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Biomedical subjects

Stephen Tomlinson

Publications and source records attributed to Stephen Tomlinson.

18 recordsLinked to original sources

Complement-dependent P-selectin expression and injury following ischemic stroke.

The mechanisms that contribute to inflammatory damage following ischemic stroke are poorly characterized, but studies indicate a role for both complement and P-selectin. In this study, we show that compared with wild-type mice, C3-deficient mice showed significant improvement in survival, neurological deficit, and infarct size at 24 h after middle cerebral artery occlusion and reperfusion. Furthermore, P-selectin protein expression was undetectable in the cerebral microvasculature of C3-deficient mice following reperfusion, and there was reduced neutrophil influx, reduced microthrombus formation, and increased blood flow postreperfusion in C3-deficient mice. We further investigated the use of a novel complement inhibitory protein in a therapeutic paradigm. Complement receptor 2 (CR2)-Crry inhibits complement activation at the C3 stage and targets to sites of complement activation. Treatment of normal mice with CR2-Crry at 30 min postreperfusion resulted in a similar level of protection to that seen in C3-deficient mice in all of the above-measured parameters. The data demonstrate an important role for complement in cerebrovascular thrombosis, inflammation, and injury following ischemic stroke. P-selectin expression in the cerebrovasculature, which is also implicated in cerebral ischemia and reperfusion injury, was shown to be distal to and dependent on complement activation. Data also show that a CR2-targeted approach of complement inhibition provides appropriate bioavailability in cerebral injury to enable complement inhibition at a dose that does not significantly affect systemic levels of serum complement activity, a potential benefit for stroke patients where immunosuppression would be undesirable due to significantly increased susceptibility to lung infection.

Animals↗

Defining the CD59-C9 binding interaction.

CD59 is a membrane glycoprotein that regulates formation of the cytolytic membrane attack complex (MAC or C5b-9) on host cell membranes. It functions by binding to C8 (alpha chain) and C9 after their structural rearrangement during MAC assembly. Previous studies indicated that the CD59 binding site in C9 was located within a 25-residue disulfide-bonded loop, and in C8alpha was located within a 51-residue sequence that overlaps the CD59 binding region of C9. By peptide screens and the use of peptides in binding assays, functional assays, and computer modeling and docking studies, we have identified a 6-residue sequence of human C9, spanning residues 365-371, as the primary CD59 recognition domain involved in CD59-mediated regulation of MAC formation. The data also indicate that both C8alpha and C9 bind to a similar or overlapping site on CD59. Furthermore, data from CD59-peptide docking models are consistent with the C9 binding site on CD59 located at a hydrophobic pocket, putatively identified previously by CD59 mutational and modeling studies.

Binding Sites↗

Interference with the complement system by tumor cell membrane type-1 matrix metalloproteinase plays a significant role in promoting metastasis in mice.

Neoplasms have developed strategies to protect themselves against the complement-mediated host immunity. Invasion- and metastasis-promoting membrane type-1 (MT1) matrix metalloproteinase (MMP) is strongly associated with many metastatic cancer types. The relative importance of the individual functions of MT1-MMP in metastasis was, however, unknown. We have now determined that the expression of murine MT1-MMP in murine melanoma B16F1 cells strongly increased the number of metastatic loci in the lungs of syngeneic C57BL/6 mice. In contrast, MT1-MMP did not affect the number of metastatic loci in complement-deficient C57BL/6-C3-/- mice. Our results indicated, for the first time, that the anticomplement activity of MT1-MMP played a significant role in promoting metastasis in vivo and determined the relative importance of the anticomplement activity in the total metastatic effect of this multifunctional proteolytic enzyme. We believe that our results shed additional light on the functions of MT1-MMP in cancer and clearly make this protease a promising drug target in metastatic malignancies.

Animals↗

Complement plays an important role in spinal cord injury and represents a therapeutic target for improving recovery following trauma.

