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Background: An elevated IgE level and increased production of T(H2) cytokines are factors associated with poor prognosis in HIV infection. We report a pediatric long-term survivor of vertically acquired HIV infection with a normal CD4 count and a low viral burden despite the lack of antiretroviral therapy and a phenotype resembling hyper-IgE syndrome., Objective: We sought to characterize the patient's T(H1) versus T(H2) cytokine profile and anti-HIV-specific immune responses., Methods: Supernatants collected from cultures of peripheral blood T cells stimulated with phorbol myristate acetate plus ionomycin were assayed for T(H1) and T(H2) cytokines by means of ELISA. Specific IgE antibodies were determined by immunoblot. HIV-specific cytotoxic T-lymphocyte responses were measured from cell lysis by fresh T cells of autologous B-lymphoblastoid cells expressing recombinant HIV proteins., Results: Patient CD4(+) T cells secreted significantly more T(H2) cytokines, IL-4 (P <.003) and IL-5 (P <.03), than HIV-infected and seronegative control cells. No difference was noted in T(H1) cytokine production. IgE specific for HIV gp160, p24, p17, and p66 proteins and Aspergillus fumigatus was detected in patient sera. Despite predominance of T(H2) cytokines, HIV-specific cytotoxic T-lymphocyte activity was vigorous., Conclusions: The patient demonstrated predominantly T(H2) cytokine production in vitro. Unlike other patients with HIV who have hyper-IgE and increased T(H2) cytokine production, our patient has maintained HIV-specific immune responses, a low viral load, and a normal CD4 count without antiretroviral therapy. These findings support a diagnosis of primary hyper-IgE syndrome. Presence of anti-HIV-specific IgE may represent a protective mechanism against HIV replication in our patient.

Urokinase (UK) type plasminogen activator is a serine protease produced by activated human monocytes. Despite the well-documented roles played by UK in cell-mediated immunity in healthy humans, the roles played by UK in the derangements of cell-mediated immune responses observed in HIV disease remain largely undefined. In these studies the numbers of peripheral blood lymphocytes and monocytes bearing surface UK (UK+) as well as serum levels of UK (flow microfluorimetry and ELISA, respectively) were determined in children with AIDS and in healthy HIV-negative children. The effects of exogenous UK on lymphocyte activation (cell cycle analysis using living cells) and surface marker (CD3, CD4, CD8, and CD19) expression (flow microfluorimetry using fixed cells) were also studied. Data are expressed as percent total cells. Numbers of UK+ lymphocytes in children with AIDS were similar to those observed in healthy children. In contrast, numbers of UK+ peripheral blood monocytes were dramatically decreased (> 70%) in the children with AIDS. However, serum levels of UK were increased (nearly threefold) in these children. When lymphocytes from these children were cultured with soluble UK, numbers of cells in S phase of cell cycle appeared suppressed. Incubation of fixed lymphocytes from either a child with AIDS or from a healthy child with exogenous UK appeared to increase numbers of cells expressing CD3. Incubation with UK had no effect on expression of any other surface marker (CD4, CD8, or CD19) using cells from the child with AIDS. In contrast, incubation with UK appeared to decrease (fivefold) numbers of cells expressing CD19 and increase numbers of cells expressing CD4 and CD8 only when fixed lymphocytes from a healthy HIV-negative child were used. The results suggest important roles for UK in regulation of lymphocyte surface markers in general and in CD3- and CD19-dependent lymphocyte activation pathways specifically. Furthermore, these studies add to a widening body of evidence implicating UK dysregulation in the pathogenesis of HIV disease and may point to pharmacological opportunities involving UK to delay or prevent progression of HIV infection into full-blown AIDS.

