Sequential events in the pathogenesis of streptococcal cell wall-induced arthritis and their modulation by bis(5-amidino-2-benzimidazolyl)methane (BABIM).

This report builds on the authors' earlier discovery of bis(5-amidino-2-benzimidazolyl)methane (BABIM) as a strong suppressive agent for streptococcal cell wall fragment-induced arthritis in the Lewis rat. As a synthetic inhibitor of trypsinlike proteases, BABIM opens up a new route to the control of inflammatory joint disease. To gain a deeper insight into the function of the compound, the authors have now studied its influence on the sequential development of the joint changes and the associated lesions in spleen and liver. Bis(5-amidino-2-benzimidazolyl)methane is shown to block acute synovitis, to retard and reduce granuloma formation in spleen and liver, to decrease neutrophilic leukocytosis, and to diminish hemopoietic hyperplasia in the bone, and thus also to mitigate the distinctive osteoclastic and chondroclastic events. The compound does not interfere with the splenic immune response, the temporary rise in hepatocytic mitotic activity, or the organ deposition of streptococcal cell walls.     

Effect of FUT-175, a new synthetic protease inhibitor, on the development of lupus nephritis in (NZB x NZW) F1 mice.

FUT-175 (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulphonate), a new synthetic protease inhibitor, was administrated to (NZB x NZB) F1 mice in order to examine its influence on the development of autoimmune diseases. A dose (400 mg/kg of body weight) of FUT-175 has both prophylactic and curative effects on the development of lupus nephritis: mice showed a significantly low percentage of proteinuria, a marked decrease in BUN levels, and the lowest degree of glomerular damages. Dexamethasone had almost the same effect as FUT-175 (400 mg/kg), but it was slightly less effective than FUT-175. These results suggest that the administration of FUT-175 may become a viable strategy for the treatment of human autoimmune diseases.     

Nephritogenic potential of sheep antibodies against glomerular basement membrane laminin in the rat

Antilaminin antibodies have been shown to bind to laminin within the glomerular basement membrane (GBM) and mesangium of experimental animals but have induced little or no glomerular injury. We used a sheep antiserum to murine Englebreth-Holm-Swarm sarcoma laminin (sheep antilaminin) to further study the potential of antilaminin antibodies to cause glomerular injury. Intravenous injections of sheep antilaminin into rats produced intense linear GBM deposits of sheep IgG but consistently failed to induce heterologous phase proteinuria as previously shown. In addition, no autologous phase injury appeared even after preimmunization with sheep IgG (N = 4) or passive administration of rat anti-sheep IgG (N = 3) (mean urine protein less than 4 mg/24 hours up to 16 days). GBM deposits of rat C3 in vivo were absent despite the ability of both sheep antilaminin and rat anti-sheep IgG to fix human and rat C3 in vitro as determined by an indirect immunofluorescent assay. In contrast, when kidneys containing sheep antilaminin were transplanted into naive recipients that were passively immunized with rat anti-sheep IgG, severe proteinuria occurred (range 7 to 109 mg/24 hours on day 2; 49 to 350 mg/24 hours on day 5 posttransplantation) in association with glomerular deposition of C3. Histological evaluation at day 5 showed a severe proliferative glomerulonephritis with infiltrating polymorphonuclear and mononuclear leukocytes. Electron microscopy showed endothelial and epithelial cell detachment from the GBM and inflammatory cell adherence to denuded GBM. Epithelial foot process effacement and cytoplasmic absorption droplets were also noted. Identical kidneys transplanted into nonimmunized recipients or immunized recipients depleted of complement had significantly less (p less than 0.05) proteinuria (nonimmunized: 5 to 18 mg/24 hours on day 2, 4 to 9 mg/24 hours on day 5; complement-depleted: 6 to 13 mg/24 hours on day 2, 4 to 27 mg/24 hours on day 5) and no glomerular complement fixation was seen in these animals. Thus, severe glomerular injury can be induced by a focused, amplified, complement-dependent immune attack on glomerular laminin. In contrast, the widespread distribution of laminin and antilaminin probably dilutes the total glomerular immune reaction and precludes effective complement fixation and glomerular injury during the autologous phase in nontransplanted kidneys. A similar explanation might account for the lack of glomerular injury in previous studies that utilized antisera to known GBM constituents.     

