Screening of blood donors for idiopathic CD4+ T-lymphocytopenia.

BACKGROUND: The recent recognition of idiopathic CD4+ T-lymphocytopenia (ICL) had led to concern that an unknown immunodeficiency virus may be transmissible by transfusion. STUDY DESIGN AND METHODS: To evaluate the prevalence and significance of low CD4+ values among blood donors, CD4+ data on 2030 blood donors who were negative for antibody to human immunodeficiency virus type 1 (HIV-1) were compiled. Those with CD4+ values below ICL cutoffs (< 300 CD4+ T cells/microL, or < 20% CD4+ T cells) were recalled for follow-up investigations. Serial CD4+ data on 55 homosexual men who seroconverted during prospective follow-up and data on 139 anti-HIV-1-positive blood donors initially evaluated in 1986 were reviewed as well. RESULTS: Five seronegative donors (0.25%) had absolute CD4+ counts < 300 cells per microL and/or < 20 percent. On follow-up, all five donors had immunologic findings within normal ranges, lacked HIV risk factors, and tested negative for HIV types 1 and 2 and human T-lymphotropic virus type I and II infections by antibody and polymerase chain reaction assays. Four of five donors reported transient illness shortly after their low CD4+ count donations. The median interval from HIV-1 seroconversion to an initial CD4+ value below ICL CD4+ cutoffs was 63 months for infected homosexual men. Of 139 HIV-1-infected blood donors studied 1 to 2 years after seropositive donations, 34 (24%) had CD4+ counts < 300 cells per microL and/or < 20 percent. CONCLUSION: Low CD4+ counts are rare among anti-HIV-1-negative volunteer blood donors and are generally associated with transient illnesses. If any unknown virus progresses similarly to HIV-1, CD4+ count donor screening would be a poor surrogate for its detection.

Early detection of antibody to human immunodeficiency virus type 1 by using an antigen conjugate immunoassay correlates with the presence of immunoglobulin M antibody.

Sequential plasma samples obtained from 16 individuals who seroconverted were tested for the presence of antibody to human immunodeficiency virus type 1 (HIV-1) by an antigen conjugate enzyme immunoassay (EIA) and a conventional antibody conjugate assay. In 11 of these individuals, the antigen conjugate assay detected antibody to HIV-1 2 to 11 days (mean, 5.5 days) earlier than the antibody conjugate assay. In 11 individuals, HIV-1 p24 antigen was detected a median of 6.5 days (range, 3 to 14 days) prior to positivity by the antigen conjugate EIA. Using class-specific probes, we determined the profiles of immunoglobulin M (IgM), IgG, and IgA antibodies for each individual and correlated these profiles with the EIA signals from both assays. In general, the appearance of IgM exhibited a peak at about 1 week postseroconversion, which was followed by gradually declining levels. Absorbance levels for IgG antibody, however, rose steadily and reached a plateau after 3 to 5 weeks. The levels of IgA were generally low and variable. In contrast to the progressive increase in EIA absorbance observed by the antibody conjugate assay, the antigen conjugate assay displayed a rapid early rise in absorbance which generally coincided with the transient expression of IgM antibody. The subsequent gradual increase coincided with rising levels of IgG. Because the configuration of the antigen conjugate EIA allows for an increased sensitivity for IgM compared with that for other classes of immunoglobulins, these results suggest that earlier detection of antibody to HIV-1 is due to the detection of IgM antibody during the early phase of seroconversion.

Phorbol ester-induced differentiation of U937 cells enhances attachment to fibronectin and distinctly modulates the alpha 5 beta 1 and alpha 4 beta 1 fibronectin receptors.

Monocyte interaction with fibronectin (Fn) involves specific cell surface receptors and results in cell attachment and differentiation. We have studied the regulation of these receptors using the promonocytic cell line U937 and its PMA-induced differentiation as a model. We recently reported that U937 cells interact with two sites in Fn, RGD and CS-1, via two independent receptors (O. C. Ferreira, A. Garcia-Pardo, and C. Bianco (1990) J. Exp. Med. 171, 351). In this study we have determined the effects of PMA on the interaction of U937 cells with both sites in Fn. PMA-U937 cells showed an enhanced attachment to Fn and to an RGD-containing 80-kDa Fn fragment. This enhancement paralleled a two- to threefold increase in the surface expression of the RGD-dependent receptor alpha 5 beta 1. An anti-alpha 5 beta 1 mAb completely inhibited cell adhesion to Fn and to the 80-kDa fragment. alpha 5 beta 1 receptors from untreated and PMA-treated U937 cells were isolated on 80-kDa-Sepharose columns and shown to contain a similar complex of 152/125-kDa proteins, although proteins from PMA-treated cells had slightly faster mobility on SDS-gels. In contrast, the total number of PMA-U937 cells adhering to a 38-kDa Fn fragment (containing the CS-1 site) was lower when compared to that of untreated cells. This decrease was accompanied by a 50% loss of cell surface alpha 4 beta 1, the specific receptor for CS-1. Our results indicate that differentiation of U937 cells enhances adhesion to Fn primarily by up-regulating the alpha 5 beta 1 Fn receptor. PMA also induces a down-regulation of alpha 4 beta 1, suggesting that these two integrins play different roles during monocyte differentiation.