SDZ 280.636, a nontoxic diacyl glycerol derivative of muramyl dipeptide (MDP), a component of the inner bacterial cell wall, which is suitable for use in man, suppressed hapten specific IgE antibody forming cell (AFC) responses in spleen, serum levels of hapten specific IgE and hapten specific immediate hypersensitivity (i.h.) responses in skin, when fed to mice at the peak of a hapten specific IgE AFC response. In addition, serum levels of IL-6 appeared increased while IFN gamma was decreased. To induce these IgE responses, BALB/c mice were injected i.p. with BPO-KLH (benzylpenicilloyl-keyhole limpet hemocyanin) (10 micrograms) in aluminum hydroxide gel (alum) on days 0, 21 and 42. Mice were fed (gavage) with either MDP or SDZ 280.636 (1.0 or 10 mg/kg) on day 44, or on days 44, 46 and 48, and killed on days 46 or 50. Numbers of BPO specific AFC in spleen, and serum levels of BPO specific immunoglobulins (IgG1, IgE and IgA) were determined (ELISPOT assay, ELISA). In addition, BPO specific IH responses were measured in these animals. Mice were injected in the right pinna with BPO-BSA (0.1 microgram) and in the left pinna with an equal volume of saline (0.05 ml). At 2 hr, pinnae were measured using a micrometer caliper. We found that 1 feeding with either MDP or SDZ 280.636 abrogated IgE AFC responses and dramatically suppressed serum levels of IgE, both in isotype specific fashion, and suppressed IH responses (> 50%). 3 feedings with SDZ 280.636 also abrogated IgE AFC responses and further decreased serum levels of IgE. In contrast to SDZ 280.636, 3 treatments with MDP had opposite effects in that IgE AFC responses and serum levels of IgE dramatically increased. A single treatment with SDZ 280.636 appeared to increase serum levels of IL-6 up to three fold, while IFN gamma levels decreased. Our data suggest that SDZ 280.636 may be useful in the therapeutic and prophylactic management of human atopic disease such as allergic rhinitis, asthma, and other atopic diseases.

To characterize the cellular basis of IgE responses in HIV-positive (HIV+) children, we obtained central (bone marrow [BM], thymus) and peripheral (Peyer's patches [PP], mesenteric [MLN], and other lymph nodes [OLN], spleen), lymphoid organs from two children with AIDS (females, 2 and 8 years old), and from a non-HIV-infected trauma victim (female, 5 years old) at autopsy. PP were obtained from one of the HIV+ children (2 yr old) and from the non-infected child, but no PP were detected in small intestine of the 8-yr-old HIV+ child. Numbers of lymphocytes bearing surface IgE, CD19, CD3, CD4, and CD8 in lymphoid organs were determined (flow cytometry) and evaluated for expression of epsilon-specific (E) mRNA (RT-PCR). Thymus and MLN of the HIV+ child without PP contained high numbers of IgE+ (34% and 41%, respectively) and CD19+ (32% and 28%, respectively) cells; IgE+ cells were not found in any other organ. In contrast, in the HIV+ child with PP, IgE+ cells were detected in all organs, except BM. The thymus of this child contained fewer CD19+ cells (7%). However, in both HIV+ children, all lymphoid organs, including thymus, contained E mRNA. Because numbers of IgE+ cells often far exceeded numbers of CD19+ B cells, and because CD8+ T cells predominated in all organs, some of the IgE+ cells were probably CD8+ T cells with cytophilic IgE and may include IgE-specific regulatory and/or memory T cells. IgE responses were not detected in the healthy trauma victim nor were B cells found in thymus. The data suggest that during HIV infection, IgE+ B cells may be found in thymus and that synthesis of IgE may occur in all lymphoid organs except BM.

Elevated serum Ige was detected in 26% (7 of 30) of children with HIV infection. The majority of children with elevated IgE were of one ethnic group (Puerto Rican) (4 of 7), compared with only 9% (2 of 23) in the normal to low IgE group (p = 0.02). Most of the children with elevated IgE had decreased circulating CD4+ T cells (5 of 7 or 71%); but none had opportunistic infections, and none failed to thrive. Although similar numbers of children with normal to low IgE had decreased circulating CD4+ T cells (19 of 23 or 83%), this group had opportunistic infections (6 of 23 or 26%) and failure to thrive (7 of 30 or 30%). There was no difference in incidence of allergic symptoms between groups. IgE antibody against HIV protein was detected by Western blot technique in the sera of three children with elevated serum IgE. Thus we have identified a group of children with HIV infection and elevated serum IgE of predominantly one ethnic group, who are without opportunistic infections or failure to thrive, some of whom produce HIV-specific IgE. This suggests that IgE may play a protective (perhaps late compensatory) role in HIV disease in genetically predisposed individuals.