C3 dependent, C5 independent immune complex glomerulopathy in the mouse

This study examines the role of complement in a murine model of accelerated nonproliferative immune complex glomerulopathy. Two C5 deficient strains (DBA/2J and B10.D2oSnJ) as well as normocomplementemic mice consistently develop heavy proteinuria and glomeruli show loss of normal visceral epithelial cell architecture within 4 days of intravenous antigen administration. In contrast, animals depleted of C3 with cobra venom factor fail to develop proteinuria and retain discrete foot processes. Semiquantitative evaluation of antigen and antibody in glomeruli shows equivalent deposition in mice from all groups. The localization of these deposits, however, is different in C3-depleted mice. There is extensive accumulation of deposits along the subepithelial aspect of the glomerular basement membrane of normocomplementemic and C5 deficient mice while deposits in glomeruli of C3-depleted animals accumulate in the subendothelial region and do not cross the glomerular basement membrane. These data demonstrate that in this model, glomerular injury is dependent on complement components generated up thru C3 but not C5 or latter components. In addition, our data suggest that C3 is important in the movement of immune complexes across the glomerular basement membrane. Although the mechanism by which complement is mediating injury in this model is not known, it does not appear to involve an inflammatory cell infiltrate or the terminal complement components.     

Current perspective on antithrombin drugs

Thrombin is a multifunctional serine protease, which is involved in blood coagulation and thrombosis, inflammation and wound repair in tissue injury. Its role in the amelioration of inflammatory tissue injury has been investigated. Protease-activated cell surface receptors (PARs) when activated by thrombin result in the production of proinflammatory mediators. In the kidney, these PARs are expressed on the glomerular epithelium and the vascular endothelium. The significant impact of thrombin inhibition on the development of crescentic glomerular nephritis is discussed.     

Cyclosporin A inhibits acute serum sickness nephritis in rabbits.

The effect of the immunosuppressive agent cyclosporin A (Cy A) on the renal injury in acute serum sickness was examined in rabbits. Serum sickness was induced in 23 untreated NZW rabbits by a single intravenous injection of bovine serum albumin (BSA) 250 mg/kg with E. coli endotoxin (5 micrograms/kg): BSA was eliminated after 8.6 +/- 0.16 days (mean +/- s.e. (mean]; proteinuria occurred in 19 (84%) and glomerular proliferation in 20 (87%) rabbits. When Cy A (15 or 25 mg/kg) was given daily by intramuscular injection, starting either 2 days before or at the time of induction of acute serum sickness, proteinuria was profoundly reduced and glomerular proliferation was inhibited. Even when rabbits were first treated with Cy A (25 mg/kg) 5 days after the induction of disease proteinuria and glomerular proliferation were similarly inhibited. When the treated animals were compared with controls there were no differences in the following: time to elimination of BSA, amount or size of circulating immune complexes, fall in serum C3 at immune elimination, or deposition of immune reactants in the glomeruli. These results show that Cy A inhibits the renal injury of acute serum sickness and indicate that T cells may play a role in mediating the nephritis in this condition.     

The mechanism of action of corticosteroids on glomerular injury in acute serum sickness in rabbits.

Macrophages have recently been identified as the predominant mediators of the glomerular injury in acute serum sickness (AcSS) in rabbits. Corticosteroids have been shown to prevent this lesion, but the mechanism of this effect is unknown. As corticosteroids are potent anti-macrophage agents, the effect of prednisolone treatment (2 mg/kg/day) on glomerular macrophage accumulation and injury was assessed in rabbits developing AcSS. Eleven untreated animals all developed a proliferative endocapillary glomerulonephritis (mean 71.7 +/- 1.9 sem cells per glomerular cross section, c/gcs) with glomerular macrophage accumulation (46.3 +/- 5.7 macrophages per glomerulus, macs/glom) and proteinuria (555 +/- 379 mg/24 h). Eight animals were treated with prednisolone commencing not more than 48 h prior to immune elimination (IE). Glomerular injury was markedly attenuated with significantly less cellular proliferation (49.1 +/- 2.1 c/gcs, P less than .005), fewer macrophages within glomeruli (10.5 +/- 7.7 macs/glom, P less than .005) and minimal proteinuria (19.3 +/- 5.5 mg/24 h, P less than 0.01). Treatment did not alter the amount of circulating BSA-anti-BSA immune complex; its time of IE (11.1 +/- 0.4 days treated, 11.4 +/- 0.4 days untreated) its renal deposition (2.36 +/- 0.64 micrograms BSA/g renal cortex treated, 2.66 +/- 0.52 mg BSA/g renal cortex untreated) or its glomerular localization. These results indicate that prednisolone treatment can effectively reduce the glomerular injury of AcSS. This effect is not dependent on any alteration of immune complex formation or deposition, but involves reduction of macrophage accumulation at the inflammatory site.     

