The role of macrophages in the generation of T-helper cells. II. The genetic control of the macrophage-T-cell interaction for helper cell induction with soluble antigens.

Helper cell induction to nonparticle antigens in vitro requires the cooperation of T cells and macrophages, but does not occur if the macrophages are allogenic. The reasons for this were investigated. Malfunction of allogenic macrophages was excluded by cultures with their syngenic T cells; suppressor cell induction was excluded by admixture experiments. Thus, T cells and macrophages only cooperated if they were genetically similar. The genetic locus (loci) involved was mapped. Using congenic lines differing only at the H-2 complex, the genetic control of T-macrophage interaction was localized in the H-2 region. Mice with intra H-2 recombinants were used to map the T-macrophage interaction locus in the I-A region of the H-2 complex (formerly known as poly-D, L-ala-poly-L-lys. Recombinants were also used to exclude the presence of another T-macrophage locus either the K, I-B, or I-C, SS-Slp, or D regions of the H-2 complex. Genetic restrictions for T-macrophage interaction in helper cell induction was shown in mice of the H-2-k, d, b, q, s genotypes as well as in H-2 recombinants. The possible mechanisms and significance of this genetic restriction are discussed.

Nature of T-cell macrophage interaction in helper-cell induction in vitro. II. Two stages of T-helper-cell differentiation analyzed in irradiation and allophenic chimeras.

The genetic restriction in the T-cell-macrophage-like cell interaction in helper cell induction was investigated with allophenic and irradiation chimeras of various types. Using T cells from P leads to F1 chimeras, there was a restriction of cooperation with the parental haplotype accessory cells, unless the chimeric mice were repopulated with macrophages of the opposite haplotype before priming. T cells from primed or unprimed F1 leads to P chimeras only cooperated with recipient type accessory cells. These observations led to the hypothesis that there are two stages in the genesis of immunocompetence of T helper cells, one dependent on the thymus, and the other on peripheral macrophage-like cells. Purified T cells from P1 + P2 leads to F1 irradiation chimeras behaved in an unexpected manner in the unprimed state, preferring to cooperate with their own haplotype macrophages. This self preference was lost after antigen priming in vivo and was not noted in allophenic chimeras. This loss of self preference was restricted to the haplotypes represented in the chimeras, and did not extend to third party haplotypes. While these in vitro induced helper cells from chimeric mice show clear genetic restrictions at the T-cell macrophage-like cell interaction, there was no evidence for a matching T-B genetic restriction.

Regression of basal cell carcinoma by intralesional interferon-alpha treatment is mediated by CD95 (Apo-1/Fas)-CD95 ligand-induced suicide.

Basal cell carcinoma (BCC) is the most common skin cancer in humans, and although metastasis rarely occurs, the tumor cells are nevertheless able to invade and destroy the surrounding tissue. Intralesional injection of IFN-alpha has been found to be highly effective in inducing BCC regression by an unknown mechanism. We show that in untreated patients, BCC cells express CD95 ligand, but not the receptor, which may allow tumor expansion by averting the attack of activated CD95 receptor-positive lymphoid effector cells. The CD95 ligand of BCC cells is functional as CD95-positive cells incubated on BCC cryosections become apoptotic and are lysed. In IFN-alpha-treated patients BCC cells express not only CD95 ligand but also CD95 receptor, whereas the peritumoral infiltrate that mainly consists of CD4+ T cells predominantly contains CD95 receptor and only few CD95 ligand-positive cells. Thus, in treated patients BCC most likely regresses by committing suicide through apoptosis induction via CD95 receptor-CD95 ligand interaction.

Pathways of tumour cell killing by activated macrophages: both tumour cell membrane and nucleus can be the primary target.

Plasma membrane and nucleus can be primary targets of tumour cell killing by activated macrophages (AMø). Necrotic-type cytotoxicity with loss of membrane integrity and cytoplasmic swelling was expressed by AMø from normal and from perforin-deficient mice, indicating that perforin was not involved. Incubation with AMø consistently triggered the release of thymidine from prelabelled targets, whereas chromatin condensation and small DNA fragments were only occasionally detected. It is shown by means of Pulsed-Field Gel Electrophoresis that DNA degradation in target cells is a slowly progressing process that may stop at any time, indicating that nuclear-type killing doesnot necessarily lead to the formation of low molecular weight fragments. Neither Fas nor the p55 tumour necrosis factor receptor appear to be involved in signalling nuclear-type killing. Accordingly, AMø do mediate membrane- and nuclear-type killing but the mechanisms differ from those identified in T cell cytotoxicity.

