Inhibition of HIV-1 infectivity through an innate mechanism involving naturally occurring IgM anti-leukocyte autoantibodies

In prior studies, we show that naturally occurring IgM anti-leukocyte autoantibodies (IgM-ALA) bind to CD3, CD4, CCR5, and CXCR4 receptors. These observations prompted us to determine whether IgM-ALA have a role in inhibiting HIV-1 infectivity by inhibiting viral entry into cells. We show that purified IgM, but not IgG, from individual sera of both normal and HIV-1 infected individuals is highly inhibitory (>95%) to HIV-1 viral infectivity both in vitro using PHA plus IL-2 activated PBL and in vivo using the human PBL-SCID mouse. Inhibition was observed with physiological doses of purified serum IgM and even after IgM was added 3 days postinfection in the in vitro assays. Absorbing purified serum IgM either with leukocytes or immobilized recombinant CD4 significantly decreased (>80%) the inhibitory effect on HIV-1 infectivity. IgM inhibited by >90% syncytia formation with the X4-IIIB infected SupT-1 cells indicating therefore that IgM inhibits viral attachment to core-receptors. IgM mediated anti-HIV-1 activity was highly specific as only certain IgM-ALA, obtained from human B cell clones inhibited HIV-1. IgM from certain HIV-1 infected individuals were not inhibitory to some R5-HIV-1 viral strains indicating that certain HIV-IgM may lack Abs reactive to strain specific coreceptor epitopes. These data indicate that an innate immune mechanism which is present from birth i.e., IgM-ALA, has a role in inhibiting HIV-1 viral entry into cells. Validation of this data with other in vivo models will be needed to determine whether in vivo administration or enhancement of IgM-ALA, e.g., through a vaccine, could prolong the asymptomatic state in HIV-1 infected individuals.     

Naturally occurring human IgM antibody that binds B7-DC and potentiates T cell stimulation by dendritic cells

A human IgM Ab, serum-derived human IgM 12 (sHIgM12), is identified that binds mouse and human dendritic cells (DC), inducing dramatic immunopotentiation following treatment of the mouse DC in vitro. Competition, transfection, and knockout studies identified the ligand on mouse DC as the costimulatory molecule family member B7-DC. Potent T cell responses are stimulated by Ag-pulsed DC treated with the sHIgM12 Ab in vitro and upon adoptive transfer of Ab-treated Ag-pulsed DC into animals. The multivalent structure of pentameric IgM provides the potential for cross-linking cell surface targets, endowing the soluble Abs with biological potential not normally associated with immune function. The ability of the sHIgM12 Ab to potentiate the immune response is dependent on the multimeric structure of IgM, as bivalent monomers do not retain this property. Furthermore, pretreatment of DC with IgM monomers blocks subsequent potentiation by intact IgM pentamers, an indication that cross-linking of B7-DC on the cell surface is critical for potentiation of Ag presentation. These findings imply that, in addition to known costimulatory roles, B7-DC can function as a receptor for signals delivered by cells expressing B7-DC ligands.     

Natural IgM anti-leukocyte autoantibodies attenuate excess inflammation mediated by innate and adaptive immune mechanisms involving Th-17

Little is known about the function of natural IgM autoantibodies, especially that of IgM anti-leukocyte autoantibodies (IgM-ALA). Natural IgM-ALA are present at birth and characteristically increase during inflammatory and infective conditions. Our prior clinical observations and those of other investigators showing fewer rejections in renal and cardiac allografts transplanted into recipients with high levels of IgM-ALA led us to investigate whether IgM-ALA regulate the inflammatory response. In this article, we show that IgM, in physiologic doses, inhibit proinflammatory cells from proliferating and producing IFN-γ and IL-17 in response to alloantigens (MLR), anti-CD3, and the glycolipid α-galactosyl ceramide. We showed in an IgM knockout murine model, with intact B cells and regulatory T cells, that there was more severe inflammation and loss of function in the absence of IgM after renal ischemia reperfusion injury and cardiac allograft rejection. Replenishing IgM in IgM knockout mice or increasing the levels of IgM-ALA in wild-type B6 mice significantly attenuated the inflammation in both of these inflammatory models that involve IFN-γ and IL-17. The protective effect on renal ischemia reperfusion injury was not observed using IgM preadsorbed with leukocytes to remove IgM-ALA. We provide data to show that the anti-inflammatory effect of IgM is mediated, in part, by inhibiting TLR-4-induced NF-κB translocation into the nucleus and inhibiting differentiation of activated T cells into Th-1 and Th-17 cells. These observations highlight the importance of IgM-ALA in regulating excess inflammation mediated by both innate and adaptive immune mechanisms and where the inflammatory response involves Th-17 cells that are not effectively regulated by regulatory T cells.     