Streptococcal cell wall-induced systemic disease. Beneficial effects of trans-bis(5-amidino-2-benzimidazolyl)ethene, a novel, macrophage-directed anti-inflammatory agent.

Previously bis(5-amidino-2-benzimidazolyl)methane (BABIM) was identified as a strong inhibitor of the multisystem inflammatory disease induced in Lewis rats by injection of streptococcus group A cell wall-derived peptidoglycan polysaccharide (PG-APS). A BABIM derivative, trans-bis(5-amidino-2-benzimidazolyl)ethene (BBE), has attracted attention because of striking qualitative and quantitative differences in its activities when compared with the parent compound. BBE could control destructive tibial osteitis and necrotizing granulomatous splenitis and hepatitis, regardless if given in a preventive or curative mode. The compound had little effect on synovitis, however. BABIM, on the other hand, was active against synovitis and osteitis, but not against splenic granuloma formation. To be effective, it needed to be applied in a preventive mode. BBE caused a characteristic enlargement of PG-APS-laden splenic and hepatic macrophages suggesting that those cells represent targets of the inhibitor. BBE may be a powerful tool for the study of granulomatous lesions.     

Complement: a unique innate immune sensor for danger signals

The complement (C) inflammatory cascade is part of the phylogenetically ancient innate immune response and is crucial to our natural ability to ward off infection. It has three critical physiologic activities: (i) defending against microbial infections by triggering the generation of a membranolytic complex (C5b9 complex) at the surface of the pathogen and C fragments (named opsonins, i.e., C1q, C3b and iC3b) which interact with C cell surface receptors (CR1, CR3 and CR4) to promote phagocytosis. Soluble C anaphylatoxins (C4a, C3a and C5a) greatly control the local pro-inflammatory response through the chemotaxis and activation of leukocytes; (ii) bridging innate and adaptive immunity (essentially through C receptor type 2, CR2, expressed by B cells) and (iii) disposing of immune complexes and the products of the inflammatory injury (i.e., other danger signals, e.g., toxic cell debris and apoptotic corpses) to ensure the protection and healing of the host. The regulatory mechanisms of C are finely balanced so that, on the one hand, the deposition of C is focused on the surface of invading microorganisms and, on the other hand, the deposition of C on normal cells is limited by several key C inhibitors (e.g., CD46, CD55 and CD59). Knowledge of the unique molecular and cellular innate immunological interactions that occur in the development and resolution of pathology should facilitate the design of effective therapeutic strategies to fight selectively against intruders.     

Modulation of the immune response in pristane-induced lupus by expression of activation and inhibitory Fc receptors

