Pig but not human interferon-γ initiates human cell-mediated rejection of pig tissue in vivo

Split-thickness pig skin was transplanted on severe combined immunodeficient mice so that pig dermal microvessels spontaneously inosculated with mouse microvessels and functioned to perfuse the grafts. Pig endothelial cells in the healed grafts constitutively expressed class I and class II major histocompatibility complex molecules. Major histocompatibility complex molecule expression could be further increased by intradermal injection of pig interferon-γ (IFN-γ) but not human IFN-γ or tumor necrosis factor. Grafts injected with pig IFN-γ also developed a sparse infiltrate of mouse neutrophils and eosinophils without evidence of injury. Introduction of human peripheral blood mononuclear cells into the animals by intraperitoneal inoculation resulted in sparse perivascular mononuclear cell infiltrates in the grafts confined to the pig dermis. Injection of pig skin grafts on mice that received human peripheral blood mononuclear cells with pig IFN-γ (but not human IFN-γ or heat-inactivated pig IFN-γ) induced human CD4+ and CD8+ T cells and macrophages to more extensivley infiltrate the pig skin grafts and injure pig dermal microvessels. These findings suggest that human T cell-mediated rejection of xenotransplanted pig organs may be prevented if cellular sources of pig interferon (e.g., passenger lymphocytes) are eliminated from the graft.

The neuroprotective agent SR 57746A abrogates experimental autoimmune encephalomyelitis and impairs associated blood–brain barrier disruption: Implications for multiple sclerosis treatment

Experimental autoimmune encephalomyelitis (EAE) is a T cell autoimmune disorder that is a widely used animal model for multiple sclerosis (MS) and, as in MS, clinical signs of EAE are associated with blood–brain barrier (BBB) disruption. SR 57746A, a nonpeptide drug without classical immunosuppressive properties, efficiently protected the BBB and impaired intrathecal IgG synthesis (two conventional markers of MS exacerbation) and consequently suppressed EAE clinical signs. This compound inhibited EAE-induced spinal cord mononuclear cell invasion and normalized tumor necrosis factor α and IFN-γ mRNA expression within the spinal cord. These data suggested that pharmacological intervention aimed at inhibiting proinflammatory cytokine expression within the central nervous system provided protection against BBB disruption, the first clinical sign of EAE and probably the key point of acute MS attacks. This finding could lead to the development of a new class of compounds for oral therapy of MS, as a supplement to immunosuppressive agents.

Identification of functional domains on human interleukin 10

Interleukin 10 (IL-10) is a recently described natural endogenous immunosuppressive cytokine that has been identified in human, murine, and other organisms. Human IL-10 (hIL-10) has high homology with murine IL-10 (mIL-10) as well as with an Epstein–Barr virus genome product BCRFI. This viral IL-10 (vIL-10) shares a number of activities with hIL-10. IL-10 significantly affects chemokine biology, because human IL-10 inhibits chemokine production and is a specific chemotactic factor for CD8+ T cells. It suppresses the ability of CD4+ T cells, but not CD8+ T cells, to migrate in response to IL-8. A nonapeptide (IT9302) with complete homology to a sequence of hIL-10 located in the C-terminal portion (residues 152–160) of the cytokine was found to possess activities that mimic some of those of hIL-10. These are: (i) inhibition of IL-1β-induced IL-8 production by peripheral blood mononuclear cell, (ii) inhibition of spontaneous IL-8 production by cultured human monocytes, (iii) induction of IL-1 receptor antagonistic protein production by human monocytes, (iv) induction of chemotactic migration of CD8+ human T lymphocytes in vitro, (v) desensitization of human CD8+ T cells resulting in an unresponsiveness toward rhIL-10-induced chemotaxis, (vi) suppression of the chemotactic response of CD4+ T human lymphocytes toward IL-8, (vii) induction of IL-4 production by cultured normal human CD4+ T cells, (viii) down-regulation of tumor necrosis factor-α production by CD8+ T cells, and (ix) inhibition of class II major histocompatibility complex antigen expression on IFN-γ-stimulated human monocytes. Another nonapeptide (IT9403) close to the NH2-terminal part of hIL-10 did not reveal cytokine synthesis inhibitory properties, but proved to be a regulator of mast cell proliferation. In conclusion, we have identified two functional domains of IL-10 exerting different IL-10 like activities, an observation that suggests that relatively small segments of these signal proteins are responsible for particular biological functions.

