Mechanisms involved in the expression of Jones-Mote hypersensitivity: II. Lymph node morphology and in vitro correlates

Lymph node morphology, in vitro lymphocyte transformation, and inhibition of macrophage migration were studied at varying intervals after sensitization for Jones-Mote hypersensitivity (JMH) with ovalbumin (OA) in Freund's incomplete adjuvant (FIA). The effect of cyclophosphamide (CY, 300 mg/kg), given 3 days before sensitization with OA in FIA, was also studied in an attempt to clarify further its role in increasing the intensity of skin reactions and its effect on the passive transfer of skin reactivity described in the preceding paper. There were increased numbers of large pyroninophilic cells in paracortical areas of draining lymph nodes and increased in vitro DNA synthesis, by lymph node cells, in animals treated with CY 3 days before sensitization with OA in FIA. There was no inhibition of macrophage migration of PEC from animals sensitized with OA in FIA, whether or not these guinea pigs had been treated with CY before sensitization.     

A sensitive and specific tritium release assay for dopamine-beta-hydroxylase (DbetaH) in serum.

The release of tritium from [7-³H₂]dopamine was investigated as a possible procedure for the assay for dopamine-β-hydroxylase (DβH) in rat and human serum. The release was found to have the same characteristics as those deseribed previously for DβH in serum; for example, an optimum rate of reaction at pH 5.0 or an enhancement of release with agents such as Cu²⁺ ions and N-ethylmaleimide which are known to inactivate endogenous inhibitors of DβH in serum. Tritium release was blocked by the DβH inhibitor fusaric acid but not by inhibitors of other dopamine-metabolizing enzymes in serum. Incubation of ¹⁴C-labeled dopamine along with [7-³H₂]dopamine revealed that, under the standard assay conditions, the formation of [¹⁴C]norepinephrine was accompanied by release of one of the two tritium atoms on the 7-carbon. It was concluded that the procedure provided a simple and sensitive assay of DβH activity in serum.     

Induction of anti-hapten antibody response by hapten-isologous carrier conjugate. II. Specificity of hapten-reactive helper T cells.

Anti-hapten antibody production was elicited by the immunization of hapten-isologous carrier conjugate (PAB-MGG) in mice. Spleen and lymph node cells taken from these primed mice could demonstrate their helper activity for anti-DNP antibody production when transferred intravenously into 600R X-irradiated recipient mice along with DNP-primed B cells and the double hapten conjugated carrier, DNP-MGG-PAB. Isologous carrier (MGG)-primed cells could not demonstrate this helper activity. Accordingly, helper cells reactive for a haptenic group are considered to develop by the immunization of hapten-isologous carrier conjugate. Hapten-reactive helper activity was also induced by the immunization of other hapten-isologous carrier conjugates, e.g., MAB-MGG, PABS-MGG or PAB-MSA. These hapten-reactive helper cells were T lymphocytes, as the helper activity of PAB-MGG-primed cells was completely abolished by in vivo ATS-treatment. Helper activity of PAB-MGG-primed cells for DNP-primed B cells was also demonstrated through the double hapten conjugated heterologous carrier DNP-HGG-PAB to be the same as with DNP-MGG-PAB, but weakly through DNP-KLH-PAB. As HGG but not KLH resembles MGG in composition, almost all hapten-reactive helper T cells can be considered to recognize not only haptenic groups but also physicochemical properties of the hapten-conjugated carrier site. However, these helper T cells could discriminate structural differences among related haptenic groups, because PAB-MGG-primed cells clearly responded to DNP-MGG-PAB to demonstrate their helper activity for DNP-primed B cells, but responded only weakly to DNP-MGG-PABS or DNP-MGG-MAB. When the specificity restrictions of T and B cells to the same haptenic group were compared by responsiveness measured after the antigenic stimulation (B cell function by anti-hapten antibody production and T cell function by helper activity), differences were noted, as PAB-MGG-primed T cells could respond not only to DNP-MGG-PAB but also fairly well to DNP-MGG-MAB to demonstrate their helper activity, but PAB-MGG-primed B cells responded to only PAB-MGG. Thus, hapten specificity appears to be much more restricted for B cells than T cells. The difference of this responsivity between B cells and helper T cells was thought to derive from the specificity difference of B cell and helper T cell receptors rather than from any sensitivity differences of the experimental procedure. The differences in the specificity restrictions of receptors of B and helper T cells were discussed in the light of hapten-specificity.     