Naturally occurring alkylresorcinols that mediate DNA damage and inhibit its repair

A study of di- and trihydroxyalkylbenzenes and bis(dihydroxyalkylbenzenes) revealed that several compounds were capable of both mediating Cu(2+)-dependent DNA cleavage and strongly inhibiting DNA polymerase beta. The most potent DNA polymerase beta inhibitors were bis(dihydroxyalkylbenzenes) 5 and 6; compounds 3 and 4 were also reasonably potent. The length of the alkyl substituent was found to be a critical element for DNA polymerase beta inhibition, since compounds 1 and 2 had shorter substituents than 3 and were completely inactive. Lineweaver-Burk plots revealed that 3, 4, and 6 exhibited mixed inhibition of DNA polymerase beta with respect to both activated DNA and dTTP. Unsaturated bis(dihydroxyalkylbenzene) 5 was a pure noncompetitive inhibitor with respect to both substrates and associated avidly with the enzyme whether or not it was in complex with its substrate(s). Copper(II)-mediated DNA cleavage was the most pronounced for the trihydroxyalkylbenzene 3, consistent with an earlier report [Singh, U. S., Scannell, R. T., An, H., Carter, B. J., and Hecht, S. M. (1995) J. Am. Chem. Soc. 117, 12691-12699]. Unsaturated bis(dihydroxyalkylbenzene) 5 was the next most active DNA cleaving agent, followed by the dihydroxyalkylbenzene 4. The saturated bis(dihydroxyalkylbenzene) (6) did not cleave DNA well in a cell-free system under the conditions studied but nonetheless potentiated the effects of bleomycin to the greatest extent in cell culture studies. Interestingly, compound 5 produced a reduction in the numbers of viable cells when incubated in the presence of bleomycin and a further reduction in the numbers of viable cells in the presence of both bleomycin and Cu(2+). The same effect was noted to a lesser extent for compound 3 but not for 4 or 6.     

T cell clone-specific alloantisera that inhibit or stimulate antigen-induced T cell activation

Pigeon cytochrome c-specific, IL 2-secreting T cell hybrids have been used for immunizations to generate alloantibodies against the T cell antigen receptor on these cells. The B10.A-derived cloned T cell hybrid 2B4 was emulsified in complete Freund's adjuvant and injected i.p. into several F1 strains of mice. After boosting the recipient animals with cells in incomplete Freund's adjuvant, malignant ascites developed. In the (BALB/c X AKR)F1 or (AKR X BALB/c)F1 strains, these ascites consistently contained material that specifically inhibited the antigen-induced IL 2 secretion of only the immunizing 2B4 cell. The inhibitory material was antibody that specifically bound and could be absorbed only by 2B4. A similar immunization was performed with the cell 2C2. Although this cell apparently has similar antigenic fine specificity as 2B4, high concentrations of ascites generated by 2C2 immune animals inhibited antigen-induced IL 2 release only from 2C2. At lower concentrations of ascites, this preparation synergized with antigen to increase the IL 2 release, again only from 2C2. This antibody preparation did not affect concanavalin A-induced IL 2 release. The most likely explanation for these data is that the ascites contain antibodies that react with the antigen-specific receptor on the T cell hybrids.     

Naturally occurring Tyzzer's disease in cotton-top tamarins (Saguinus oedipus)

We noted naturally occurring infection with Clostridium piliforme (Tyzzer's disease) in 2 captive-reared cotton-top tamarins (Saguinus oedipus). Spontaneous Tyzzer's disease has been reported in multiple species of laboratory, domestic, and wild animals but is extremely rare in humans and nonhuman primates. Distinct from idiopathic colitis, which is common in cotton-top tamarins, these 2 tamarins had severe, transmural, necrotizing typhlocolitis accompanied by myocarditis and hepatitis. Abundant bacteria compatible with C. piliforme, the etiologic agent of Tyzzer's disease, were present adjacent to lesions in the cecum-colon, liver, and heart. Therefore, colitis caused by C. piliforme, although rare, should be included as a differential diagnosis in cotton-top tamarins and as a cause of postnatal mortality in this species.     

