David E. Justus

Conserved surface-exposed K/R-X-K/R motifs and net positive charge on poxvirus complement control proteins serve as putative heparin binding sites and contribute to inhibition of molecular interactions with human endothelial cells: a novel mechanism for evasion of host defense.

ABSTRACT Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP—through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells—is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.

The inflammation modulatory protein (IMP) of cowpox virus drastically diminishes the tissue damage by down-regulating cellular infiltration resulting from complement activation

Vaccinia virus (VV) and other pathogenic poxviruses encode for a complement control protein. The VV complement control protein or VCP, was one of the first soluble microbial proteins postulated to have an active role in the immunomodulation of the host defense. Since then, 2 other poxviruses, including variola virus and cowpox virus (CPV), were found to have corresponding proteins. Based upon earlier studies which demonstrated the role of the CPV complement control protein in modulating the specific tissue responses in BALB/c and congenic-matched C5-sufficient and C5-deficient mice [1], the CPV equivalent has been renamed the inflammation modulatory protein (IMP), so as to specifically reflect its function. In this study, the in vivo cellular response of mice injected with CPV or a recombinant virus lacking the IMP sequence (CPV-IMP) was examined using a connective tissue air pouch model. Microscopic examination revealed that CPV-IMP caused a significant mononuclear cell infiltration into the connective tissue and adjacent dermal tissue of the skin. To characterize IMP`s ability to regulate the observed cellular infiltration through both complement derived and non-complement derived chemotactic factors, footpad and skin connective tissue of C3 knockout mice and footpad of MIP-1 α knockout mice received injections of CPV and CPV-IMP. In comparison to the matched control, significantly greater footpad specific swelling response was seen in C3 -/- mice injected with CPV. This indicates an important role for C3 in poxvirus pathogenesis. However, MIP-1 alpha -/- mice injected with CPV-IMP recovered earlier than mice injected with CPV alone. This indicates that the function of IMP in vivo in mice with a complete repertoire of immune components is to limit cellular infiltration by down regulating the complement derived chemotactic analphylotoxins, thereby modulating the inflammatory response contributing to a diminished tissue pathology and preservation of viral habitat.

Molecular mimicry of the inflammation modulatory proteins (IMPs) of poxviruses: evasion of the inflammatory response to preserve viral habitat.

Microorganisms encode numerous immunomodulators that resemble, in structure and function, molecules captured over the millennia from their hosts [G. J. Kotwal J. Leukoc. Biol. 62, 415–429]. The vaccinia virus complement control protein (VCP) was the first soluble microbial protein to have a postulated role in the immunomodulation and evasion of host defense [G. J. Kotwal and B. Moss Nature 355, 176–179]. Purified bioactive VCP has been shown to bind to C3 and C4, block the complement cascade at multiple sites [G. J. Kotwal et al. Science 250, 827–830; R. Mckenzie, G. J. Kotwal et al. J. Infect. Dis. 166, 1245–1250] and exhibit a greater potency than the human complement 4b binding protein, C4b‐BP [G. J. Kotwal, Am. Biotech. Lab. 9,76]. The importance of this protein to poxviruses was further demonstrated in rabbits and guinea pigs through the use of recombinant virus lacking an intact DNA coding for VCP [Isaacs, G. J. Kotwal, and B. Moss Proc. Natl. Acad. Sci. 89, 628–672]. Studies in mice have shown that the homolog of VCP in cowpox virus (CPV), referred to as the inflammation modulatory protein (IMP) can, in a mouse model, significantly diminish the specific footpad swelling response [C. G. Miller, S. N. Shchelkunov, and G. J. Kotwal Virol. 229, 126–133]. To determine the precise cellular changes at the site of infection, BALB/c mice were subcutaneously injected (in the backs) with CPV or a recombinant virus lacking IMP, CPV‐IMP. Differences in histology were observed by staining the adjoining skin tissue sections with hematoxylin & eosin or by removal of the connective tissue and staining with May‐Grunwald‐Geimsa. All mice that were injected with the CPV‐IMP experienced severe tissue destruction and formation of nodular lesions compared with the mice injected with CPV. Microscopic examination indicated significantly greater cellular infiltration and destruction of skeletal muscle cells in the sections of connective tissue and adjoining skin tissue, respectively, of the mice injected with the CPV‐IMP [G. J. Kotwal et al. Mol. Cell. Biochem. in press]. Thus IMP preserves the tissue at the site of infection (viral habitat). In this review, we present evidence for molecular mimicry and evolutionary relationship to other homologs of IMP and discuss their relationships with other IMPs such as the poxviral chemokine and cytokine receptor‐like proteins. J. Leukoc. Biol. 64: 68–71; 1998.