Initiation of an inflammatory cascade following traumatic spinal cord injury (SCI) is thought to cause secondary injury and to adversely impact functional recovery, although the mechanisms involved are not well defined. We report on the dynamics of complement activation and deposition in the mouse spinal cord following traumatic injury, the role of complement in the development of SCI, and the characterization of a novel targeted complement inhibitor. Following traumatic injury, mice deficient in C3 had a significantly improved locomotor score when compared with wild-type controls, and analysis of their spinal cords revealed significantly more tissue sparing, with significantly less necrosis, demyelination, and neutrophil infiltration. Wild-type mice were also treated with CR2-Crry, a novel inhibitor of complement activation that targets to sites of C3 deposition. A single intravenous injection of CR2-Crry 1 hour after traumatic injury improved functional outcome and pathology to an extent similar to that seen in C3-deficient animals. CR2-Crry specifically targeted to the injured spinal cord in a distribution pattern corresponding to that seen for deposited C3. As immunosuppression is undesirable in patients following SCI, targeted CR2-Crry may provide appropriate bioavailability to treat SCI at a dose that does not significantly affect systemic levels of serum complement activity.

Animals↗

Complement-mediated mechanisms in anti-GD2 monoclonal antibody therapy of murine metastatic cancer.

The role of complement in antibody therapy of cancer is in general poorly understood. We used the EL4 syngeneic mouse model of metastatic lymphoma to investigate the role of complement in immunotherapy directed against GD2, a target of clinical relevance. IgG2a and IgM anti-GD2 therapy protected EL4-challenged mice from metastases and prolonged survival. Expression of CD59, an inhibitor of direct complement-mediated cytotoxicity (CMC), effectively protected EL4 cells from CMC in vitro but did not affect the outcome of monoclonal antibody therapy. Protection by IgG therapy was also unaffected in mice deficient in C3 or complement receptor 3 (CR3) but was almost completely abrogated in FcgammaR I/III-deficient mice. These data indicate a crucial role for antibody-dependent cell-mediated cytoxicity (ADCC). However, at lower doses of IgG, therapeutic effect was partially abrogated in C3-deficient mice, indicating complement-mediated enhancement of ADCC at limiting IgG concentration. In contrast to IgG, the therapeutic effect of IgM was completely abrogated in C3-deficient mice. High level expression of CD59 on EL4 did not influence IgM therapy, suggesting IgM functions by complement-dependent cell-mediated cytotoxicity (CDCC), a mechanism thought to be inactive against tumor cells. Thus, IgG and IgM can operate via different primary mechanisms of action, and CDCC and complement-dependent enhancement of ADCC mechanisms are operative in vivo. The effects of complement can be supplemental to other antibody-mediated mechanisms and likely have increased significance at limiting antibody concentration or low antigen density.

Animals↗

Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection.

Previous studies indicate a pivotal role for complement in mediating both local and remote injury following ischemia and reperfusion of the intestine. Here, we report on the use of a mouse model of intestinal ischemia/reperfusion injury to investigate the strategy of targeting complement inhibition to sites of complement activation by linking an iC3b/C3dg-binding fragment of mouse complement receptor 2 (CR2) to a mouse complement-inhibitory protein, Crry. We show that the novel CR2-Crry fusion protein targets sites of local and remote (lung) complement activation following intestinal ischemia and reperfusion injury and that CR2-Crry requires a 10-fold lower dose than its systemic counterpart, Crry-Ig, to provide equivalent protection from both local and remote injury. CR2-Crry has a significantly shorter serum half-life than Crry-Ig and, unlike Crry-Ig, had no significant effect on serum complement activity at minimum effective therapeutic doses. Furthermore, the minimum effective dose of Crry-Ig significantly enhanced susceptibility to infection in a mouse model of acute septic peritonitis, whereas the effect of CR2-Crry on susceptibility to infection was indistinguishable from that of PBS control. Thus, compared with systemic inhibition, CR2-mediated targeting of a complement inhibitor of activation improved bioavailability, significantly enhanced efficacy, and maintained host resistance to infection.

Animals↗

Insights into the human CD59 complement binding interface toward engineering new therapeutics.