Assay of proteolytic enzymes by the fluorescence polarization technique.

Neuraminidase substrates of high specific activity (>300 μCi/μmol) were prepared by reduction of sialyllactose with NaB³H₄, followed by separation of the 2 → 3 and 2 → 6 isomers of [³H]sialyllactitol by paper chromatography. Hydrolysis of sialyllactitol by neuraminidase was monitored by measuring the radioactivity in the neutral reaction product, which was separated from the charged substrate by passage over a small anion exchange column. The assay was applied to the neuraminidase activity of cultured human skin fibroblasts. The K_m was found to be 1.1 mm for both substrates; the pH optimum, 4.0; the 2 → 3 isomer was hydrolyzed twice as fast as the 2 → 6. In several genetic disorders associated with neuraminidase deficiency, the activity toward both isomers was reduced almost completely (mucolipidoses I and II; Goldberg syndrome), or only partially (mucolipidosis III; adult myoclonus syndrome); however, the relative activity towards the two isomers remained approximately the same in all cases.     

Letter: Myosin filaments showing a 430 A axial repeat periodicity

Synthetic myosin filaments with regular projections at intervals of 430 Å along their entire lengths have been observed using the electron microscope. The filaments were formed following dialysis in or rapid dilution to 0.3 m-KC1, 0.01 m-imidazole or phosphate buffer, pH 7.0. It is suggested that these filaments are made up of myosin molecules staggered by 430 Å.     

Increased expression of HLA-DR antigens in hydrocortisone-treated monocytes

Human peripheral blood monocytes incubated overnight with hydrocortisone had an increased expression of HLA-DR antigens. This change was noted as an increased proportion of DR-positive staining monocytes at greater fluorescence intensities as determined on a fluorescence-activated cell sorter. Hydrocortisone treatment of monocytes did not alter the expression of another Ia antigen on monocytes, HLA-DS. Neither did hydrocortisone treatment alter the expression of either Mac 120 antigen or monocyte .2 antigen on monocytes. Thus, the effect of hydrocortisone on monocyte DR antigens may be somewhat selective. Hydrocortisone also caused an increase in monocyte cell size aftr 3 to 4 days as compared to untreated controls.     

Secretion of proteoglycans by chondrocytes. Influence of colchicine, cytochalasin B, and beta-D-xyloside.

Chondrocytes obtained from epiphyseal cartilage of fetal guinea pigs or ear cartilage of young rabbits were cultured in monolayer. The influence of colchicine, cytochalasin B, and p-nitrophenyl-β-d-xylopyranoside on secretion of proteoglycans was investigated. Radioactive sulfate was used as a precursor. As observed previously in other systems, β-d-xylosides initiated the synthesis of free chondroitin sulfate chains, competing with the endogenous proteoglycan core protein acceptor. The molecular weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycans synthesized in the absence of xyloside. The size of the chondroitin sulfate chains synthesized on the xyloside was inversely related to the concentration of this compound. This finding suggests that the chain length is dependent on the ratio between available acceptor and chain-lengthening enzymes or precursors. Cytochalasin B, a microfilament-modifying agent, inhibited proteoglycan synthesis, without any effect on secretion. Cells treated with cytochalasin B could be stimulated with β-d-xyloside to synthesize free chondroitin sulfate chains to the same relative degree as cells with intact microfilaments. Colchicine, an antimicrotubular agent, partially inhibited synthesis and secretion of proteoglycan. However, cells treated with colchicine could be stimulated with β-d-xyloside to synthesize and secrete free chondroitin sulfate chains to about the same relative degree as cells with intact microtubules. The data suggest that microtubules may have a facilitatory rather than an obligatory role in the secretion of proteoglycans and that at least part of the effect of colchicine is located at or after the site of glycosaminoglycan synthesis.     