Naturally occurring Sarcocystis infection in domestic cats (Felis catus)

Equine protozoal myeloencephalitis is an important neurological disease of horses in the United States. Consequently, there is an active research effort to identify hosts associated with the primary causative agent, Sarcocystis neurona. The purpose of this study was to determine whether the domestic cat (Felis catus) is a natural host for S. neurona. Muscle sections from 50 primarily free-roaming domestic cats were examined for the presence of sarcocysts. Serum from cats in this group and another group of 50 free-roaming cats were evaluated for the presence of S. neurona antibody. Sarcocysts were found in five of 50 (10%) cats, and S. neurona antibody in five of 100 (5%) cats. Morphological, molecular (including ribosomal RNA genes), and biological characterisation of these sarcocysts showed that they were not S. neurona or S. neurona-like. Sarcocysts found in the cats were identified morphologically as Sarcocystis felis, a common parasite of wild felids. The life cycle of S. felis is not known, and prior to this study, no molecular marker for S. felis existed. Although cats were found to be infected with S. felis sarcocysts, serological data provided evidence of possible infection with S. neurona as well. Further work is needed to determine the role of the domestic cat in the life cycle of S. neurona.     

Naturally occurring chlorophyll derivatives inhibit aflatoxin B1-DNA adduct formation in hepatoma cells

The inhibitory effects of four chlorophyll derivatives (chlorophyllide [Chlide] a and b and pheophorbide [Pho] a and b) on aflatoxin B1 (AFB1)-DNA adduct formation, and on the modulation of hepatic glutathione S-transferase (GST) were evaluated in murine hepatoma (Hepa-1) cells. Enzyme-linked immunosorbent assay showed that pretreatment with Chlide or Pho significantly reduced the formation of AFB1-DNA adducts, and that Pho was the most potent inhibitor. However, wash-out prior to adding AFB1 totally eliminated inhibition by Childe and partially eliminated inhibition by Pho, indicating that the inhibitory effect of Chlide, and to some extent Pho, was mediated through direct trapping of AFB1. Furthermore, spectrophotometric analysis showed that Pho treatment could increase GST activity in Hepa-1 cells. These observations indicate that the chlorophyll derivatives studied may attenuate AFB1-induced DNA damage in the Hepa-1 cell by direct trapping of AFB1. Pho provided additional protection not only by direct trapping, but also by increasing GST activity against hepatic AFB1 metabolites.     

Purines inhibit poly(ADP-ribose) polymerase activation and modulate oxidant-induced cell death.

Purines such as adenosine, inosine, and hypoxanthine are known to have potent antiinflammatory effects. These effects generally are believed to be mediated by cell surface adenosine receptors. Here we provide evidence that purines protect against oxidant-induced cell injury by inhibiting the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Upon binding to broken DNA, PARP cleaves NAD+ into nicotinamide and ADP-ribose and polymerizes the latter on nuclear acceptor proteins such as histones and PARP itself. Overactivation of PARP depletes cellular NAD+ and ATP stores and causes necrotic cell death. We have identified some purines (hypoxanthine, inosine, and adenosine) as potential endogenous PARP inhibitors. We have found that purines (hypoxanthine > inosine > adenosine) dose-dependently inhibited PARP activation in peroxynitrite-treated macrophages and also inhibited the activity of the purified PARP enzyme. Consistently with their PARP inhibitory effects, the purines also protected interferon gamma + endotoxin (IFN/LPS) -stimulated RAW macrophages from the inhibition of mitochondrial respiration and inhibited nitrite production from IFN/LPS-stimulated macrophages. We have selected hypoxanthine as the most potent cytoprotective agent and PARP inhibitor among the three purine compounds, and investigated the mechanism of its cytoprotective effect. We have found that hypoxanthine protects thymocytes from death induced by the cytotoxic oxidant peroxynitrite. In line with the PARP inhibitory effect of purines, hypoxanthine has prevented necrotic cell death while increasing caspase activity and DNA fragmentation. As previously shown with other PARP inhibitors, hypoxanthine acted proximal to mitochondrial alterations as hypoxanthine inhibited the peroxynitrite-induced mitochondrial depolarization and secondary superoxide production. Our data imply that purines may serve as endogenous PARP inhibitors. We propose that, by affecting PARP activation, purines may modulate the pattern of cell death during shock, inflammation, and reperfusion injury.     