CD59 is a 77-amino acid membrane glycoprotein that plays an important role in regulating the terminal pathway of complement by inhibiting formation of the cytolytic membrane attack complex (MAC or C5b-9). The MAC is formed by the self assembly of C5b, C6, C7, C8, and multiple C9 molecules, with CD59 functioning by binding C5b-8 and C5b-9 in the assembling complex. We performed a scanning alanine mutagenesis screen of residues 16-57, a region previously identified to contain the C8/C9 binding interface. We have also created an improved NMR model from previously published data for structural understanding of CD59. Based on the scanning mutagenesis data, refined models, and additional site-specific mutations, we identified a binding interface that is much broader than previously thought. In addition to identifying substitutions that decreased CD59 activity, a surprising number of substitutions significantly enhanced CD59 activity. Because CD59 has significant therapeutic potential for the treatment of various inflammatory conditions, we investigated further the ability to enhance CD59 activity by additional mutagenesis studies. Based on the enhanced activity of membrane-bound mutant CD59 molecules, clinically relevant soluble mutant CD59-based proteins were prepared and shown to have up to a 3-fold increase in complement inhibitory activity.

Alanine↗

Complement inhibitors targeted to the proximal tubule prevent injury in experimental nephrotic syndrome and demonstrate a key role for C5b-9.

In glomerular diseases of diverse etiologies, dysfunction of the glomerular barrier to protein passage results in proteinuria, and proteinuria is considered an independent risk factor that plays a direct role in inflammation, interstitial fibrosis, and renal failure. The mechanism by which proteinuria leads to nephrotoxic injury is unclear, but a role for complement in mediating interstitial damage appears likely. We describe a strategy for Ag-specific targeting of complement inhibitors using a single chain Ab fragment and show that complement inhibitors targeted to the tubular epithelium protect against tubulointerstitial injury and renal dysfunction in a rat model of puromycin-induced nephrosis. The targeting of systemically administered complement inhibitors markedly enhanced their efficacy and obviated the need to systemically inhibit complement, thus reducing the risk of compromising host defense and immune homeostasis. Targeted inhibition of complement activation by Crry, and of membrane attack complex (MAC) formation by CD59 was equally therapeutic, demonstrating that the MAC plays a key role in proteinuria-induced tubulointerstitial injury. CD59 activity was dependent on its being targeted to the site of complement activation, and this is the first report of specific inhibition of the MAC in vivo after systemic administration of inhibitor. The data establish the MAC is a valid target for pharmaceutical intervention in proteinuric disorders and provide an approach to investigate the role of the MAC in complement-dependent disease under clinically relevant conditions.

Animals↗

Non-proteolytic, receptor/ligand interactions associate cellular membrane type-1 matrix metalloproteinase with the complement component C1q.

Membrane type-1 matrix metalloproteinase (MT1-MMP), a prototypic member of the membrane-tethered MMP family, is an essential component of a cellular proteolysis apparatus. Recognition of protein cleavage targets followed by proteolysis is a main function of MT1-MMP. For the first time, however, we present evidence that MT1-MMP and other structurally related membrane MMPs bind C1q, the recognition unit of the first component of complement C1 that initiates activation of the classical pathway of complement. These interactions involve the catalytic domain of MT1-MMP and the C1q globular domain. In silico modeling followed by mutagenesis and the in vitro and cell-based binding studies showed that the His(171)-Glu-Lys-Gln-Ala-Asp(176) and Val(223)-Arg-Asn(224) peptide sequences of MT1-MMP are directly involved in the binding with C1q. These sequence regions are spatially distant from the active site of the protease. As a result, the catalytically active and the catalytically latent forms of cellular MT1-MMP are both efficient in binding with C1q. In agreement, despite the MT1-MMP/C1q interactions, C1q is totally resistant to MT1-MMP proteolysis. The discovery of the unconventional, receptor/ligand-like interactions of MT1-MMP with C1q, an essential component of immunity, is a significant step toward a more complete understanding of the role of this membrane-tethered protease in cancer.

