD91.

We recently have identified CD91 as a receptor for the heat shock protein gp96. CD91 was identified initially as a receptor for alpha(2)-macroglobulin (alpha(2)M). Gp96 and alpha(2)M are both ligands for CD91. Because gp96-chaperoned peptides can prime CD8(+) T cell responses and are re-presented by APCs, we tested alpha(2)M for similar properties. Our studies show that alpha(2)M binds peptides in vitro and that the peptides, chaperoned by alpha(2)M, efficiently prime peptide-specific CD8(+) T cell responses in mice immunized with alpha(2)M-peptide complexes. Furthermore, peptides chaperoned by alpha(2)M, like those chaperoned by gp96, can be re-presented by CD91(+) APCs on their MHC I molecules. These studies demonstrate that alpha(2)M molecules, like the heat shock protein molecules, are T cell adjuvants that can channel exogenous Ags into the endogenous pathway of Ag presentaion. The remarkable similarities between an intracellular chaperone and an extracellular serum chaperone may have interesting physiological ramifications.

Effects of protease inhibitors and antioxidants on In vitro survival of porcine primordial germ cells.

One of the problems associated with in vitro culture of primordial germ cells (PGCs) is the large loss of cells during the initial period of culture. This study characterized the initial loss and determined the effectiveness of two classes of apoptosis inhibitors, protease inhibitors, and antioxidants on the ability of porcine PGCs to survive in culture. Results from electron microscopic analysis and in situ DNA fragmentation assay indicated that porcine PGCs rapidly undergo apoptosis when placed in culture. Additionally, alpha(2)-macroglobulin, a protease inhibitor and cytokine carrier, and N:-acetylcysteine, an antioxidant, increased the survival of PGCs in vitro. While other protease inhibitors tested did not affect survival of PGCs, all antioxidants tested improved survival of PGCs (P: < 0.05). Further results indicated that the beneficial effect of the antioxidants was critical only during the initial period of culture. Finally, it was determined that in short-term culture, in the absence of feeder layers, antioxidants could partially replace the effect(s) of growth factors and reduce apoptosis. Collectively, these results indicate that the addition of alpha(2)-macroglobulin and antioxidants can increase the number of PGCs in vitro by suppressing apoptosis.

Involvement of alpha-2-macroglobulin receptor in clearance of interleukin 8-alpha-2-macroglobulin complexes by human alveolar macrophages.

The purpose of this study was to determine if interleukin 8 (IL-8) in complex with alpha2-macroglobulin (alpha-2-M) can be taken up by human alveolar macrophages. First, we demonstrated that human alveolar macrophages have receptors for alpha-2-M but not IL-8. The binding of(125)I-labeled alpha-2-M to the cells was specific and saturable, whereas(125)I-labeled recombinant human IL-8 (rhIL-8) did not bind to macrophages. However,(125)I-rhIL-8-alpha-2-M complexes bound to macrophages, and unlabeled alpha-2-M competed for the binding. We then cultured the cells in the presence of(125)I-rhIL-8-alpha-2-M complexes,(125)I-rhIL-8 alone or buffer for 24 h. Macrophages were lysed, and the released radioactivity measured. IL-8 concentrations in supernatants and cells were also measured using an IL-8 ELISA. When the macrophages were incubated with(125)I-rhIL-8-alpha-2-M complexes there was a significant amount of IL-8 associated with the cells. However, this was not the case when the cells were incubated with(125)I- rhIL-8 alone suggesting that only these complexes were taken-up by human alveolar macrophages. Furthermore, the clearance of complexes was specifically inhibited by a monoclonal antibody against the 515-kDa subunit of the alpha-2-M receptor (alpha-2-MR) but not by an isotopic mouse IgG1. The study shows an important clearance mechanism for IL-8 in the lung.

A modified human alpha 2-macroglobulin derivative that binds tumor necrosis factor-alpha and interleukin-1 beta with high affinity in vitro and reverses lipopolysaccharide toxicity in vivo in mice.