Cell-mediated immunity in experimental filariasis: lymphocyte reactivity to filarial stage-specific antigens and to B- and T-cell mitogens during acute and chronic infection.

To study immunological responses in chronic filarial infections, a model utilizing inbred Lewis rats infected with Brugia pahangi was developed. Microfilaria were found in the bloodstream of over 90% of the rats by 16 weeks of infection. Using in vitro lymphocyte blastogenesis, cell-mediated immune responses of blood, splenic, and mesenteric node lymphocytes were followed during 1.5 years of infection. Lymphocyte responses to antigen prepared from infective stage filarial larvae were detectable in the early weeks of infection, whereas responses to microfilarial antigen only developed late as microfilaremia waned. Lymphocyte responses to antigen from adult filaria vacillated during the infection. With the mitogens, phytohemagglutinin, pokeweed mitogen, and bacterial lipopolysaccharide, periods of B and T-cell hyporesponsiveness were demonstrable. Between 16 and 36 weeks of infection node lymphocytes from many rats were unresponsive to all mitogens and antigens. The model of B. pahangi in inbred rats offers advantages for immunological studies of filarial infections.     

Antigen-specific human B-cell responses: modulation by immunoregulatory T-cell subsets

In vitro T-cell requirements for and modulation of human B-cell responses were studied in individuals immunized in vivo to the protein antigen keyhole limpet hemocyanin or tetanus toxoid. T cells were required for antibody synthesis in both antigen-driven and pokeweed mitogen (PWM)-driven cultures. T cells were separated into T4+ and T8+ subpopulations using monoclonal antibodies, and their modulation of antibody synthesis was studied. T4+ cells functioned as helper cells in both antigen-driven and PWM-driven cultures in a dose-dependent manner. Whereas T8+ cells suppress both total and specific immunoglobulin secretion in PWM-stimulated cultures, in antigen-stimulated cultures T8+ cells do not suppress unless activated by another cell population present in peripheral blood mononuclear cells (PBMNC). This cellular requirement was further investigated by prestimulation of cells prior to addition to optimally stimulated antigen-driven cultures of PBMNC or B cells, monocytes, and helper T cells. No suppression of these optimally stimulated cultures was seen when T8+ cells were precultured with antigen or PWM. However, after 3-5 days preculture of total T cells with PWM or antigen and then selection of T4+ cells, these cells were able to induce fresh autologous T8+ cells to suppress optimally stimulated antigen-driven cultures. Addition of a precultured mixture of T8+ cells with 20% T4+ cells also resulted in antigen-induced suppression. These data indicate that T8+ cells can suppress antigen-driven cultures but require the presence of preactivated T4+ cells for induction of this suppression of antigen-specific T-cell-dependent human B-cell responses.     

Differential effect of monoclonal anti-DR antibody on monocytes in antigen- and mitogen-stimulated responses: Mechanism of inhibition and relationship to interleukin 1 secretion

A differential role for DR antigens on monocytes in antigen-stimulated as opposed to mitogen-stimulated human lymphocyte responses has been observed. A monoclonal anti-DR antibody used to treat monocytes caused inhibition of antigen-induced T-cell responses and of T-cell-dependent B-cell responses. However, anti-DR antibody treatment of monocytes did not inhibit mitogen-induced responses. Anti-DR treatment of monocytes did not induce suppression, as antigen-induced responses could be reconstituted with untreated monocytes. Anti-DR treatment of monocytes did not merely block interleukin 1 (IL-1) secretion since addition of IL-1 could not restore antigen-induced responses. Monoclonal anti-DR antibody did not directly inhibit monocyte secretion of IL-1. DR-negative monocytes, selected by antibody and complement, could not present antigen, even though they were capable of secreting IL-1. Thus, this monoclonal anti-DR antibody sterically blocks antigen presentation by monocytes without induction of suppression or inhibition of IL-1 secretion. Monocyte DR antigens appear essential for stimulation of antigen-induced responses, but DR antigens on monocytes may not be essential for mitogen-stimulated responses and do not appear to be related to the ability of monocytes to secrete IL-1.     