Cytotoxic T lymphocytes and viral turnover in HIV type 1 infection

To understand the role of the immune system in limiting HIV type 1 replication, it is critical to know to what extent the rapid turnover of productively infected cells is caused by viral cytopathicity or by immune-mediated lysis. We show that uncultured peripheral blood mononuclear cells of many patients contain cytotoxic T lymphocytes (CTL) that lyse target cells—at plausible peripheral blood mononuclear cell-to-target ratios—with half-lives of less than 1 day. In 23 patients with CD4 counts ranging from 10 to 900 per μl, the average rate of CTL-mediated lysis corresponds to a target cell half-life of 0.7 day. We develop mathematical models to calculate the turnover rate of infected cells subjected to immune-mediated lysis and viral cytopathicity and to estimate the fraction of cells that are killed by CTL as opposed to virus. The models provide new interpretations of drug treatment dynamics and explain why the observed rate of virus decline is roughly constant for different patients. We conclude that in HIV type 1 infection, CTL-mediated lysis can reduce virus load by limiting virus production, with small effects on the half-life of infected cells.

Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation

Liver-specific and nonliver-specific methionine adenosyltransferases (MATs) are products of two genes, MAT1A and MAT2A, respectively, that catalyze the formation of S-adenosylmethionine (AdoMet), the principal biological methyl donor. Mature liver expresses MAT1A, whereas MAT2A is expressed in extrahepatic tissues and is induced during liver growth and dedifferentiation. To examine the influence of MAT1A on hepatic growth, we studied the effects of a targeted disruption of the murine MAT1A gene. MAT1A mRNA and protein levels were absent in homozygous knockout mice. At 3 months, plasma methionine level increased 776% in knockouts. Hepatic AdoMet and glutathione levels were reduced by 74 and 40%, respectively, whereas S-adenosylhomocysteine, methylthioadenosine, and global DNA methylation were unchanged. The body weight of 3-month-old knockout mice was unchanged from wild-type littermates, but the liver weight was increased 40%. The Affymetrix genechip system and Northern and Western blot analyses were used to analyze differential expression of genes. The expression of many acute phase-response and inflammatory markers, including orosomucoid, amyloid, metallothionein, Fas antigen, and growth-related genes, including early growth response 1 and proliferating cell nuclear antigen, is increased in the knockout animal. At 3 months, knockout mice are more susceptible to choline-deficient diet-induced fatty liver. At 8 months, knockout mice developed spontaneous macrovesicular steatosis and predominantly periportal mononuclear cell infiltration. Thus, absence of MAT1A resulted in a liver that is more susceptible to injury, expresses markers of an acute phase response, and displays increased proliferation.

Kinetics of cytokine expression during primary human immunodeficiency virus type 1 infection.

In the present study, we have determined the kinetics of constitutive expression of a panel of cytokines [interleukin (IL) 2, IL-4, IL-6, IL-10, interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha)] in sequential peripheral blood mononuclear cell samples from nine individuals with primary human immunodeficiency virus infection. Expression of IL-2 and IL-4 was barely detected in peripheral blood mononuclear cells. However, substantial levels of IL-2 expression were found in mononuclear cells isolated from lymph node. Expression of IL-6 was detected in only three of nine patients, and IL-6 expression was observed when transition from the acute to the chronic phase had already occurred. Expression of IL-10 and TNF-alpha was consistently observed in all patients tested, and levels of both cytokines were either stable or progressively increased over time. Similar to IL-10 and TNF-alpha, IFN-gamma expression was detected in all patients; however, in five of nine patients, IFN-gamma expression peaked very early during primary infection. The early peak in IFN-gamma expression coincided with oligoclonal expansions of CD8+ T cells in five of six patients, and CD8+ T cells mostly accounted for the expression of this cytokine. These results indicate that high levels of expression of proinflammatory cytokines are associated with primary infection and that the cytokine response during this phase of infection is strongly influenced by oligoclonal expansions of CD8+ T cells.