Effect of antiretroviral therapy on viral load, CD4 cell count, and progression to acquired immunodeficiency syndrome in a community human immunodeficiency virus-infected cohort. Swiss HIV Cohort Study.

OBJECTIVE: To examine the effect of different antiretroviral treatment regimens on viral load, CD4 lymphocyte counts, and rates of progression to clinical acquired immunodeficiency syndrome events among treatment-naive human immunodeficiency virus (HIV)-infected patients enrolled in a large community cohort study. METHODS: Based in 7 outpatient clinics, the Swiss HIV Cohort Study is a cohort with national coverage. Virological, immunologic, and clinical results of 755 treatment-naive patients (median age, 36 years; 28.2% female) who initiated antiretroviral therapy between July 1, 1995, and June 30, 1997, were analyzed. Patients started undergoing monotherapy with 1 reverse transcriptase inhibitor (RTI), combination therapy with at least 2 RTIs, or highly active antiretroviral therapy (HAART) with RTIs and protease inhibitors. RESULTS: Antiretroviral treatment led to a mean reduction of viremia of 1.8 log10 copies per milliliter with HAART, 1.2 log10 copies per milliliter with RTI combination therapy, and 0.4 log10 copies per milliliter with monotherapy. Virological failure, defined as less than 1 log10 reduction per milliliter in viremia, was present in 45 (20%) patients undergoing HAART, 180 (38%) undergoing RTI combination therapy, and 47 (82%) undergoing monotherapy. The proportion of patients reaching undetectable viremia was 12% (n = 7) for monotherapy, 41% (n = 197) for RTI combination therapy, and 63% (n = 137) for HAART. Similar gains of CD4 cells were achieved with RTI combination therapy and HAART. Kaplan-Meier estimates of progression rates to a new acquired immunodeficiency syndrome event at 18 months were 13.6% (monotherapy), 4.7% (RTI combination therapy), and 3.9% (HAART). CONCLUSIONS: The rate of virological failure of antiretroviral treatments was high in this population of treatment-naive patients, even among patients receiving combination regimens. Clinical progression rates were, however, low in patients treated with RTI combination therapy and HAART.

Spontaneous hypoglycemia associated with autoimmunity specific to human insulin.

A 55-yr-old woman with a history of Graves' disease experienced attacks of postprandial hypoglycemia for 6 yr. An insulinoma could not be confirmed by repeated fasting tests and by surgical pancreas revision. Extracted pancreatic insulin was chemically normal. Fasting plasma total insulin (1.22 nM = 183 microU/ml) and proinsulin (0.48 nM) were elevated and greatly increased after oral glucose. Glucose-clamp studies revealed delayed insulin clearance. Plasma free-insulin levels were normal. Insulin-binding antibodies were detected by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay with human insulin as ligand but not with pork or beef insulins. Analysis with a modified ELISA suggested a monotypic and monoclonal human insulin autoantibody, which showed a restriction to the lambda-light chain. T-lymphocytes (predominantly helper) demonstrated increased responsiveness to beef, pork, and human insulins by proliferation assay. A T-lymphocyte line showed exclusively human insulin specificity. All this indicated cellular and humoral anti-human insulin autoimmunities. Clinically, the cause of hypoglycemia associated with elevated total insulin and proinsulin was misdiagnosed as atypical insulinoma. The study of total and free plasma insulin levels and sensitive antibody assays specific to human insulin were necessary to correctly diagnose autoimmune hypoglycemia.

The Fas receptor in HIV infection: expression on peripheral blood lymphocytes and role in the depletion of T cells.