Amino Acid Sequence↗

Cellular membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves C3b, an essential component of the complement system.

Neoplasms have developed numerous strategies to protect themselves against the host immune system. Membrane type-1 matrix metalloproteinase (MT1-MMP) is strongly associated with many cancer types and is up-regulated in the aggressive, metastatic neoplasms. During the past few years, there has been an increasing appreciation of the important, albeit incompletely understood, role of MT1-MMP in cancer. We have discovered, using cell-free and cell-based assays in vitro, that MT1-MMP proteolysis specifically targets C3b, an essential component of the complement propagation pathway. MT1-MMP proteolysis liberates the deposited C3 activation fragments from the cell surface. The shedding of these cell-deposited opsonins by MT1-MMP inhibits the complement cascade and protects breast carcinoma MCF7 cells from direct complement-mediated injury in the in vitro tests. The functional link associating MT1-MMP with the host immune system, heretofore unrecognized, may empower tumors with an escape mechanism that contributes to the protection against the host anti-tumor immunity as well as to the survival of invading and metastatic malignant cells in the bloodstream.

Blotting, Western↗

Inhibition of a complement regulator in vivo enhances antibody therapy in a model of mammary adenocarcinoma.

Membrane-bound complement regulatory proteins provide tumor cells with protection from antibody and complement in vitro. However, complement regulators are widely expressed on normal tissue, and inhibiting the function of complement regulatory proteins on tumor cells in vivo has not been investigated due to the absence of appropriate tumor-targeting strategies. Using a mouse model of rat mammary adenocarcinoma, we demonstrate that tumor-specific targeting of a complement regulator with a blocking antibody has functional consequences with regard to both complement deposition on tumor cells and the efficacy of monoclonal antibody therapy. Rat adenocarcinoma 13762 cells express Crry, a widely expressed rodent regulator of complement activation, and are recognized by C595 MAb, an anti-MUC1 MAb in clinical trials. Anti-rat Crry 5I2 MAb and F(ab)(2) enhanced complement deposition on C595 MAb-sensitized 13762 cells in vitro. In vivo, C595 MAb bound to 13762 tumors, albeit not specifically, but was not therapeutic when administered after tumor challenge. However, the coadministration of 5I2 MAb with C595 MAb resulted in enhanced complement deposition and significantly delayed tumor onset and reduced tumor growth; 5I2 MAb alone also enhanced complement deposition and reduced tumor growth but less effectively than when combined with C595 MAb; 5I2 MAb alone did not directly activate mouse complement, but its inhibitory effect on Crry enhanced complement deposition following complement activation by both the alternative pathway and by natural IgM reactive to 13762 cells present in mouse serum. Our proof of principle study shows that inhibiting the function of a tumor-expressed complement regulatory protein enhances immune-mediated clearance of tumor cells and improves prospects for successful immunotherapy. The results justify further research and development of targeting strategies to inhibit or downregulate complement regulatory proteins on tumor cells.

Adenocarcinoma↗

Mouse complement receptor-related gene y/p65-neutralized tumor vaccine induces antitumor activity in vivo.

Two mouse tumor cell lines, Meth A (BALB/c mouse-derived fibrosarcoma) and MM46 (C3H/He mouse-derived mammary tumor), were shown to express high levels of complement receptor-related gene y/p65 (Crry/p65), a membrane-bound complement-regulatory protein. Inhibiting the complement-regulatory activity of Crry/p65 with mAb 5D5 induced high levels of C3 deposition on in vivo tumor-derived Meth A and MM46 cells. To determine the effect of Crry/p65 blockade and increased C3 deposition on in vivo tumor growth, Meth A and MM46 cells were treated with 5D5 mAb and injected into BALB/c and C3H/He mice, respectively. Pretreating MM46 cells with 5D5 mAb significantly suppressed their tumorigenicity when injected s.c. Pretreatment with 5D5 mAb had a modest effect on Meth A s.c. tumor growth. Because complement is involved in the induction of an immune response, we investigated the effect of Crry/p65 blockade and increased C3 deposition on the immunogenicity of the tumor cells in a vaccination protocol. Vaccination of mice with irradiated Meth A cells pretreated with 5D5 mAb protected mice from subsequent challenge. In contrast, vaccination with irradiated Meth A cells without pretreatment was not protective. Survival was correlated with a high titer IgM response and specific CTL activity. These data demonstrate that the functional inhibition of Crry/p65 on tumor cells affects tumor growth and immunogenicity, and that the complement deposition resulting from this inhibition can act in concert with antitumor effector mechanisms to elicit potent antitumor immunity in vivo.