Inflammatory skin disease in transgenic mice that express high levels of interleukin 1 alpha in basal epidermis.

Resting epidermal keratinocytes contain large amounts of interleukin 1 (IL-1), but the function of this cytokine in the skin remains unclear. To further define the role of IL-1 in cutaneous biology, we have generated two lines of transgenic mice (TgIL-1.1 and TgIL-1.2) which overexpress IL-1 alpha in basal keratinocytes. There was high-level tissue-specific expression of transgene mRNA and protein and large quantities of IL-1 alpha were liberated into the circulation from epidermis in both lines. TgIL-1.1 mice, which had the highest level of transgene expression, developed a spontaneous skin disease characterized by hair loss, scaling, and focal inflammatory skin lesions. Histologically, nonlesional skin of these animals was characterized by hyperkeratosis and a dermal mononuclear cell infiltrate of macrophage/monocyte lineage. Inflammatory lesions were marked by a mixed cellular infiltrate, acanthosis, and, in some cases, parakeratosis. These findings confirm the concept of IL-1 as a primary cytokine, release of which is able to initiate and localize an inflammatory reaction. Furthermore, these mice provide the first definitive evidence that inflammatory mediators can be released from the epidermis to enter the systemic circulation and thereby influence, in a paracrine or endocrine fashion, a wide variety of other cell types.

Protective immune responses induced by secretion of a chimeric soluble protein from a recombinant Mycobacterium bovis bacillus Calmette-Guérin vector candidate vaccine for human immunodeficiency virus type 1 in small animals.

A recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) vector-based vaccine that secretes the V3 principal neutralizing epitope of human immunodeficiency virus (HIV) could induce immune response to the epitope and prevent the viral infection. By using the Japanese consensus sequence of HIV-1, we successfully constructed chimeric protein secretion vectors by selecting an appropriate insertion site of a carrier protein and established the principal neutralizing determinant (PND)-peptide secretion system in BCG. The recombinant BCG (rBCG)-inoculated guinea pigs were initially screened by delayed-type hypersensitivity (DTH) skin reactions to the PND peptide, followed by passive transfer of the DTH by the systemic route. Further, immunization of mice with the rBCG resulted in induction of cytotoxic T lymphocytes. The guinea pig immune antisera showed elevated titers to the PND peptide and neutralized HIVMN, and administration of serum IgG from the vaccinated guinea pigs was effective in completely blocking the HIV infection in thymus/liver transplanted severe combined immunodeficiency (SCID)/hu or SCID/PBL mice. In addition, the immune serum IgG was shown to neutralize primary field isolates of HIV that match the neutralizing sequence motif by a peripheral blood mononuclear cell-based virus neutralization assay. The data support the idea that the antigen-secreting rBCG system can be used as a tool for development of HIV vaccines.

Autoimmunity in Chagas disease cardiopathy: biological relevance of a cardiac myosin-specific epitope crossreactive to an immunodominant Trypanosoma cruzi antigen.