In vivo red cell destruction by anti-Lu6.

An example is presented of an IgG1, anti-Lu6, that reacted by indirect antiglobulin test and was capable of destroying antigen-positive red cells in vivo. Two methods for the measurement of red cell survival, 51Cr labeling and flow cytometry, gave the same result: 20 percent of the test dose of Lu:6 red cells was destroyed in the first hour after injection and 80 percent in the first 24 hours. The clinical relevance of the antibody was correctly predicted by an in vitro monocyte monolayer assay. The finding that this example of anti-Lu6 was clinically significant should not be taken to mean that all antibodies directed against high-incidence Lutheran and Lutheran system-related antigens will behave similarly. When such antibodies are encountered, in vivo and/or in vitro studies to assess their clinical significance are necessary before rare blood is used for transfusion.

Carbohydrates on human Fc gamma receptors. Interdependence of the classical IgG and nonclassical lectin-binding sites on human Fc gamma RIII expressed on neutrophils.

To explore the molecular basis for the ability of aggregated IgG to block the phagocytosis by human polymorphonuclear leukocytes of Con A-opsonized E and of nonopsonized Escherichia coli with mannose-binding adhesins, we examined specific aspects of the glycoprotein structure of both the 40- to 43-kDa receptor for the Fc portion of IgG (Fc gamma RII) and the 50- to 78-kDa receptor for the Fc portion of IgG (Fc gamma RIIIPMN) from human polymorphonuclear leukocytes. Fc gamma RIIIPMN isolated by both mAb and ligand affinity chromatography, but not Fc gamma RII, binds Con A in Western blots. This binding is specifically inhibitable by alpha-methylmannoside. Digestion of Fc gamma RIIIPMN by recombinant endoglycosidase H, which is specific for high mannose-type (Con A-binding) oligosaccharides, alters the epitope recognized by mAb 3G8 in or near the IgG ligand-binding site of the receptor. Similarly, the ability of Fc gamma RIIIPMN to bind human IgG ligand is sensitive to endoglycosidase H digestion. Our data indicate that ligands other than the classical IgG opsonins can bind to human Fc gamma RIIIPMN per se through lectin-carbohydrate interactions. Furthermore, Fc gamma RIIIPMN contains a high mannose type oligosaccharide chain which contributes importantly to the integrity of the classical IgG ligand-binding site. Thus, specific glycosylations of the receptor are important for both classical and nonclassical engagement of Fc gamma RIII and may play a role in determining the properties of the ligand-binding site.

Mouse thymic virus (MTLV). A mammalian herpesvirus cytolytic for CD4+ (L3T4+) T lymphocytes.

Mouse thymic virus (MTLV; ICTV designation murid herpesvirus 3) infects developing T lymphocytes of neonatal mice, causing thymic necrosis and acute immunosuppression. Infected animals shed virus indefinitely. In the present report, two-color flow cytometric analysis of T lymphocyte subpopulations defined by the markers CD4 (L3T4) and CD8 (Lyt-2) was used to determine whether MTLV was lytic for a specific thymocyte population. At peak necrosis (8-11 d after infection), numbers of CD4+8+ cells in the thymus were reduced by 80% or more as compared with controls, and CD4+8- cells were reduced by greater than 98%. The major survivors were CD4-8+ and CD4-8- lymphocytes. These data indicate that the CD4 bearing lymphocyte is a primary target for cytolysis during MTLV infection. Possible parallels between MTLV and a newly described lymphotropic human herpesvirus, human herpesvirus 6 (HHV-6/HBLV), are also suggested.

The development of antibody to the interferon-induced indoleamine 2,3-dioxygenase and the study of the regulation of its synthesis.