Naturally occurring melioidosis in a colonized rhesus monkey (Macaca mulatta)

An aged wild-caught male rhesus monkey (Macaca mulatta), maintained in a research facility for 10 years, developed bilateral pelvic limb paralysis without other signs of disease. Unresponsive to therapy, the monkey was killed and necropsied. Chronic inflammation with osteolysis of thoracic vertebrae 10-13 was observed. Pseudomonas pseudomallei was cultured and identified from cerebrospinal fluid obtained at the site of the thoracic lesion. This Gram-negative bacterium can cause infection in animals and man and may remain latent for years before the appearance of clinical signs.     

Naturally occurring Yersinia enterocolitica septicemia in patas monkeys (Erythrocebus patas)

Two cases of Yersinia enterocolitica septicemia occurred in a breeding group of 22 adult patas monkeys (Erythrocebus patas). Affected animals had acute clinical signs of depression, weakness, dehydration, hypothermia, hepatomegaly and pronounced leukopenia. Both animals died a few hours after treatment was initiated. Gross necropsy findings included jaundice, fluid in body cavities, hepatomegaly, splenomegaly, multiple white foci within the liver and spleen, generalized lymph node enlargement and numerous mucosal ulcerations in the colon. Primary histopathological lesions were multifocal hepatic necrosis, splenic necrosis, chronic ulcerative enteritis and diaphragmatic myositis with necrosis and edema. Yersinia enterocolitica was cultured from the liver, spleen, lung, jejunum and rectum. Wild rodents, particularly mice, may have been a source of infection for these animals, as the monkeys were housed in a rural, indoor-outdoor facility. A preliminary culture survey showed that some clinically normal patas monkeys harbored the organism in their intestinal tracts.     

Naturally occurring Bacillus piliformis infection (Tyzzer's disease) in guinea pigs

Two juvenile male Hartley guinea pigs (Cavia porcellus) were found dead 36 hours after receipt from a commercial source. Both guinea pigs had dependent, subcutaneous edema and excess serous fluid in their thoracic and abdominal cavities. Their livers were mottled and the cecal walls were reddened focally. Histopathologic exam revealed nests of Bacillus piliformis within the absorptive epithelial cells of the ileum, cecum and colon. Vegetative organisms and spore-like structures were observed in the cytoplasm of intestinal epithelial cells by electron microscopy. A diagnosis of Tyzzer's disease was made.     

Phosphatidylinositol (4,5) bisphosphate controls T cell activation by regulating T cell rigidity and organization

Here we investigate the role of Phosphatidylinositol (4,5) bisphosphate (PIP(2)) in the physiological activation of primary murine T cells by antigen presenting cells (APC) by addressing two principal challenges in PIP(2) biology. First, PIP(2) is a regulator of cytoskeletal dynamics and a substrate for second messenger generation. The relative importance of these two processes needs to be determined. Second, PIP(2) is turned over by multiple biosynthetic and metabolizing enzymes. The joint effect of these enzymes on PIP(2) distributions needs to be determined with resolution in time and space. We found that T cells express four isoforms of the principal PIP(2)-generating enzyme phosphatidylinositol 4-phosphate 5-kinase (PIP5K) with distinct spatial and temporal characteristics. In the context of a larger systems analysis of T cell signaling, these data identify the T cell/APC interface and the T cell distal pole as sites of differential PIP(2) turnover. Overexpression of different PIP5K isoforms, as corroborated by knock down and PIP(2) blockade, yielded an increase in PIP(2) levels combined with isoform-specific changes in the spatiotemporal distributions of accessible PIP(2). It rigidified the T cell, likely by impairing the inactivation of Ezrin Moesin Radixin, delayed and diminished the clustering of the T cell receptor at the cellular interface, reduced the efficiency of T cell proximal signaling and IL-2 secretion. These effects were consistently more severe for distal PIP5K isoforms. Thus spatially constrained cytoskeletal roles of PIP(2) in the control of T cell rigidity and spatiotemporal organization dominate the effects of PIP(2) on T cell activation.     