Heart tissue destruction in chronic Chagas disease cardiopathy (CCC) may be caused by autoimmune recognition of heart tissue by a mononuclear cell infiltrate decades after Trypanosoma cruzi infection. Indirect evidence suggests that there is antigenic crossreactivity between T. cruzi and heart tissue. As there is evidence for immune recognition of cardiac myosin in CCC, we searched for a putative myosin-crossreactive T. cruzi antigen. T. cruzi lysate immunoblots were probed with anti-cardiac myosin heavy chain IgG antibodies (AMA) affinity-purified from CCC or asymptomatic Chagas disease patient-seropositive sera. A 140/116-kDa doublet was predominantly recognized by AMA from CCC sera. Further, recombinant T. cruzi protein B13--whose native protein is also a 140- and 116-kDa double band--was identified by crossreactive AMA. Among 28 sera tested in a dot-blot assay, AMA from 100% of CCC sera but only 14% of the asymptomatic Chagas disease sera recognized B13 protein (P = 2.3 x 10(-6)). Sequence homology to B13 protein was found at positions 8-13 and 1442-1447 of human cardiac myosin heavy chain. Competitive ELISA assays that used the correspondent myosin synthetic peptides to inhibit serum antibody binding to B13 protein identified the heart-specific AAALDK (1442-1447) sequence of human cardiac myosin heavy chain and the homologous AAAGDK B13 sequence as the respective crossreactive epitopes. The recognition of a heart-specific T. cruzi crossreactive epitope, in strong association with the presence of chronic heart lesions, suggests the involvement of crossreactivity between cardiac myosin and B13 in the pathogenesis of CCC.

The α1 domain of HLA-G1 and HLA-G2 inhibits cytotoxicity induced by natural killer cells: Is HLA-G the public ligand for natural killer cell inhibitory receptors?

We have investigated the protective role of the membrane-bound HLA-G1 and HLA-G2 isoforms against natural killer (NK) cell cytotoxicity. For this purpose, HLA-G1 and HLA-G2 cDNAs were transfected into the HLA class I-negative human K562 cell line, a known reference target for NK lysis. The HLA-G1 protein, encoded by a full-length mRNA, presents a structure similar to that of classical HLA class I antigens. The HLA-G2 protein, deduced from an alternatively spliced transcript, consists of the α1 domain linked to the α3 domain. In this study we demonstrate that (i) HLA-G2 is present at the cell surface as a truncated class I molecule associated with β2-microglobulin; (ii) NK cytolysis, observed in peripheral blood mononuclear cells and in polyclonal CD3− CD16+ CD56+ NK cells obtained from 20 donors, is inhibited by both HLA-G1 and HLA-G2; this HLA-G-mediated inhibition is reversed by blocking HLA-G with a specific mAb; this led us to the conjecture that HLA-G is the public ligand for NK inhibitory receptors (NKIR) present in all individuals; (iii) the α1 domain common to HLA-G1 and HLA-G2 could mediate this protection from NK lysis; and (iv) when transfected into the K562 cell line, both HLA-G1 and HLA-G2 abolish lysis by the T cell leukemia NK-like YT2C2 clone due to interaction between the HLA-G isoform on the target cell surface and a membrane receptor on YT2C2. Because NKIR1 and NKIR2, known to interact with HLA-G, were undetectable on YT2C2, we conclude that a yet-unknown specific receptor for HLA-G1 and HLA-G2 is present on these cells.

TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines

We have previously identified a cellular protein kinase activity termed TAK that specifically associates with the HIV types 1 and 2 Tat proteins. TAK hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II in vitro in a manner believed to activate transcription [Herrmann, C. H. & Rice, A. P. (1995) J. Virol. 69, 1612–1620]. We show here that the catalytic subunit of TAK is a known human kinase previously named PITALRE, which is a member of the cyclin-dependent family of proteins. We also show that TAK activity is elevated upon activation of peripheral blood mononuclear cells and peripheral blood lymphocytes and upon differentiation of U1 and U937 promonocytic cell lines to macrophages. Therefore, in HIV-infected individuals TAK may be induced in T cells following activation and in macrophages following differentiation, thus contributing to high levels of viral transcription and the escape from latency of transcriptionally silent proviruses.