Hydrocortisone-mediated inhibition of monocyte antigen presentation: Dissociation of inhibitory effect and expression of DR antigens

The suppressive effects of hydrocortisone (HC) on the human immune system are well known. The mediation of the immunosuppressive effects of HC on lymphocyte responses via inhibition of monocyte function has been examined by monocyte-dependent, antigen-induced lymphocyte proliferation. Monocytes that were first treated with HC and then washed were unaffected in their subsequent ability to present antigen. However, there was a dramatic inhibition of lymphocyte proliferative responses if HC was present while monocytes were pulsed with antigen. This was directly related to the dose of HC present. HC-mediated inhibition of monocyte antigen presentation could not be overcome by the addition of interleukin-1 (IL-1) to the cultures, and thus inhibition of monocyte IL-1 secretion cannot totally account for the inhibition of monocyte antigen presentation. Although HC inhibits monocyte antigen presentation, HC increases the expression of HLA-DR antigens on monocytes. Other monocyte stimulants, including lipopolysaccharide (LPS), lymphokine, and gamma interferon, were examined for their effect on monocyte DR expression and their effect on monocyte antigen presentation. No correlation was found between the ability to increase monocyte DR antigen expression and the effect on antigen presentation. While HC, lymphokine, and gamma interferon all increased the expression of DR antigens on monocytes, HC, LPS, and lymphokine, but not gamma interferon, inhibited monocyte antigen presentation. Although HC can exert profound immunosuppressive effects via monocytes, it is not the only mechanism of inhibition. HC added to cultures after monocytes had been pulsed with antigen was also inhibitory.     

Development and characterization of interleukin-2-independent antigen-specific human T cell clones that produce multiple lymphokines

The development of antigen-specific T lymphocyte lines and clones has greatly facilitated the investigation of T-cell recognition of and response to foreign antigens. In the present study, human antigen-specific helper T cell lines and clones which are completely independent of exogenous interleukin-2 (IL-2) have been developed by cyclic restimulation with the soluble antigen keyhole limpet hemocyanin (KLH) to which the T cell donor had previously been immunized. These T cells uniformly bear the OKT4 phenotype and were shown to require both histocompatible antigen-presenting cells (APC) and antigen for optimal proliferation. The T cell line was composed of a highly antigen-specific and clonable T cell population. Following four cycles of antigen stimulation, limiting dilution cloning analysis showed a Poisson distribution of clonable T cells with a precursor frequency of 0.62, and from 88 to 92% of viable clones were specific for the stimulating antigen. Individual clones were obtained which recognized KLH with either DR 1 (one parental Ia haplotype of the donor) or DR 2 (the other parental Ia haplotype) allogeneic APC, but not both. Following stimulation with KLH, the T cell clones produced IL-2. Peak amounts of IL-2 were assayable in the first 6 to 24 hr after stimulation. In contrast, virtually no IL-2 was detectable in supernatants at 72 to 96 hr, suggesting autoutilization by the proliferating T cells. In addition, some clones were also capable of producing both B cell growth factor and IL-2 following KLH stimulation. These IL-2-independent T cells appeared to be derived from a discrete Leu 8-negative subclass of T4⁺ cells and expressed the full complement of Ia antigen of the donor. Thus, soluble antigen-specific human helper T cell clones have been produced which can be maintained in the absence of exogenous IL-2, elaborate their own growth factors and other immunoregulatory lymphokines, and show fine DR-related restriction to either one or the other parental DR haplotypes in antigen-stimulated proliferative responses.     

Colony-forming B cells in F1 hybrid and transplanted CBA/N mice.