OBJECTIVE: To analyse the role of the apoptosis-inducing Fas receptor in the depletion of CD4+ and CD8+ T cells in HIV-infected individuals. METHODS: Peripheral blood lymphocytes (PBL) obtained from HIV-infected subjects of all 1993 Centers for Disease Control and Prevention (CDC) stages and from non-infected controls were examined. A two-colour cytofluorometry was employed using monoclonal antibodies against Fas receptor (CD95) in combination with the surface markers CD4, CD8, CD28, CD26 and CD45RO. CD4+ and CD8+ T-cell-enriched PBL were used as target cells to assess their susceptibility to lysis by CD4+ cytotoxic T lymphocytes (CTL) which kill via the Fas pathway. RESULTS: Fas+PBL are more elevated in HIV-infected individuals than in HIV-negative controls and increase significantly from CDC stages A to C. Whereas Fas+CD4+ and Fas-CD4+ T-cell populations decline in parallel with the progression of HIV infection, the Fas+CD8+, but not of the Fas-CD8+ fraction, significantly increases. The Fas+CD8+ lymphocytes are susceptible to Fas-mediated lysis as they are efficiently killed by Fas-ligand+CD4+CTL. CONCLUSION: The Fas receptor may contribute, but not as a unique cause, to the decline of CD4+ T cells in HIV-infected individuals. This and the significant increase of the number of Fas+ CD8+ T cells indicates that Fas-mediated immune regulation is disturbed.

A single-nucleotide polymorphism in the sterol-regulatory element-binding protein 1c gene is predictive of HIV-related hyperlipoproteinaemia.

A single-nucleotide polymorphism (3'322C/G) was identified in the gene encoding a key cholesterol/triglyceride regulator, sterol-regulatory element-binding protein 1c (SREBP-1c). Although it did not alter the amino acid sequence, SREBP-1c-3'322C/G was predictive of highly active antiretroviral therapy-related hyperlipoproteinaemia. Increases in cholesterol were less frequently associated with homozygous SREBP-1c-3'322G (genotype 22) than with heterozygous/homozygous SREBP-1c-3'322C (genotypes 11/12) and correlated with leptin and insulin increases, particularly in genotype 11/12 carriers. A functional mutation linked to SREBP-1c-3'322C/G or messenger RNA conformation differences may explain our findings.

Ultraviolet light downregulates CD95 ligand and TRAIL receptor expression facilitating actinic keratosis and squamous cell carcinoma formation.

Long-term ultraviolet light exposure of human skin epidermis in Caucasians is associated with an increased risk for the development of melanoma and nonmelanoma skin cancers. Ultraviolet radiation not only induces DNA damage in epidermal cells, it also interferes with skin homeostasis, which is maintained by a unique distribution pattern of apoptosis-inducing and apoptosis-preventing molecules. We demonstrate that, beside CD95 ligand, TRAIL and TRAIL receptors also function as important sensors in the human epidermis preserving skin integrity and preventing cell transformation. Ultraviolet irradiation extensively changes the expression pattern of some of these molecules, diminishing their sensor function. In particular, CD95 ligand and to a somewhat lesser extent TRAIL receptors are downregulated upon ultraviolet light exposure. CD95 ligand downregulation is not due to protein degradation as in situ hybridization experiments strongly support a transcriptional regulation. The downregulation of these molecules with sensor function increases the risk that aberrant cells are less efficiently eliminated. This concept is supported by the fact that the expression of these molecules is also low or absent in actinic keratosis, a precancerous state that has developed as the consequence of long-term ultraviolet exposure. Progression to invasive neoplasms is then accompanied by an upregulation of CD95 ligand and a downregulation of CD95 and of the TRAIL receptors. The high expression of CD95 ligand, TRAIL, and FLIP in squamous cell carcinoma may then contribute to the immune escape of the tumor, whereas the lack of expression of CD95 and TRAIL receptors prevents autolysis of the tumor.

Quantitative anti-p24 determinations can predict the risk of vertical transmission. Swiss HIV and Pregnancy Collaborative Study Group.

Quantitative serum antibody to p24 was evaluated as a predictor of risk of vertical transmission of human immunodeficiency virus type 1 (HIV-1) infection. HIV-positive mothers, 13 with HIV-infected children and 24 with noninfected children were investigated during pregnancy and at the time of delivery. A statistically significant difference in anti-p24 titers was found between the mothers with infected and those with noninfected children independent of whether antibodies were measured during pregnancy or at the time of delivery. High anti-p24 levels correlated with a low risk of vertical transmission, whereas low anti-p24 titers were associated with an increased risk of vertical transmission. Although the number of CD4+ T-cells was lower and neopterin and beta-2 microglobulin values were higher in the group of mothers with infected children than in the noninfected group, no statistical significance was achieved due to the small sample size.