Altered homeostasis in Fcgamma receptor (FcgammaR) expression has been implicated in the induction of both immune complex-mediated glomerulonephritis and autoantibody production in systemic lupus erythematosus. FcgammaRI and III are required for immune complexes to activate inflammatory cells, thereby inciting tissue injury. In contrast, FcgammaRIIB functions as a negative regulator of immune complex-mediated inflammation and autoantibody production. We investigated the role of FcgammaRI/III versus FcgammaRIIB on pristane-induced lupus in mice. FcgammaRI/III and FcgammaRIIB-deficient ((-/-)) and control ((+/+)) BALB/c mice were injected with either pristane or PBS. Proteinuria and glomerular immune deposits were evaluated 9 months after treatment and serial sera were analysed for total IgG levels and lupus-specific autoantibodies. The incidence of nephritis was higher in pristane-treated FcgammaRIIB(-/-) mice than pristane-treated FcgammaRI/III(-/-) and (+/+) mice. Hypergammaglobulinaemia and spontaneous anti-DNA/chromatin autoantibody production were associated with interleukin (IL)-6 over-expression in FcgammaRIIB(-/-) mice and were augmented further by pristane treatment when compared to both FcgammaRI/III(-/-) and (+/+) mice. Lack of either FcgammaRIIB or FcgammaRI/III had little effect on both anti-nRNP/Sm and anti-Su production induced by pristane. Our results confirm that spontaneous autoimmunity occurs in the absence of FcgammaRIIB. Moreover, the lupus-like syndrome induced by pristane in BALB/c mice was regulated by opposing activating and inhibitory FcgammaRs. Activating FcgammaRs were required for significant proteinuria and unbridled activation in the absence of FcgammaRIIB dramatically exacerbated glomerular inflammatory responses. FcgammaRIIB may be a key modulator that suppresses cell activation in the inflammatory immune response in systemic lupus erythematosus in humans.     

Enhancement of respiratory syncytial virus-induced cytopathology by trypsin, thrombin, and plasmin.

A series of proteases of diverse substrate specificity were tested for their effect on respiratory syncytial virus-induced cytopathology. Three of the enzymes, thrombin, plasmin, and trypsin, were able to augment significantly the fusion of virus-infected A549 cells. On a concentration basis, thrombin was the most active promoter, followed by plasmin and then trypsin. Hirudin, a specific thrombin inhibitor, blocked the fusion-enhancing property of thrombin, yet had no influence on the basal rate of fusion in the absence of the enzyme. By contrast, the amidine-type inhibitors of trypsin-like proteases, bis(5-amidino-2-benzimidazolyl)-methane (BABIM), blocked not only the thrombin effect, but also the fusion in the thrombin-free controls. The suppressive activity of BABIM was observed at concentrations so low as to exclude any direct inhibitory effect on thrombin itself. These results make it seem very likely that thrombin advances cell fusion by activating a BABIM-sensitive protease. Plasmin and trypsin can be expected to act in a similar manner.     

Role of serine proteases in inflammation: Bowman–Birk protease inhibitor (BBI) as a potential therapy for autoimmune diseases

Serine proteases, a sub-category of the protease family, participate in various physiologic and pathologic conditions. Serine proteases are involved in different arms of the immune system and play an important role in inflammation. They have been evaluated as therapeutic targets in several inflammatory diseases. The Bowman–Birk protease inhibitor (BBI), a soybean-derived serine protease inhibitor, is resistant to temperature and acidic conditions. These characteristics make it a good candidate for oral administration, with no major side effects. In addition, the therapeutic effect of BBI has been shown in inflammatory diseases and cancer. We have demonstrated the immunoregulatory and anti-inflammatory effects of BBI in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Here we review the role of serine proteases in inflammatory diseases, with emphasis on the potential of BBI as a novel oral therapy for multiple sclerosis.     

Macrophages in Lupus Nephritis: Exploring a potential new therapeutic avenue

Lupus nephritis (LN) is a serious complication of systemic lupus erythematosus (SLE) that occurs in about half of patients. LN is characterized by glomerular deposition of immune complexes, leading to subendothelial, mesangial and subepithelial electron dense deposits, triggering immune cell infiltration and glomerular as well as tubulointerstitial injury. Monocytes and macrophages are abundantly present in inflammatory lesions, both in glomeruli and the tubulointerstitium. Here we discuss how monocytes and macrophages are involved in this process and how monocytes and macrophages may represent specific therapeutic targets to control LN.     