Naturally occurring human autoantibodies recognize a fetal brain antigen identified as microtubulus associated protein 1B

We have recently reported naturally occurring autoantibodies against a large fetal brain antigen (FBA). Now we describe the process of purification and identification of this particular FBA. The brains of newborn rabbits were solubilized and purified with preparative gel electrophoresis. The protein fractions were concentrated and desalted and the fractions were tested by a known positive serum. On membrane digestion of the FBA-band gave a twelve amino acid sequence that resulted in best identity score for mouse, rat and human microtubule-associated protein (MAP) 1B: a member of the microtubule-associated protein family. Monoclonal anti-MAP1B recognized a band in immunoblots of the brain homogenate and of the partially purified fractions with the same electrophoretic mobility as that recognized by a known anti-FBA positive serum. When adult rabbit brain was used as an antigen, the anti-MAP1B failed to recognize any bands on immunoblots. MAP lB has not been previously known as an autoantigen, even though many structural proteins of the neuronal cytoskeleton are known to be targets of naturally occurring autoantibodies. MAP 1B is a functionally important regulatory protein in the developing brain; thus autoantibodies against MAP1B may affect the normal development.     

CTLA-4 (CD152) can inhibit T cell activation by two different mechanisms depending on its level of cell surface expression

CTLA-4 (CD152) engagement results in down-regulation of T cell activation. Two mechanisms have been postulated to explain CTLA-4 inhibition of T cell activation: negative signaling and competitive antagonism of CD28:B7-mediated costimulation. We assessed the contributions of these two mechanisms using a panel of T cell lines expressing human CTLA-4 with mutations in the cytoplasmic region. Under conditions of B7-independent costimulation, inhibition of IL-2 production following CTLA-4 engagement required the CTLA-4 cytoplasmic region. In contrast, under B7-dependent costimulation, inhibition of IL-2 production by CTLA-4 engagement was directly proportional to CTLA-4 cell surface levels and did not require its cytoplasmic region. Thus, CTLA-4 down-regulates T cell activation by two different mechanisms-delivery of a negative signal or B7 sequestration-that are operational depending on the levels of CTLA-4 surface expression. These two mechanisms may have distinct functional outcomes: rapid inhibition of T cell activation or induction of T cell anergy.     

T cell activation via Leu-23 (CD69)

The CD69 (Leu-23) activation Ag is a phosphorylated 28 to 32-kDa disulfide-linked homodimer that is rapidly induced after lymphocyte activation. CD69 is not present on the surface of peripheral blood resting T cells, but is constitutively expressed by CD3bright thymocytes. Activation of protein kinase C (PKC) by stimulation of the TCR/CD3 or by phorbol esters directly induces CD69 expression on T cells. In the attempt to elucidate the function of CD69 we investigated the ability of the CD69 glycoprotein to transmit an activation signal. Cross-linking of CD69 by mAb induced a prolonged elevation of intracellular [Ca2+], mostly due to an influx of extracellular Ca2+. This signal alone was unable to effectively activate PKC. When PKC was simultaneously activated by PMA, stimulation of CD69 induced IL-2 and IFN-gamma gene expression, enhancement of CD25 expression, and ultimately IL-2-dependent T cell proliferation. Both CD4+ and CD8+ peripheral T cells responded to CD69-mediated activation. Stimulation of CD69 induced proliferation of thymocytes as well as peripheral T cells, but both required independent PKC activation by PMA. Cyclosporin A, which does not prevent PKC-induced CD69 expression, completely suppressed CD69-induced IL-2 and IFN-gamma gene expression. Although the signal delivered by the CD69 initiates T cell proliferation, it is unable to trigger cytotoxicity programs in CD69+-activated T cells or T cell clones.     

BTNL2, a butyrophilin-like molecule that functions to inhibit T cell activation

B7 family members regulate T cell activation and tolerance. Although butyrophilin proteins share sequence homology with the B7 molecules, it is unclear whether they have any function in immune responses. In the present study, we characterize an MHC class II gene-linked butyrophilin family member, butyrophilin-like 2 (BTNL2), the mutation of which has been recently associated with the inflammatory autoimmune diseases sarcoidosis and myositis. Mouse BTNL2 is a type I transmembrane protein with two pairs of Ig-like domains separated by a heptad peptide sequence. BTNL2 mRNA is highly expressed in lymphoid tissues as well as in intestine. To characterize the function of BTNL2, we produced a BTNL2-Ig fusion protein. It recognized a putative receptor whose expression on B and T cells was significantly enhanced after activation. BTNL2-Ig inhibited T cell proliferation and TCR activation of NFAT, NF-kappaB, and AP-1 signaling pathways. BTNL2 is thus the first member of the butyrophilin family that regulates T cell activation, which has implications in immune diseases and immunotherapy.