Interferon (IFN) treatment of cells induces the synthesis of several new proteins. Antibody to the IFN-induced 42,000 dalton protein has been prepared and used in the study of this protein. The synthesis of the 42,000 dalton protein is dependent on de novo RNA synthesis, as its induction can be blocked if actinomycin D and the IFN are added to the cells simultaneously. Several lines of evidence suggest that the IFN-induced 42,000 dalton protein is the IFN-induced indoleamine 2,3-dioxygenase (IDO). These are as follows: 1) antibody to the 42,000 dalton protein neutralizes the activity of the IDO; 2) examination of a variety of cell lines reveals a correlation between the presence of this protein and the presence of the IDO; and 3) the induction of both the IDO and the 42,000 dalton protein is blocked under conditions in which the IFN treatment is performed in the presence of cycloheximide, and actinomycin D is added to the cells prior to the removal of the cycloheximide. A study of a variety of cell lines has revealed that the induction of the IDO occurs primarily in response to IFN-gamma. Peripheral blood mononuclear cells (PBMC) were the only cell population in which IFN-alpha and IFN-gamma were observed to produce the IDO. The IDO activity induced in IFN-alpha and IFN-gamma treated PBMC is neutralized by antibody to the 42,000 dalton protein, thus demonstrating that the IDO activity induced in these cells by IFN-alpha and IFN-gamma is mediated by the same molecule or antigenically related molecules. Fractionation of the PBMC populations reveals that it is the monocyte that produces the IFN-induced IDO.

A monoclonal antibody to a human neutrophil-specific plasma membrane antigen. Effect of the antibody on the C3bi-mediated adherence by neutrophils and expression of the antigen during myelopoiesis.

We have used mice selectively tolerized to antigens of human lymphocytes by treatment with cyclophosphamide to raise an mAb, BH2-C6, that reacts with a plasma membrane antigen specific for human neutrophils. This specificity is demonstrated by indirect immunofluorescence microscopy, cytochemical analysis of fluorescence-positive and -negative cell populations separated by flow cytometry, and by the selective, complement-mediated killing of mAb BH2-C6-treated neutrophils. Additional evidence for the neutrophil specificity of mAb BH2-C6 is shown by immunoelectron microscopy, which demonstrates a lack of reactivity with human eosinophils. Immunoblotting of SDS-PAGE-separated proteins of polymorphonuclear leukocytes with 125I-labeled BH2-C6 identifies protein with an average molecular mass of 157 kD. Binding studies show that, at saturation, neutrophils bind 214,000 molecules of 125I-BH2-C6 per cell. Addition of mAb BH2-C6 to neutrophils significantly reduces the number of C3bi-opsonized sheep erythrocytes (EIgMC3bi) bound by these cells. This reduction is partly reversed by the presence of soybean trypsin inhibitor (SBTI), indicating that at least one part of this inhibition is due to BH2-C6-stimulated secretion of a serine protease that may affect ligand binding. Cytochemical analysis of normal human bone marrow cells sorted by cytofluorimetry identifies the promyelocyte as the precursor cell that first expresses BH2-Ag on the plasma membrane. Using the leukemic cell line HL-60, we demonstrate that only inducers of granulocytic differentiation, cis-retinoic acid, and dimethyloxazolidine stimulate the expression of BH2-Ag. These results show that the expression of BH2-Ag during myelomonocytic differentiation is a property uniquely possessed by cells committed to the neutrophilic lineage.

T cell immunophenotypes and DR antigen expression in intravenous drug users. Relationship to human immunodeficiency virus serology.

Thirty-six intravenous drug users were studied for peripheral blood mononuclear cell (PBMC) immunophenotypes and human immunodeficiency virus (HIV) serological profiles. This population has a high risk for developing HIV infection. Half (18/36) were HIV antibody (Ab) negative (-) and half were positive (+). Total T lymphocytes (CD3+ and CD2+) were not different between HIV Ab-negative and HIV-positive groups. Unactivated T(CD3+DR-) cells/mm3 were less (p = 0.003) in HIV Ab-positive patients (1,467 +/- 628) compared to HIV Ab-negative patients (2,190 +/- 695). T-helper (CD4+) cells/mm3 were also less in HIV Ab-positive patients (762 +/- 344 vs. 1,161 +/- 419, p = 0.005). The most significant difference was in activated T lymphocyte CD3+DR+) percentages where the mean was 9.6% in those HIV Ab-positive compared to 3.8% in seronegatives (p less than 0.001). Preliminary studies showed that in vitro naloxone treatment of PBMC had no effects on immunophenotypic expression except for CD3+DR+ lymphocytes, where a significant reduction was observed in the HIV Ab-positive group (p = 0.022) but not in the HIV ab-negative group. These findings suggest that in certain populations, activated T cells may be an early manifestation of HIV infection.

Blockade of clearance of immune complexes by an anti-Fc gamma receptor monoclonal antibody.