Myeloperoxidase-dependent generation of hypochlorite-modified proteins in human placental tissues during normal pregnancy

Myeloperoxidase (MPO), which is released from cytoplasmic granules of activated phagocytes by a degranulation process, reacts with H(2)O(2) (generated during the oxidative burst) and chloride ions to generate hypochlorous acid/hypochlorite (HOCl/OCl(-)). HOCl, a strong oxidant, in turn reacts with proteins to form HOCl-modified proteins. The presence of these cytotoxic chloramines during inflammatory conditions, eg, atherosclerosis and glomerular and tubulointerstitial injury, suggested that chloramines are powerful oxidants that can have profound biologic effects. In the present study, immunoreactive MPO was identified in fetal membranes and the basal plate and in maternal and fetal blood cells of human placental tissues. Monocytes/macrophages represent the major cell source for MPO in human placental tissues. Immunohistochemical findings revealed that HOCl-modified proteins are present in normal human term placenta but not during the first trimester of pregnancy (Weeks 7 to 12). HOCl-modified proteins were localized in areas formed by fetally derived cells as well as maternal decidual tissues, ie, areas where fetal extravillous trophoblast cells invade the maternal tissue and stimulate the maternal immune system. HOCl-modified proteins, products of the MPO-H(2)O(2)-chloride system in vivo, were not present intracellularly, but immunoreactivity for HOCl-modified proteins was cell-associated and/or present in the extracellular matrix. Extravillous trophoblast cells, which may also exert phagocytic activities, showed no intracellular immunoreactivity for MPO or HOCl-modified proteins. The present findings indicate that the generation of HOCl-modified proteins during normal pregnancy is a physiologic rather than a pathophysiologic process.     

C3 and C4 gene expression and interferon-gamma-mediated regulation in human glomerular mesangial cells.

The glomerular mesangial cell (GMC) plays a key role in the maintenance of glomerular structure and function and in the mediation of glomerular injury. To explore the potential of this cell to produce complement and react to local inflammatory signals, we studied the synthesis and regulation of the third and fourth components of complement in cultured human GMC. Using metabolic labelling and immunoprecipitation, we found that C3 and C4 polypeptide chains were synthesized and secreted by GMC. Interferon-gamma (IFN-gamma) led to an increase in C4 protein synthesis, but not C3 synthesis. There was a corresponding increase in C4 mRNA in IFN-gamma-activated cells, but no increase in C3 mRNA, as determined by semi-quantitative polymerase chain reaction (PCR) estimation. These results demonstrate that human GMC can synthesize C3 and C4 proteins, and that regulation of expression of the C4 gene is mediated by IFN-gamma. We hypothesize that GMC production of complement could influence the clearance of immune aggregates by the kidney and the mediation of glomerular injury.     

Structural characterization of the mesangial cell type IV collagenase and enhanced expression in a model of immune complex-mediated glomerulonephritis.

Secretion of glomerular cell-derived matrix metalloproteinases (MMPs) and their specific inhibitors, TIMP-1,2, may play an important role in the turnover of the glomerular extracellular matrix under basal and pathologic conditions. A 66-68 kd MMP secreted by cultured mesangial cells (MC) with activity against Type IV collagen and gelatin was purified and shown by amino-acid sequence analysis to be identical with a Type IV collagenase/gelatinase secreted by certain transformed tumor cell lines. The expression of the mesangial MMP in vivo was limited within the kidney to a small subset of the intrinsic glomerular mesangial cell population. After induction of acute anti-Thy 1.1 glomerulonephritis, there was a large increment in the number of Type IV collagenase-secreting MC, temporally coincident with the development of mesangial hypercellularity. The expression of the MMP inhibitor protein, TIMP-1, was not changed over this period. Ultrastructural studies localized the mesangial MMP to areas of evolving mesangiolysis and at sites of glomerular basement membrane disruption. Enhanced expression of the mesangial cell-derived Type IV collagenase may contribute to the evolution of glomerular injury in this model of immune complex-mediated glomerulonephritis or may be involved in the extensive matrix remodeling process that accompanies this form of glomerular injury.     

Polyvinyl alcohol-polyvinyl pyrrolidone thin films provide local short-term release of anti-inflammatory agents post spinal cord injury