Use of the CTL44 cell line, a derivative of CTL/L cells, to identify and quantify mouse interleukin-4 by bioassay.

The isolation and characterization of a new and excellent indicator cell line for murine interleukin-4 (IL-4) bioassays, CTL44, is described. CTL44, a subline of CTL/L cells, is vigorously responsive to murine IL-4, but hyporesponsive to IL-2, requiring > 6 ng/ml (approximately 120 U/ml) of human IL-2 or > 80 U/ml of mouse IL-2 to induce IL-2 dependent proliferation. In CTL44 both IL-4 receptor mRNA accumulation and cell surface expression are detected, whereas IL-2 receptor expression appears to be absent. CTL44 cells maintained in IL-4 containing medium grow rapidly in a non-adherent fashion, and can be stored frozen in liquid nitrogen without loss of function.

Astrocytes as antigen-presenting cells. Part II: Unlike H-2K-dependent cytotoxic T cells, H-2Ia-restricted T cells are only stimulated in the presence of interferon-gamma.

Various studies strongly suggest that astrocytes are potent immune-regulating cells. They can be activated to release prostaglandin E, interleukin-1- and interleukin-3-like factors. Cocultivation of antigen-specific T cell lines and astrocytes results in induction of Ia on astrocytes and antigen-specific proliferation of T cells. In the current study, astrocytes were found to be incapable of serving as stimulator cells when unprimed T lymphocytes were used as responders in syngeneic or allogeneic lymphocyte reactions. However, when interferon-gamma (IFN-gamma) was added, astrocytes became Ia positive and potent stimulators in both syngeneic or allogeneic lymphocyte responses. In the presence of IFN-gamma, astrocytes presented antigens to Ia-restricted T hybridoma cells; in contrast hapten was presented to Kb-restricted cytotoxic cloned T cells by astrocytes in the absence of IFN-gamma. Thus, cultured astrocytes do function directly as accessory cells in class I antigen-dependent T cell activation, whereas Ia induction by IFN-gamma is necessary to enable them to present antigen to class II antigen-restricted T cells.

The immunomodulatory role of CD4-positive cytotoxic T-lymphocytes in health and disease.

Among the CD4-positive (CD4+) T-lymphocytes a population exists which expresses cytolytic activity. These 'killer' cells belong to the T helper type 1 (Th1) subset and if activated, express Fas-ligand (FasL) which induces apoptosis in Fas-positive target cells. The major targets of these CD4+ cytotoxic T-lymphocytes (CTL) are cells of the immune system, such as T, B cells and macrophages which express Fas upon activation. Thus, CD4+ CTL play a major immunoregulatory part through the elimination of activated myeloid and lymphoid cells during and upon completion of an immune response. In certain diseases, such as in HIV-infection and some autoimmune disorders, the functional activity of CD4+ CTL is disturbed preferentially at the level of FasL-Fas interaction, further emphasizing their important immunoregulatory role. Furthermore, Fas-ligand expressing tumors can evade the attack of Fas-positive CD4+ CTL and other effector cells, thereby giving them an opportunity to expand.

Downregulation of Bcl-2, but not of Bax or Bcl-x, is associated with T lymphocyte apoptosis in HIV infection and restored by antiretroviral therapy or by interleukin 2.