The plasma protein alpha 2-macroglobulin (alpha 2M) has been reported to bind the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta), which play a central role in the pathogenesis of chronic inflammatory disorders, including Crohn's disease and rheumatoid arthritis. In this study, we chemically modified alpha 2M to stabilize a conformation of the protein (termed MAC, Macroglobulin Activated for Cytokine binding) with greatly increased TNF-alpha- and IL-1 beta-binding activity. The equilibrium dissociation constant (KD) for the binding of TNF-alpha to MAC was 80 +/- 20 nM, reflecting a 100-fold increase in affinity compared with native alpha 2M. To test the ability of MAC to neutralize proinflammatory cytokines in vivo, we treated mice with lipopolysaccharide (LPS) by intravenous injection. When MAC (2.5 mg) was administered by intraperitoneal injection 1 hour before the LPS, 12 of 12 mice survived and were without signs of toxicity at 5 days. None of the mice survived in the untreated control group (0/26) or in the group treated with 2.5 mg of unmodified alpha 2M (0/4). MAC also prevented the large increase in expression of inducible nitric oxide synthase in the liver, kidneys, and heart of LPS-treated mice. A novel property of MAC, compared with previously studied anticytokine agents, was its ability to reverse LPS toxicity in 12 of 24 mice when administered after the plasma level of TNF-alpha was elevated. These studies demonstrate that a naturally occurring protein, alpha 2M, can be modified so that it acquires the properties of clinically active monoclonal antibodies. Thus, MAC may have therapeutic potential in the control of chronic inflammatory disorders.

Inhibition of dopamine and choline acetyltransferase concentrations in rat CNS neurons by rat alpha 1- and alpha 2-macroglobulins.

Previous studies have implicated human alpha-2-macroglobulin (alpha2M) as a potential regulator of neuronal development and function. Rat alpha-1-macroglobulin (alpha1M) and acute-phase alpha-2-macroglobulin (alpha2M) are murine homologues of human alpha2M. In this report, we tested the effect of intracranially infused serotonin-activated rat alpha1M (5HT-alpha1M) on the concentration of dopamine (DA) in the corpus striatum in vivo and the effect of 5HT-activated rat alpha1M and alpha2M on the choline acetyltransferase (ChAT) activity upon embryonic basal forebrain neurons in culture. The results show that direct infusion of 0.65 nmole rat 5HT-alpha1M into the adult rat corpus striatum produced a consistent attenuation upon striatal DA concentrations. This decrease was particularly prominent at 5-7 days post-infusion. In addition, rat 5HT-alpha1M and rat 5HT-alpha2M, like human 5HT-alpha2M, all significantly inhibited ChAT activity of embryonic rat cerebral cortex neurons. Although normal human alpha2M and rat alpha2M were either marginally or insignificantly inhibitory in this preparation, normal rat alpha1M dose-dependently inhibited ChAT activity. These results demonstrate that monoamine-activated alpha-macroglobulins from rat depress dopaminergic and cholinergic neurotransmitter systems in the CNS, and this suggests a potential regulatory role of these alpha-macroglobulins in neurotransmitter metabolism.

Adjuvant-free in vivo targeting. Antigen delivery by alpha 2-macroglobulin enhances antibody formation.

The proteinase "inhibitor" alpha 2-macroglobulin (alpha 2M) is able to entrap and form covalent linkages with diverse proteins during a transient proteinase-activated state. These complexes are rapidly endocytosed after binding to receptors present on macrophages and other cells. We have previously shown that compared to free hen egg lysozyme (HEL), alpha 2M-complexed HEL undergoes enhanced macrophage uptake, processing, and presentation to T hybridoma clones in vitro. Inasmuch as it is not clear whether T hybridoma responses accurately reflect primary immune responses in vivo, we studied antibody production in rabbits using two Ag complexed with either human alpha 2M (H alpha 2M) or a homologous protein purified from rabbit plasma, alpha 1-macroglobulin (R alpha 1M). Pathogen-free NZW rabbits received s.c. injections with adjuvant-free preparations of free HEL or porcine pancreatic elastase (PPE), H alpha 2M-HEL-PPE complexes, R alpha 1M-HEL-PPE complexes, or mixtures of the uncomplexed proteins. Complexing the Ag to alpha 2M resulted in 10 to 500-fold higher IgG titers compared to uncomplexed controls. Injection of Ag complexed to either H alpha 2M or R alpha 1M resulted in levels of anti-HEL IgG comparable to those elicited by emulsification in CFA. Inasmuch as inflammatory proteinases such as neutrophil elastase can initiate covalent complex formation with alpha 2M, we propose that "proteinase-activated" alpha 2M may mediate receptor-enhanced Ag uptake by macrophages, resulting in augmented Ag processing and antibody production.