Clearance of immune complexes by the mononuclear phagocyte system is important for maintaining normal host defenses against bacterial and viral assault (1), but also contributes to the pathogenesis of a variety of immune- mediated diseases . For example, removal from the circulation of IgG-coated erythrocytes and platelets by the MPS is the sine qua non of immune-mediated cytopenias (2, 3). On the other hand, abnormally decreased removal by the MPS of smaller, soluble immune complexes may play a role in the pathogenesis of immune complex-mediated tissue damage found in such autoimmune diseases as SLE (4). Although the physicochemical nature and the size of immune complexes can influence rates of clearance and sites of deposition (reviewed in 5), interactions between immune complexes and the MPS in vivo are poorly understood. The inability to directly measure binding or internalization of immune complexes by cells in the liver and spleen has made the analysis of the molecular basis of immune complex clearance very difficult . Receptors for the Fc portion of IgG (FcgammaR) and for complement (CR) undoubtedly play a role in the removal of immune complexes, but the relative importance of these receptors is not known.

Production of plasminogen activator by migrating cephalic neural crest cells.

Neural crest cells migrate extensively during embryonic development and differentiate into a wide variety of cell types. Our working hypothesis is that during migration, embryonic cells secrete proteases which modify local microenvironments, thereby facilitating directed cellular movements. In this communication, we report studies on the migration of cephalic neural crest cells in the avian embryo. We demonstrate that these cells produce high levels of the serine protease, plasminogen activator (PA), at the time of their initial migration from the neural tube and during their migration to and colonization of the developing head and neck.

Production of plasminogen activator in cultures of superior cervical ganglia and isolated Schwann cells.

Plasminogen activator has been implicated in tissue remodeling and cell migration during embryogenesis. In the developing nervous system, these processes are evident in the migration of neurons, axonal extension, Schwann cell migration, and the ensheathment and myelination of nerves. We have studied the production of plasminogen activator in cultures of superior cervical ganglia under conditions in which both neurons and glia are present. We have found that a principal source of the enzyme in these cultures is the glial cells and that the enzyme could not be detected at the growing tips of neurites. Plasminogen activator is also produced by Schwann cells isolated from neonatal rat sciatic nerve. The production of the enzyme by these cells is stimulated 6- to 10-fold by cholera toxin. Isolated Schwann cells and glial cells in the ganglion explant cultures produce the tissue form of plasminogen activator, a form of the enzyme not often found in nonmalignant cells. Preliminary experiments suggest that neuronal-glial interactions may regulate enzyme production by Schwann cells.

The cholinergic system of the primitive streak chick embryo.

The presence of neurotransmitters at stages of embryonic development prior to neurulation has been demonstrated in several systems. Although the functions of these molecules at early stages of embryogenesis have not been ascertained, it is possible that they are involved in aspects of cell migration, regulation of the synthesis of macromolecules, intercellular communication, and in the transmission of positional information during gastrulation. As an initial approach to the resolution of questions concerning the function of transmitters during early development, we have begun a study of the cholinergic system in the primitive streak chick embryo (Hamburger-Hamilton stages 3 + to 5). We have found that the chick embryo: (1) can use exogenously applied choline for the synthesis of acetylcholine; (2) possesses a true acetylcholinesterase, which is predominantly in the form of the 4-6s monomer; and (3) can take up exogenous choline through a sodium-dependent, high-affinity choline transport system. To date we do not have any evidence for the presence of nicotinic or muscarinic receptors at the primitive streak stage.

Ontogeny of Fc receptors and complement receptor (CR3) during human myeloid differentiation.

Two different Fc receptors for IgG (Fc gamma R) have been identified on human leukocytes: a high avidity receptor (Fc gamma Rhi) present on monocytes but not on neutrophils, and a low avidity receptor (Fc gamma Rlo) present on neutrophils but not on monocytes. Fc gamma Rlo can be inhibited and the receptor precipitated by monoclonal antibody 3G8. We have used this monoclonal antibody to study the course of Fc gamma Rlo appearance on bone marrow cells, leukocytes of patients with chronic myelogenous leukemia (CML), and HL-60 and U937 cells induced to differentiate with agents such as dimethyl sulfoxide (DMSO), retinoic acid, phorbol myristate acetate, and lymphokine. We report that Fc gamma Rlo is a late differentiation antigen, first expressed at the metamyelocyte stage. Since precursors to metamyelocytes bear Fc gamma R, and the promyelocyte line HL-60 bears Fc gamma Rhi, there must be a progressive loss of Fc gamma Rhi during myeloid differentiation and the reciprocal expression of Fc gamma Rlo. Results of immunoprecipitation and polyacrylamide gel analysis of the proteins are consistent with these results. We have also studied the receptor for the C3bi complement component (CR3), which is blocked and immunoprecipitated by monoclonal antibody OKM10. During DMSO-driven differentiation of HL-60 cells, we find that CR3 is induced on all cells, whereas Fc gamma Rlo is induced on only 24% of cells, suggesting that CR3 appears earlier during differentiation than Fc gamma Rlo does.