The role of Bcl-2, Bax, and Bcl-x in the apoptosis of T lymphocytes in HIV-infected individuals was investigated. A strong correlation between Bcl-2 downregulation and spontaneous apoptosis has been reported by various groups in short-term cultures of CD8+ but not of CD4+ T lymphocytes. We describe a similar correlation in CD4+ T cells and provide an explanation why Bcl-2 downregulation in these cells has not been detected so far. In apoptotic cells not only Bcl-2, but also the CD4 surface receptors, are downregulated, preventing the detection of these cells in flow cytometric analysis. In contrast to Bcl-2, no correlation is detectable between Bax or Bcl-x expression and apoptosis. T lymphocytes of HIV-infected, but not of control, individuals display ex vivo a heterogeneous Bcl-2 expression pattern with a low and a high Bcl-2-expressing lymphocyte fraction. The proportion of low Bcl-2-expressing T cells correlates with a higher viral load in these individuals. Antiretroviral therapy significantly reduces the proportion of low Bcl-2-expressing lymphocytes, which is associated with a decrease in apoptosis. Bcl-2 downregulation and spontaneous apoptosis of T lymphocytes from HIV-infected individuals can be partially prevented by the exogeneous addition of IL-2, but not of IL-12, IL-4, or antibodies that prevent the CD95/CD95 ligand pathway of apoptosis.

Evaluation of accessory cell heterogeneity. III. Role of dendritic cells in the in vitro activation of the antibody response to soluble antigens.

Dendritic cells and macrophages obtained from spleen and peritoneal exudate were tested as accessory cells for the activation of lymphokine production by T cells, for supporting T-B cooperation and for the induction of antigen-specific T helper cells. Dendritic cells as well as macrophages were able to activate T cells for interleukin-2 secretion and functioned as accessory cells in T-B cooperation, but only macrophages induced T helper cells, which cooperate with B cells by a linked recognition interaction, to soluble antigens. Dendritic cell- and antigen-activated T cells also did not help B cells in the presence of Con A supernatants which contained various T cell- and B cell-stimulatory factors. The failure of dendritic cells to differentiate memory into functional T helper cells, but their efficient accessory cell function in T-B cooperation, where functional T helper cells are already present, can be best explained by a differential accessory cell requirement for T helper cell activation dependent on the differentiation stage of the T helper cell.

Differentiation markers and accessory function of murine macrophages derived from cultured bone-marrow stem cells.

Murine bone-marrow cells cultured in the presence of colony-stimulating factor from mouse-lung-conditioned medium give rise to macrophages which function as accessory cells in antigen-specific T helper cell induction. Virtually all Ia+ bone-marrow stem cell-derived macrophages express determinants encoded in the I-A subregion. A second set of macrophages bears I-A as well as I-E/C-endoced determinants. The products of the I-A and I-E/C subregion, but not those of the I-J subregion, are involved in T helper cell induction.

Comparison of antigen-specific I-region-associated cell interaction factors.

Two basic types of factors reacting with anti-I region (anti-Ia) antisera are compared, those derived from macrophage-like antigen presenting cells and others derived from T-lymphocytes, of either the suppressor or helper type. Despite the common property of reacting with anti-Ia antisera, the two sets of factors differ by many criteria. Macrophages, upon culture with antigen, release complexes of Ia antigen and a fragment of the original immunogen. This material is only produced by responder macrophages and thus appears to be a soluble Ir gene product. The genetic restriction of the T-macrophage interaction was investigated in chimeras, and it was found that the host environment as well as the donor genotype was of importance in determining restrictions, which were thus not really directed to "self." There was no evidence for intrinsic T-cell Ir genes, as nonresponder stem cells developed into responder T-cells in a (responder X nonresponder) F1 environment. However, these cells only responded in the presence of responder macrophages. Specific T-cell factors are different in nature. These all react with anti-Ia antisera, but the nature or function of the T-cell Ia is unknown. The basic structure involves a VARIAble region" responsible for antigen binding which, as it reacts with anti-idiotype antisera and anti-variable region framework antisera is an immunoglobulin variable region. There is also a "constant region," defined by its biological properties as well as by specific rabbit antisera. This two-region nature of specific factors is reminiscent of immunoglobulin structure and it is a reasonable hypothesis that the constant region is linked to the Ig cluster of genes.

Magnetic resonance imaging of children without sedation: preparation with simulation.