[The physicochemical anticomplement properties of alpha 2-macroglobulin for intravenous administration]. / Fiziko-khimicheskie antikomplementarnye svoistva al'fa 2-makroglobulina dlia vnutrivennogo vvedeniia.

Stabilized composition of alpha-2-macroglobulin retains its physico-chemical and biological properties for 1 year storage at (6 +/- 2) degrees C. During this term it does not change its anticomplement activity, i.e. the alpha-2-macroglobulin concentrate with the stabilizer is safe for the body and ready for intravenous administration.

Intrabursal administration of protein kinase or proteinase inhibitors: effects on ovulation in the rat.

OBJECTIVE: To test the hypothesis that inhibition of protein kinase (PK) activity or proteolysis inhibits ovulation. DESIGN: Rats were injected intrabursally with protein kinase (H9 or staurosporin) or proteinase (alpha 2-macroglobulin) inhibitors and oocyte release was evaluated. SETTING: Clinical Research Laboratory, Center for Reproductive Medicine, University of Kentucky Medical Center. PARTICIPANTS: Immature rats stimulated with pregnant mare serum gonadotropin. INTERVENTIONS: Staurosporin (1 or 10 microM), H9 (1 mM), alpha 2-macroglobulin (835 microIU of activity); or vehicle was injected into the right ovarian bursa. The left ovarian bursa remained intact. Animals immediately received human chorionic gonadotropin (hCG). MAIN OUTCOME MEASURES: Analysis of oocyte release and ovarian morphology. RESULTS: Oocyte release from the inhibitor-treated side decreased for the H9 group (8.1 +/- 1.9 fewer oocytes released, P less than 0.001) and the 10 microM staurosporin group (5.5 +/- 0.6, P less than 0.001). No change in oocyte release was observed in the 1 microM staurosporin, alpha 2-macroglobulin, or vehicle injected groups. Histologic examination of vehicle treated ovaries demonstrated numerous developing corpora lutea (CL; 20.5 +/- 2.1 CL/ovary) and a lack of preovulatory follicles. In contrast, ovaries treated with PK inhibitors contained unruptured preovulatory follicles coincident with fewer forming CL (11.5 +/- 3.5 CL/ovary). CONCLUSIONS: Inhibition of PK activity in vivo suppresses ovulation, demonstrating that protein phosphorylation plays an important intermediary role in the ovulatory process.

The effect of alpha 2 macroglobulin-proteinase complexes on macrophage Ia expression in vivo.

Alpha-2-Macroglobulin (alpha 2M) is a major plasma proteinase inhibitor. It can also regulate the function of cells of the immune system, including macrophage expression of Ia antigens in tissue culture systems. The present work was done to assess the effect of alpha 2M-trypsin complexes (alpha 2M-t) on macrophage Ia expression in vivo. Bacillus Calmette-Guerin-infected mice were injected intraperitoneally with 100nM alpha 2M-t, phosphate buffered saline (PBS), or bovine serum albumin (BSA) in PBS. The peritoneal cells were harvested by lavage from 3 to 6 days after injection. Differential cell counts were performed, and macrophage Ia antigen expression determined by indirect immunofluorescence. Injection of either alpha 2M-t or BSA solutions tended to increase the number of total cells and lymphocytes harvested, without changing the number of macrophages harvested. alpha 2M-t injection reduced the proportion of macrophages which were Ia positive from 60 to 37% on day 3 after injection, and to 20% Ia positive on day 6. The reduction in Ia positive macrophages was statistically significant when compared to either PBS or BSA injected groups. In summary, in vivo exposure to alpha 2M-t can alter macrophage function. alpha 2M-proteinase complexes formed during the course of coagulation or inflammation may play a physiologic role as regulators of the immune response.

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.