Immunosuppression in Malaria: Effect of Hemozoin Produced by Plasmodium berghei and Plasmodium falciparum

To a considerable degree, malaria-induced immunosuppression has been attributed to an inhibition of macrophage accessory cell function. In this study hemozoin, a plasmodium hemoglobin degradation product which readily accumulates in phagocytic cells and tissues during infection, was examined for its influence on immune responses. Hemozoin-laden liver and splenic macrophages from Plasmodium berghei-infected mice, displayed accessory cell dysfunction which was likely due to hemozoin loading by these phagocytic cells. This indicated by the observation that hemozoin obtained from livers and spleens of infected mice as well as from Plasmodium falciparum cultures greatly inhibited splenic plaque-forming cell responses to sheep red blood cells. The results of the present study strongly suggest that the inhibition of macrophage accessory cell activity is due, at least in part, to the uptake and accumulation of hemozoin in their cytoplasms.

Modulation of Gamma Interferon-Induced Major Histocompatibility Complex Class II Gene Expression by Porphyromonas gingivalis Membrane Vesicles

ABSTRACT Gamma interferon (IFN-γ)-induced endothelial cells actively participate in initiating immune responses by interacting with CD4+ T cells via class II major histocompatibility complex (MHC) surface glycoproteins. Previously, Porphyromonas gingivalis membrane vesicles were shown to selectively inhibit IFN-γ-induced surface expression of HLA-DR molecules by human umbilical cord vascular endothelial cells. In this study, we demonstrated an absence of HLA-DRα mRNA from IFN-γ-induced cells in the presence of P. gingivalis membrane vesicles by using reverse transcriptase-PCR and Southern blotting. Vesicles also prevented transcription of the gene encoding class II transactivator, a transactivator protein required for IFN-γ-induced expression of MHC class II genes. In addition, the effects of vesicles on IFN-γ signal transduction involving Jak and Stat proteins were characterized by using immunoprecipitation and Western blot analyses. Jak1 and Jak2 proteins could not be detected in endothelial cells treated with membrane vesicles. Consequently, IFN-γ-induced phosphorylation of Jak1, Jak2, and Stat1α proteins was prevented. The class II-inhibitory effect of the membrane vesicles could be eliminated by heating vesicles at 100°C for 30 min or by treating them with a cysteine proteinase inhibitor. This indicates that the cysteine proteinases were most likely responsible for the absence of Jak proteins observed in vesicle-treated cells. The observed increased binding of radiolabeled IFN-γ to vesicle-treated cells suggests that vesicles may also modulate the IFN-γ interactions with the cell surface. However, no evidence was obtained demonstrating that vesicles affected the expression of IFN-γ receptors. Thus, P. gingivalis membrane vesicles apparently inhibited IFN-γ-induced MHC class II by disrupting the IFN-γ signaling transduction pathway. Vesicle-inhibited class II expression also occurred in other IFN-γ-inducible cells. This suggested that the ability of P. gingivalis membrane vesicles to modulate antigen presentation by key cells may be an important mechanism used by this particular bacterium to escape immunosurveillance, thereby favoring its colonization and invasion of host tissues.

Detection of the membrane-retained carboxy-terminal tail containing polypeptides of the amyloid precursor protein in tissue from Alzheimer's disease brain.

A major hallmark of Alzheimers disease (AD) is the presence of extracellular amyloid plaques consisting primarily of amyloid beta peptide (A beta) which is derived from a larger beta-amyloid precursor protein (APP). APP is processed via secretory and endosomal/lysosomal pathways by a group of proteases called secretases. During the processing of APP, the carboxy-terminal tail fragment has been suggested to remain within the cell. To investigate the fate of this fragment, we generated an antibody specific for a nine amino acid residue, the sequence of which was derived from the carboxy-terminal putative cytoplasmic tail of APP. Computer analysis of the entire APP gene, searching for regions of greatest antigenicity, surface probability, hydrophilicity, and presence of beta turns, indicated that the cytoplasmic tail region is an immunodominant region of APP. The peptide coupled to keyhole limpet hemocyanin protein, produced a very high titer antibody (1:1 x 10(6)). To evaluate the specificity of the antibody, immunoprecipitation of in vitro transcribed and translated DNA encoding the carboxy-terminal amino acids of APP in wheat germ extract was carried out. A single immunoprecipitated band of the correct size was seen by SDS-PAGE. The antibody was also able to specifically detect the accumulation of the stable C-terminal tail containing fragments of APP in neurites of the amygdala and hippocampus regions of the human brain tissue from AD subjects, but did not react with age-matched control normal brain tissue. The localization of the C-terminal tail of APP within the brain tissue of AD patients underscores the likely importance of the C-terminus in the pathogenesis of AD.