The conditions for evaluation of suppressor cell regulation of the pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) responses of peripheral blood (PB) B cells in normal individuals using allogeneic cocultures is described. In 14 separate experiments, after preincubation with concanavalin A (Con A) for 2 days, PB cells suppressed the PWM-induced anti-sheep erythrocyte (SRBC) PFC response of fresh allogeneic PB cells to 17% of the expected PFC response (P < 0.05). In addition, control cells incubated for 2 days in the absence of Con A suppressed the PWM- induced PFC response of allogeneic cells in 6 of 14 experiments to the same extent as did the Con A-generated cells (P < 0.01). It was found that unstimulated control cells (without Con A activation) from normal subjects who themselves were nonresponders to PWM stimulation (< 50 PFC/10⁶ cells) usually suppressed the PFC response of allogeneic cells (P < 0.05), while control cells from normal subjects who consistently had a good PFC response to PWM stimulation (> 75 PFC/10⁶ cells) did not suppress the PFC response of allogeneic cells. The spontaneously occurring suppressor cell in nonresponder PB cell suspensions was sensitive to 3000-R irradiation, and the nonresponder state was not associated with a decreased blastogenic response to PWM. Thus, some normal subjects who themselves had a poor PWM-induced PFC response had irradiation-sensitive, spontaneously occurring suppressor cells which were capable of suppressing the PWM-induced PFC response of normal responders. The majority of normal subjects (90%) were good PFC responders to PWM stimulation and did not spontaneously suppress the PFC response of allogeneic cells to PWM, but did have PB cells which were capable of being activated by Con A to suppress.     

Developmental changes in the sensitivity of embryonic heart cells to tetrodotoxin and D600

During Days 4 to 7 in ovo, beating of embryonic chick hearts becomes progressively more sensitive to inhibition by tetrodotoxin, an inhibitor of fast Na⁺ channels, and progressively less sensitive to inhibition by D600, an inhibitor of slow Ca2+/Na+ channels. The developmental change in tetrodotoxin sensitivity is not retained in heart cells cultured in monolayer. In contrast, the developmental change in D600 sensitivity is retained. Veratridine-stimulated ²²Na⁺ influx mediated by fast Na⁺ channels is inhibited by tetrodotoxin (K_i = 1.6 nM) in cells prepared from either 3-day or 12-day embryos. These results suggest that young embryonic hearts contain physiologically inactive Na⁺ channels. ²²Na⁺ influx mediated by slow Ca2+/Na+ channels is inhibited by D600 with a K_i of 40 nM for cells from 3-day hearts and 8 μM for cells from 12-day hearts. Beating of heart cells in aggregate cultures is also inhibited by D600. Aggregates which have reactivated after inhibition by tetrodotoxin are 10-fold more sensitive to inhibition by D600 than untreated controls. The results suggest that the primary developmental event is a change in slow Ca2+/Na+ channels which reduces their sensitivity to D600 and diminishes their ability to support beating without the activity of the fast Na⁺ channel.     

The relationship between immunization and circulating antigen-specific plaque-forming cells

The relationship between the appearance of cells producing antibody to tetanus toxoid (TT) in the circulation and the serum titers of anti-TT IgG following booster immunization has been studied. It was found that cells producing anti-TT antibody can be detected in the circulation in a hemolytic plaque assay using sheep red blood cells (SRBC) coated with TT by the chromic chloride method. In symmetric inhibition studies using cells from TT or keyhole limpet hemocyanin (KLH)-immune donors, the homologous antigen inhibited 100% of the PFC with no cross-inhibition. Thus, the plaque-forming cells (PFC) detected in this assay are specific for the immunizing antigen. No evidence of polyclonal B-cell activation in response to TT was found, as shown by a failure to detect any PFC against unmodified or KLH or human serum albumin-treated SRBC. In addition, the increase in total Ig-secreting cells observed in a staphylococcal protein A reverse hemolytic plaque assay was always accounted for by the number of anti-TT antibody-producing cells observed. The peak number of anti-TT antibody-producing cells varied between donors, but the kinetics of their appearance was highly reproducible--none before Day 5, peak numbers between Days 6 and 8, and a sharp decline with only rare anti-TT Ig-secreting cells in the circulation by Day 15 postimmunization. Anti-TT antibody-producing cells appeared in the circulation prior to any detectable increase in serum anti-TT antibody titers, and following the disappearance of PFC from the circulation, there was no further increase in serum IgG anti-TT levels. These observations demonstrate a marked specificity of B-cell activation on boosting with a recall antigen, and a parallelism between the appearance of activated B cells in the circulation and of IgG anti-TT synthesis by the subject as a whole.     