Animals↗

The effect of dexamethasone on human mucin 1 expression and antibody-dependent complement sensitivity in a prostate cancer cell line in vitro and in vivo.

Dexamethasone has been shown to up-regulate human mucin 1 (MUC1) expression in certain types of cancer cell lines in vitro, suggesting that this gluocorticoid may enhance MUC1-based immunotherapies. Here we investigated the effect of dexamethasone on MUC1 expression in the DU145 human prostate cancer cell line in terms of antibody-mediated complement-dependent cell lysis. Cells treated with 1 x 10-8 m dexamethasone in vitro expressed maximal levels of MUC1 after 6 days, with an approximately 3-fold increase over MUC1 levels on untreated cells. DU145 cells were highly resistant to lysis by anti-MUC1 antibody and complement, and their susceptibility to antibody and complement was unaffected by dexamethasone treatment. However, dexamethasone also induced expression of the complement inhibitor decay accelerating factor (DAF) on DU145 cells. Blocking or overcoming the function of DAF resulted in enhanced complement-dependent lysis of dexamethasone-treated cells with anti-MUC1 antibodies, indicating that the failure of dexamethasone to enhance the complement susceptibility of DU145 cells was caused by the up-regulated expression of DAF. We also investigated MUC1 expression in vivo and found that MUC1 expression was significantly up-regulated on tumour cells isolated from immune-deficient mice that had been injected with dexamethasone. However, in contrast to in vitro data, there was no difference between the levels of DAF expressed on tumour-derived DU145 cells isolated from either phosphate buffered saline (PBS)-treated or dexamethasone-treated mice, and tumour cells isolated from dexamethasone-treated mice were more sensitive to complement-mediated lysis. In the broad context of immunotherapy, the in vivo data support the use of dexamethasone as an adjunct treatment. Up-regulated DAF expression would not be a favourable outcome of dexamethasone treatment in terms of complement-dependent antibody therapy, but the in vivo data caution against extrapolation of in vitro data with regard to the modulation of complement inhibitors reported here and elsewhere.

Animals↗

Complement receptor 2-mediated targeting of complement inhibitors to sites of complement activation.

In a strategy to specifically target complement inhibitors to sites of complement activation and disease, recombinant fusion proteins consisting of a complement inhibitor linked to a C3 binding region of complement receptor (CR) 2 were prepared and characterized. Natural ligands for CR2 are C3 breakdown products deposited at sites of complement activation. Fusion proteins were prepared consisting of a human CR2 fragment linked to either the N terminus or C terminus of soluble forms of the membrane complement inhibitors decay accelerating factor (DAF) or CD59. The targeted complement inhibitors bound to C3-opsonized cells, and all were significantly more effective (up to 20-fold) than corresponding untargeted inhibitors at protecting target cells from complement. CR2 fusion proteins also inhibited CR3-dependent adhesion of U937 cells to C3 opsonized erythrocytes, indicating a second potential anti-inflammatory mechanism of CR2 fusion proteins, since CR3 is involved in endothelial adhesion and diapedesis of leukocytes at inflammatory sites. Finally, the in vivo validity of the targeting strategy was confirmed by the demonstration that CR2-DAF, but not soluble DAF, targets to the kidney in mouse models of lupus nephritis that are associated with renal complement deposition.

Animals↗

Expression of rat complement control protein Crry on tumor cells inhibits rat natural killer cell-mediated cytotoxicity.