OBJECTIVE: It was hypothesized that a scanner simulator that replicates the magnetic resonance imaging (MRI) environment could be used to prepare pediatric subjects for successful completion of a diagnostic-quality MRI examination without pharmacological sedation. METHOD: Sixteen healthy children, 6 to 17 years of age, were matched for age and sex with 16 psychotropic medication-naive children with obsessive-compulsive disorder. Distress was measured throughout simulation and scanning procedures using heart rate and a self-report distress scale. Ten healthy children, 6 to 17 years of age, also underwent the same actual MRI scanning procedure but did not undergo the simulation scanning procedure. RESULTS: Significant decreases in heart rate and self-reported distress level were observed in all subjects during the simulator session that were maintained to the end of the actual scanner experience. All subjects successfully completed MRI examinations without chemical restraint. Subjects who were not trained in the simulator had higher heart rates and self-reported distress levels in the actual scanner than did simulation-trained subjects. CONCLUSIONS: Simulation without pharmacological sedation successfully prepared pediatric subjects in this pilot study for high-quality MRI studies. Subject preparation may be an alternative procedure to sedation for routine MRI examination in healthy and anxious children 6 years of age and older.

Lymphoproliferative responses to Borrelia burgdorferi in patients with erythema migrans, acrodermatitis chronica atrophicans, lymphadenosis benigna cutis, and morphea.

BACKGROUND AND DESIGN: Specific humoral and cell-mediated immune responses play an important role in the pathogenesis of Lyme borreliosis. Several previous studies demonstrated that a specific cellular immune response to Borrelia burgdorferi can occur independently of a diagnostic humoral response. Little is known about T-cell reactivities against B burgdorferi in early and late cutaneous manifestations of Lyme borreliosis. We studied the lymphoproliferative response of peripheral blood mononuclear cells to B burgdorferi antigen from 99 patients (25 with erythema migrans, 16 with acrodermatitis chronica atrophicans, 13 with lymphadenosis benigna cutis, and 45 with localized scleroderma) and 21 control subjects. The results are expressed as a stimulation index (SI) (mean count per minute of triplicate cultures with stimulant divided by mean count per minute without stimulant). The serum samples from all patients and control subjects were tested for antibodies to B burgdorferi by indirect immunofluorescence assay. RESULTS: The 21 healthy seronegative controls had an SI of 3.3 +/- 2.0 (mean +/- SD). Compared with that of control subjects, the SIs were significantly elevated in patients with erythema migrans (9.8 +/- 9.1), acrodermatitis chronica atrophicans (11.8 +/- 8.2), and lymphadenosis benigna cutis (7.2 +/- 6.2). The 45 patients with localized scleroderma had elevated proliferative responses, with an SI of 6.5 +/- 7.3, but these responses did not significantly differ from those of controls. Elevated titers of antibodies to B burgdorferi were present in six (24%) of 25 patients with erythema migrans, five (38%) of 13 patients with lymphadenosis benigna cutis, and 13 (29%) of 45 patients with localized scleroderma. All 16 patients with acrodermatitis chronica atrophicans had markedly elevated antibody titers. CONCLUSIONS: Our findings show that a significant lymphoproliferative response to B burgdorferi occurs in the majority of patients with cutaneous manifestations of Lyme borreliosis. The lymphocyte proliferation assay may be of diagnostic value in patients in whom Lyme borreliosis is strongly clinically suspected and who have nondiagnostic levels of antibodies against B burgdorferi.

Ia determinants on macrophages.

It is well established that T cells cannot be activated by antigen alone but only if antigen is presented in context with I region associated (Ia) determinants. As a matter of fact, antigen-presenting cells or accessory cells, which are obligatory for the induction of any type of immune response, all share the same major characteristic of Ia expression. Thus, there seems to be a direct correlation between accessory cell function and Ia expression. Originally, Ia determinants were only detected on a few cell types, B cells and macrophages being the first. However, during the course of time, more and more cells were found to be Ia positive (Ia +) and it is possible that most cells can express Ia, if appropriately induced. The regulation of Ia expression has been best studied in macrophages, where it has been found that positive induction elements include phagocytosis and gamma-interferon, while prostaglandin E and alpha-fetoprotein tend to down-regulate the expression of Ia. The regulation of Ia expression on accessory cells is thus an integrated part of immune regulation. It is highly likely, although not yet directly proven, that the Ia molecules are the products of the immune response (Ir) genes located within the major histocompatibility complex. They may even be the mediators of the Ir genes which determine whether an immune response can take place at all and/or the extent of the response. Recently, it has been shown that not all Ia + cells are able to activate every known T cell function.(ABSTRACT TRUNCATED AT 250 WORDS)