Radioimmunoassay for pig angiotensin I converting enzyme: a comparison of immunologic with enzymatic activity.

Angiotensin I converting enzyme in body fluids and extracts of various pig tissues was measured by radioimmunoassay and by enzymic assay. Based on the ratios of enzymic to immunologic activity, the extracts could be separated into two groups. One group, with ratios around 4 U/mg, included urine and extracts from the adrenal, choroid plexus, epididymis, gall bladder, heart, liver, retina, spleen, and testis. The other group, with ratios around 12 U/mg, contained serum and extracts from lung and kidney. Explanations are offered for why one group had a lower enzymic to immunologic ratio than the other.     

Glutamine synthetase cascade: enrichment of uridylyltransferase in Escherichia coli carrying hybrid ColE1 plasmids

Colchicine-binding properties of the total cytoplasmic pool of tubulin from rat liver were evaluated in tubulin-stabilizing (TS) supernates. Microtubules were separated from free tubulin using a microtubule-stabilizing solution (MTS) and ultracentrifugation. [³H]Colchicine-binding properties of microtubule-derived tubulin were investigated in supernates prepared after resuspension of MTS pellets in TS. In TS buffer at 37 °C the colchicine-binding activity of the total cytoplasmic pool of tubulin decayed with $\text{T}\text{1}\text{2}$ of 3.39 h. Resuspended pellet tubulin decayed much more rapidly under the same conditions with a $\text{T}\text{1}\text{2}$ of 0.72 h. This rapid time decay of microtubule-derived tubulin was found to be at least partially attributable to prior microtubule-stabilizing solution exposure. Since tartrate has been reported to increase the rate of colchicine binding to tubulin, sodium tartrate (150 mm) was added to our colchicine-binding system. This addition increased the detectable [³H]colchicine binding by 10% in the total cytoplasmic preparation and by 85% in the resuspended pellet preparation. Addition of tartrate (150 mm) also resulted in a 105% increase in the $\text{T}\text{1}\text{2}$ for total cytoplasmic tubulin and a 412% increase for microtubule derived tubulin. Total cytoplasmic supernates of liver bound [³H]colchicine linearly over a wide range of tissue concentrations. However, resuspended microtubule-stabilizing solution pellet supernates in tubulin-stabilizing solution showed some increase in colchicine binding per tissue weight in the more dilute samples. Our data which demonstrate differences in colchicine-binding properties for total cytoplasmic and microtubule-derived pools of tubulin suggest that present assays for hepatic tubulin polymerization which assume identical binding properties should be interpreted with caution.     

Occurrence of molybdenum in the nicotinic acid hydroxylase from Clostridium barkeri

Molybdenum, assayed by atomic absorption spectrometry, copurifies with the selenium-containing nicotinic acid hydroxylase from Clostridium barkeri. Fluorescence spectral studies on the enzyme indicate the presence, along with flavin, of another component. The fluorescence spectra of this component obtained after the aerobic denaturation of the nicotinic acid hydroxylase are similar to the fluorescence properties reported for the “pterin-like” cofactor from xanthine oxidase and several other molybdoproteins. Nicotinic acid hydroxylase from C. barkeri contains molybdenum, selenium, iron, acid-labile sulfur, and flavin with the occurrence of a “pterin-like” cofactor also a likely component.     

Lack of B-cell participation in acute lymphocyte choriomeningitis disease of the central nervous system.

The pattern of acute lymphocytic choriomeningitis (LCM) virus-induced central nervous system (CNS) disease was studied in BALB/c mice rendered deficient in B cells and incapable of producing a humoral immune response. The CNS disease in mice treated from birth with goat anti-mouse μ-chain globulin (μ-suppressed mice) was clinically and histopathologically indistinguishable from that of controls. This result demonstrates that neither B cells nor antibody participate in the pathogenesis of acute LCM of the CNS. These observations support the view that the host cell-mediated response to viral antigens is solely responsible for acute CNS disease induced by the intracerebral inoculation of LCM virus.