Mast Cell Degranulation Associated with Sequestration and Removal of Trichinella spiralis Antigens

Mast cell degranulation which occurred following the subcutaneous injection of trichinella larval antigen into trichinella-infected mice was found to be associated with antigen binding by the released granules and with the uptake of granule antigen complexes by phagocytic cells. Similar activity could be demonstrated in vitro, and in addition it was found that living intact mastocytoma and peritoneal mast cells rapidly took up fluorescein-labeled larval antigen and stored it in their cytoplasmic granules as evidenced by perigranular fluorescence. Complex formation between heparin and trichinella antigens as well as other related and unrelated parasitic antigens could readily be demonstrated using in vitro methods. These observations strongly suggest that mast cells play a significant and important supportive role in parasitic infections.

Quantitation of Immediate and Delayed Hypersensitivity Responses in Trichinella-Infected Mice

The developmental relationships between active cutaneous or local anaphylaxis, delayed hypersensitivity and worm expulsion were quantitatively examined in Trichinella-infected mice. The onset of both types of hypersensitivities and increase in sensitivity to antigen following an initial infection correlated with the onset and rate of elimination of adult worms. Mice passively sensitized with serum containing both IgG1 and IgE antibodies, but not IgG1 alone, expelled their worms at a faster rate in comparison to the controls. On the basis of these findings it is suggested that local allergic reactions mediated by anaphylactic antibodies are involved in the development of resistance to Trichinella infection.

Mast Cell Differentiation in Cultures of T Cell-Depleted Mesenteric Lymph Node Cells from Nippostrongylus brasiliensis-lnfected Mice

Lymphoid and bone marrow cells from normal and horse serum-immunized mice and lymphoid cells from Nippostrongylus brasiliensis-infected mice were cultured on monolayers of embryonic skin fibroblasts to analyze the factors which regulate the differentiation and proliferation of mast cells in vitro. Our results indicate that T cells can regulate the development of mast cells in vitro by either enhancement or suppression. In cultures of horse serum-immune spleen cells, inducer T cells are required for mast cells to develop. However, in cultures of mesenteric lymph node cells from N. brasiliensis-infected mice, inducer T cells are not required for mast cell development. This suggests that the development of mast cells may occur at discrete interleukin-3 (IL-3)-dependent and IL-3-independent stages. Mast cell precursors in the mesenteric lymph nodes of N. brasiliensis-infected mice may have already been acted on by inducer cells in vivo to become mast cell committed. While IL-3 does not appear to be required for mast cell development, these precursors do require the presence of a connective tissue microenvironment such as embryonic skin. The precursors can be inhibited from development by interferon preparations.

Vaccinia virus complement control protein ameliorates collagen-induced arthritic mice.

The main objective of this study was to investigate the therapeutic efficiency of recombinant vaccinia virus complement control protein (rVCP) on collagen‐induced arthritis (CIA) in DBA‐1/J mice. Arthritis was induced in DBA‐1J mice by injecting bovine collagen emulsified in complete Freunds adjuvant. We used rVCP to block complement activation and investigated its effect on different aspects of CIA including osteoclast formation and bone destruction. The osteoclast‐like cells were detected using immunohistochemistry. Joint destruction was studied using X‐ray of the intact knee joints. Cartilage destruction was monitored by staining the paraffin sections with toluidine blue. ELISA was used to measure the cytokine levels in the serum. Blocking complement activation in DBA/1J arthritic mice with rVCP resulted in significant inhibition of the clinical progression of the disease and reduction in joint destruction as revealed by X‐ray analysis and toluidine blue staining of the joint sections. Inhibition of complement reduced the production of proinflammatory cytokines and the number of osteoclast‐like cells in arthritic joints. In conclusion, blocking of complement in CIA by rVCP inhibits the inflammation and the formation of osteoclast‐like cells and reduces cartilage destruction.