U94 of human herpesvirus 6 is expressed in latently infected peripheral blood mononuclear cells and blocks viral gene expression in transformed lymphocytes in culture

Human herpesvirus 6 (HHV-6) like other herpesviruses, expresses sequentially immediate early (IE), early, and late genes during lytic infection. Evidence of ability to establish latent infection has not been available, but by analogy with other herpesviruses it could be expected that IE genes that regulate and transactivate late genes would not be expressed. We report that peripheral blood mononuclear cells of healthy individuals infected with HHV-6 express the U94 gene, transcribed under IE conditions. Transcription of other IE genes (U16/17, U39, U42, U81, U89/90, U91) was not detected. To verify that U94 may play a role in the maintenance of the latent state, we derived lymphoid cell lines that stably expressed U94. HHV-6 was able to infect these cells, but viral replication was restricted. No cytopathic effect developed. Furthermore, viral transcripts were present in the first days postinfection and declined thereafter. A similar decline in the level of intracellular viral DNA also was observed. These findings are consistent with the hypothesis that the U94 gene product of HHV-6 regulates viral gene expression and enables the establishment and/or maintenance of latent infection in lymphoid cells.

CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

It has been demonstrated that CD8+ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4+ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8+ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8+ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8+ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8+ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8+ T-cell supernatants; and (iii) the CD8+ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8+ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8+ T-cell-derived HIV-suppressor factors.

The long cytoplasmic tail of gp41 is required in a cell type-dependent manner for HIV-1 envelope glycoprotein incorporation into virions

Lentiviruses, including HIV-1, have transmembrane envelope (Env) glycoproteins with cytoplasmic tails that are quite long compared with those of other retroviruses. However, mainly because of the lack of biochemical studies performed in cell types that are targets for HIV-1 infection, no clear consensus exists regarding the function of the long lentiviral Env cytoplasmic tail in virus replication. In this report, we characterize the biological and biochemical properties of an HIV-1 mutant lacking the gp41 cytoplasmic tail. We find that the gp41 cytoplasmic tail is necessary for the efficient establishment of a productive, spreading infection in the majority of T cell lines tested, peripheral blood mononuclear cells, and monocyte-derived macrophages. Biochemical studies using a high-level, transient HIV-1 expression system based on pseudotyping with the vesicular stomatitis virus glycoprotein demonstrate that in HeLa and MT-4 cells, mutant Env incorporation into virions is reduced only 3-fold relative to wild type. In contrast, gp120 levels in virions produced from a number of other T cell lines and primary macrophages are reduced more than 10-fold by the gp41 truncation. The Env incorporation defect imposed by the cytoplasmic tail truncation is not the result of increased shedding of gp120 from virions or reduced cell-surface Env expression. These results demonstrate that in the majority of T cell lines, and in primary cell types that serve as natural targets for HIV-1 infection in vivo, the gp41 cytoplasmic tail is essential for efficient Env incorporation into virions.

In vivo detection and imaging of phosphatidylserine expression during programmed cell death

One of the earliest events in programmed cell death is the externalization of phosphatidylserine, a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer. Annexin V, an endogenous human protein with a high affinity for membrane bound phosphatidylserine, can be used in vitro to detect apoptosis before other well described morphologic or nuclear changes associated with programmed cell death. We tested the ability of exogenously administered radiolabeled annexin V to concentrate at sites of apoptotic cell death in vivo. After derivatization with hydrazinonicotinamide, annexin V was radiolabeled with technetium 99m. In vivo localization of technetium 99m hydrazinonicotinamide-annexin V was tested in three models: fuminant hepatic apoptosis induced by anti-Fas antibody injection in BALB/c mice; acute rejection in ACI rats with transplanted heterotopic PVG cardiac allografts; and cyclophosphamide treatment of transplanted 38C13 murine B cell lymphomas. External radionuclide imaging showed a two- to sixfold increase in the uptake of radiolabeled annexin V at sites of apoptosis in all three models. Immunohistochemical staining of cardiac allografts for exogenously administered annexin V revealed intense staining of numerous myocytes at the periphery of mononuclear infiltrates of which only a few demonstrated positive apoptotic nuclei by the terminal deoxynucleotidyltransferase-mediated UTP end labeling method. These results suggest that radiolabeled annexin V can be used in vivo as a noninvasive means to detect and serially image tissues and organs undergoing programmed cell death.