Plasminogen in the chick embryo. Transport and biosynthesis.

As part of a program to define potential roles for plasminogen activation during development, we have studied the metabolism of plasminogen in the chick embryo. Here we report that: 1) plasminogen is present in significant quantities in the yolk of fertile, unincubated eggs; 2) the zymogen can be translocated intact, from the yolk to the developing embryonic circulation; and 3) de novo synthesis of plasminogen occurs during the early phases of embryonic life. The combination of a reservoir of the zymogen in the yolk and protein biosynthesis thus ensures the availability of a substrate for enzymes which may participate in morphogenetic events occurring throughout embryonic life.

Plasminogen activator in the bursa of Fabricius: correlations with morphogenetic remodeling and cell migrations.

The association of controlled extracellular proteolysis, mediated by plasminogen activator, with embryonic tissue remodeling and cell migration was studied in the developing bursa of Fabriculus of quail and chick embryos. We found: that the specific activity of plasminogen activator in the bursa changes as a function of developmental age; that these changes are correlated temporally with the migration of hemopoietic cells into the bursal rudiment and with the period of extensive remodeling in the bursal epithelium; that the enzyme is distributed asymmetrically in the epithelial and mesenchymal compartments of the bursa. Chick and quail plasminogen activators can be distinguished by differences in their rates of electrophoretic migration. When interspecific grafts between quail and chick embryos were analyzed in this way, we observed that hemopoietic precursor cells produce plasminogen activator during their colonization of the bursa.

Merocyanine 540 as a fluorescent probe of membranes: staining of electrically excitable cells.

With the exception of certain blood cells considered in the accompanying paper (Valinsky, Easton and Reich, 1978), merocyanine 540 (MC 540), a fluorescent membrane probe, selectively strains the membranes of a wide variety of electrically excitable cells, but not those of nonexcitable cells. This reaction is Ca2+-dependent when staining is performed in buffered iso-osmotic sucrose, Ca2+-independent when staining proceeds at high ionic strength, inhibited by La3+ and sodium Suramin, enhanced by controlled, low level photosensitization of cell-associated dye and essentially irreversible. These characteristics of the staining reaction depend upon the maintenance of both cell viability and a normal unperturbed membrane structure. Although the mechanisms involved in the staining specificity remain unknown, observation of MC 540 partitioning between benzene and water in model reactions indicates that dye transport into hydrophobic solvents is accompanied by the formation of stoichiometric complexes with cations and phospholipids. These results may suggest the existence of specific, possibly phospholipid-rich membrane domains that mediate complex formation with MC 540 in excitable cells; comparable domains either would not exist, or would be inaccessible at the external surfaces of nonexcitable cells.

Merocyanine 540 as a fluorescent probe of membranes: selective staining of leukemic and immature hemopoietic cells.

We have reported (Easton, Valinsky and Reich, 1978) that merocyanine 540 (MC 540) specifically stains a variety of living excitable cells, but not nonexcitable cells. This paper describes the exceptional permeability to MC 540 of leukemic leukocytes and immature hemopoietic precursor cells. We have used fluorescence microscopy and uptake of radioactive dye to study MC 540 staining of peripheral blood leukocytes from 80 leukemic and 34 normal individuals; leukemic leukocytes stain, whereas normal leukcytes do not. The leukocyte staining reaction differs from that previously described for excitable cells since it is independent of the ionic composition of the staining medium, kinetically complex, enhanced by light, enhanced by oxygen and essentially irreversible. Virtually all circulating nucleated cells from leukemic individuals are stained to approximately the same extent, and there is no qualitative or quantitative distinction between the various forms of leukemia. We have also found that MC 540 interacts with granulopoietic colony-forming cells (CFU-C) and with spleen colony-forming cells derived from mouse bone marrow (CFU-S). We cannot as yet identify a specific property of leukocyte plasma membranes that determines MC 540 permeability; since changes in MC 540 uptake appear to be correlated with cellular maturation during normal hemopoiesis, the retention of staining by leukemic cells, some of which appear morphologically normal, may indicate of failure in membrane maturation during leukemic blood cell development.