Spinal cord injury (SCI) triggers a large inflammatory response that results in exacerbated tissue damage. Locally delivering anti-inflammatory drugs could mitigate this secondary wave of degeneration. The mitogen-activated protein kinase family members p38 and c-Jun N-terminal kinase (JNK) play important roles in the inflammatory response and cell death. We propose that the use of polymer thin films, made of polyvinyl alcohol and polyvinyl pyrrolidone blends (PVA-PVP), can be used to provide local release of inhibitors to p38 and JNK post-SCI. Release studies performed in vitro confirmed the inhibitors could be released from the film for up to 7 days. The thin film was also tested for its surgical feasibility using a cervical contusion model of SCI in adult female rats. Films with or without the inhibitors were placed subdurally over the injury site immediately following SCI. Animals were sacrificed 5 days post-SCI and spinal cord tissue above and below the injury site was harvested. Additionally, films were removed for analysis. Scanning electron microscopy confirmed the anti-fouling properties of the PVA-PVP film. Tissue histology confirmed that the films themselves did not generate a large immune response, but they did compress the tissue slightly at its placement above the injury site. Finally, quantitative Western blot analysis determined the films loaded with p38 and JNK inhibitors delivered bioactive agents to the injury site and resulted in a significantly decreased amount of pro-cell death proteins. These data indicate that PVA-PVP films can be used to effectively deliver drugs to a SCI site. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.     

Patterns of glomerular injury in kidneys infiltrated by lymphoplasmacytic neoplasms

Glomerular injury may occur as a result of immune dysfunction in patients with remote lymphoplasmacytic neoplasms. Glomerular injury concurrent with direct infiltration of the kidney by lymphoplasmacytic neoplasms has been reported but is not extensively characterized. We identified 18 patients, all presenting with elevated serum creatinine and many with proteinuria, whose renal biopsies showed direct involvement of kidney by a variety of neoplasms, including chronic leukocytic leukemia/small lymphocytic lymphoma (n = 7), diffuse large B-cell lymphoma (n = 6), multiple myeloma (n = 4), or B-cell lymphoblastic lymphoma (n = 1). In 10 cases (55%), there was coexistent glomerular pathology: 5 of these cases, including glomerulonephritis with membranoproliferative glomerulonephritis-like pattern of injury (n = 4) and membranous nephropathy (n = 1), featured deposition of immune complexes; 2 demonstrated deposition of monoclonal immunoglobulin components: λ light chain amyloidosis (n = 1) and light chain deposition disease (n = 1); 2 showed minimal change disease; and, in 1 case, there was focal crescentic pauci-immune-type glomerulonephritis. In addition, 1 biopsy revealed diabetic nephropathy and 3 showed nonspecific ischemic changes. In the remaining 4 cases, there were no significant glomerular abnormalities. In 11 cases (61%), the diagnosis of lymphoproliferative disease was established following the kidney biopsy. Our study indicates that lymphoplasmacytic neoplasms may be first diagnosed in renal biopsies performed for evaluation of renal dysfunction with or without proteinuria. Concurrent glomerular injury may be a direct result of the lymphoplasmacytic disorder through a paraprotein deposition process resulting in amyloid or monoclonal immunoglobulin deposition disease, or may be caused indirectly through immune-mediated mechanisms, as in the cases of glomerulonephritis with membranoproliferative glomerulonephritis-like pattern of injury, membranous nephropathy, and possibly minimal change disease.     

Macrophage-derived cytokines amplify immune complex-triggered O2-. responses by rat alveolar macrophages.

Interleukin-1 (IL-1) and tumor necrosis factor (TNF) are monocyte macrophage-derived hormonelike regulatory proteins that participate in many physiologic and pathophysiologic processes. Several proinflammatory activities have been attributed to these cytokines, but their importance in anatomically compartmentalized inflammatory processes is unclear. The current in vitro studies have been designed to examine modulatory influences of these cytokines on O2-. responses of rat phagocytes implicated as effector cells in immune complex mediated lung injury. Purified human IL-1, recombinant human TNF (rTNF), and culture supernatant from zymosan-activated alveolar macrophages significantly amplified O2-. responses of immune complex-stimulated alveolar macrophages but did not enhance the responses of neutrophils. Equivalent concentrations of IL-1, rTNF, and alveolar macrophage culture supernatant had no direct stimulatory effect on alveolar macrophages as measured by O2-. production. Culture media from unstimulated alveolar macrophages exerted negligible effects on O2-. generation by immune complex-activated alveolar macrophages. These data indicate that O2- responses of immune complex alveolar macrophages can be enhanced by the presence of IL-1, TNF, or media from activated macrophages. It is possible that macrophage products may greatly amplify tissue injury through the enhancement of oxygen radical production.