Crry is a rodent membrane-bound inhibitor of complement activation and is a structural and functional analog of the human complement inhibitors decay-accelerating factor and membrane cofactor protein. We found previously that expression of rat Crry on a human tumor cell line enhances tumorigenicity in nude rats. In this study, we investigated the effect that rat Crry expressed on tumor cells has on rat cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). The expression of rat Crry on the surface of different human tumor cell lines inhibited ADCC mediated by rat natural killer (NK) cells. C3 opsonization is known to enhance NK cell-mediated cytolysis, and a potential mechanism for Crry-mediated inhibition of NK cell lysis is through Crry modulation of C3 deposition on target cells. However, the transfection of tumor cell lines with Crry enhanced their resistance to NK cell-mediated lysis in the absence of exogenous complement. The resistance of Crry-expressing tumor cells to NK cell-mediated ADCC could be reversed by treatment with anti-Crry F(ab)(2). In addition, anti-Crry F(ab)(2) enhanced the susceptibility of 13762 rat mammary adenocarcinoma cells (that endogenously express Crry) to ADCC mediated by allogeneic rat NK cells in the absence of added complement. We found no evidence that rat NK cells were a source of complement for target cell deposition during the in vitro cytolysis assay. These data suggest a novel function for rat Crry in tumor immune surveillance that may be unrelated to complement inhibition.

Adenocarcinoma↗

A tumor-expressed inhibitor of the early but not late complement lytic pathway enhances tumor growth in a rat model of human breast cancer.

Membrane-bound complement inhibitors protect host cells from inadvertent complement attack, and complement inhibitors are often up-regulated on tumors, possibly representing a selective adaptation by tumors to escape elimination by a host antitumor immune response. Relevant in vivo studies using rodent models of human cancer have been hampered by the fact that human complement inhibitors are not effective against rodent complement. Using nude rats and MCF7 cells expressing different rat complement inhibitors, a model of human breast cancer was established to investigate the role of complement and complement inhibitors in tumor progression. Expression of rat CD59, an inhibitor of the terminal cytolytic membrane attack complex of complement, had no effect on the incidence or growth rate of MCF7 tumors. In contrast, expression of rat Crry, an inhibitor of complement activation, dramatically enhanced the tumorigenicity of MCF7 cells. The expression of rat Crry on MCF7 inhibited the in vivo deposition of complement C3 fragments that serve as opsonins for receptors on phagocytes and natural killer cells. These data provide direct in vivo evidence that an inhibitor of complement activation can facilitate tumor growth by modulating C3 deposition. These data indicate an important role for complement opsonization in promoting cell-mediated antitumor immune function, a conclusion further supported by the demonstration that expression of rat Crry, but not rat CD59, on MCF7 cells inhibits rat cell-mediated cytotoxicity in vitro. Rat complement activation on MCF7 tumors was mediated by tumor-reactive antibodies present in the serum of naïve nude rats, but there was also an IgM response to MCF7 tumors, a situation with similarities to some human cancers. These data support a hypothesis that blocking complement inhibitor function on tumor cells will not only enhance monoclonal antibody-mediated immunotherapy but may also be effective at enhancing a normally ineffective humoral immune response in the absence of administered antitumor antibody.

Animals↗

Natural immunity to human African trypanosomiasis: trypanosome lytic factors and the blood incubation infectivity test.

This review focuses on the epidemiology of human African trypanosomiasis: why it occurs in humans, the current methods of surveillance, and the drugs available to treat it. Emphasis is placed on the identification of human-infective trypanosomes by the blood incubation infectivity test. This test distinguishes between trypanosomes that are non-infective for humans and those that are potentially infective. Currently the test requires incubation of parasites with human serum before injection into mice; any surviving parasites are considered human-infective. The factors in serum that kill all non-human-infective parasites are known as trypanosome lytic factors. The paper details the biochemistry of these factors and recommends standardization of the test based on current knowledge. This test can be used to screen animals with trypanosomiasis, in order to evaluate their role during endemic and epidemic human African trypanosomiasis.

Animals↗