The ability of interleukin (IL)-6 or interferon-alpha (IFN-alpha) to regulate expression of low-affinity Fc(epsilon) receptor (CD23) and serum levels of CD23 was studied in benzylpenicilloyl-keyhole limpet hemocyanin-sensitized BALB/c mice at the peak of a hapten-specific immunoglobulin E (IgE) antibody-forming cell (AFC) response. These responses are analogous to those observed in human atopic disease. To induce peak IgE responses, mice were injected intraperitoneally with BPO-KLH (10 micrograms) in aluminum hydroxide gel (alum) on days 0, 21, and 42. On day 44, mice were injected subcutaneously with IL-6 (100-1000 U) or IFN-alpha (1000-10,000 U). On day 46, numbers of CD23+ lymphocytes in Peyer's patches (PP), mesenteric lymph nodes (MLN), and spleen and levels of soluble CD23 in serum were determined (flow microfluorimetry and enzyme-linked immunosorbent assay, confirmed by competition assay). Data are expressed as percent total cells or as optical density at 490 nm. IFN-alpha treatment strongly suppressed (up to 100%) numbers of CD23+ cells exclusively in PP (i.e., numbers of CD23+ cells in MLN and spleen were unchanged) whereas serum levels of soluble CD23 were dramatically increased (60%). IL-6 treatment had no effect on either numbers of CD23+ lymphocytes or on serum levels of soluble CD23. The data suggest that the mechanism(s) by which IFN-alpha, but not IL-6, regulates IgE responses involves suppression of CD23 expression on lymphocytes in PPs and supports a central role for these organs in regulation of IgE responses in vivo.

The ability of substance P (SP) to regulate peak benzyl-penicilloyl (BPO)-specific IgE antibody-forming cell (AFC) responses in vivo and the ability of SP and other neuropeptides to regulate BPO-specific memory IgE AFC responses induced in vitro was determined. SP injected subcutaneously into BPO-keyhole limpet hemocyanin (BPO-KLH)-sensitized mice at the time of peak IgE responses suppressed these responses within 48 h (> 90%). The suppression obtained was IgE isotype-specific, dose-dependent, and transient. When spleen cells from immunized mice were cultured for 5 days with BPO-KLH, peak memory IgE AFC responses were induced in vitro. Inclusion of either SP or vasoactive intestinal peptide (VIP), but not neurotensin, serotonin, somatostatin, or gastrin, in cultures suppressed these responses in isotype-specific, dose-dependent fashion (approximately 70%). SP-, but not VIP-mediated suppression of IgE responses was abrogated by inclusion of anti-IFN gamma culture.

The ability of IL-6 or IFN alpha or antibodies to these cytokines to regulate serum levels of hapten specific immunoglobulins (IgM, IgG1, IgE, IgA) was studied in BPO-KLH (benzylpenicilloyl-keyhole limpet hemocyanin) sensitized BALB/c mice at the peak of a hapten specific IgE antibody forming cell (AFC) response. To induce peak IgE responses, mice were injected intraperitonealy (i.p.) with BPO-KLH (10 micrograms) in aluminum hydroxide gel (alum) on days 0, 21, and 42. On day 44, mice were injected s.c. with IL-6 (100-1000 U), IFN alpha (1000-10,000 U), anti-IL-6 (100-1000 neutralizing units [NU]), or anti-IFN alpha (1000-10,000 NU). On day 46, levels of BPO specific IgM, IgG1, IgE and IgA in serum were determined (ELISA). Data are expressed as micrograms/ml. IL-6 suppressed BPO specific IgE in serum in isotype specific fashion (to > 90%), increasing IgA (approximately 3 fold), and leaving IgM and IgG1 unchanged. Since removal of endogenous IL-6 with anti-IL-6 increased serum IgE, and suppressed IgG1 (approximately 50%), with IgM and IgA unchanged, this suggests that IL-6 is an isotype specific suppressor of peak IgE responses and as such may be useful in the therapeutic management of atopic disease. IFN alpha treatment increased serum IgE levels (60%), and potentiated IgA responses (> 30 fold), with IgM and IgG1 unchanged. Since removal of endogenous IFN alpha with anti-IFN alpha decreased IgE levels (approximately 50%), increasing IgA, with IgM and IgG1 unchanged, this suggests a role for IFN alpha as an isotype specific helper of peak IgE responses and in maintenance of IgA responses.

Previous studies in our laboratory have shown that substance P (SP), injected into benzylpenicilloyl-keyhole limpet hemocyanin (BPO-KLH) sensitized mice at the peak of the benzylpenicilloyl (BPO)-specific IgE response, suppressed these responses in isotype-specific fashion within 48 h. These studies also showed that SP, but not neurotensin (NT), serotonin (5-HT), somatostatin (SOM) or gastrin, suppressed BPO-specific memory IgE antibody-forming cell (AFC) responses induced in vitro, also in isotype-specific fashion. To investigate the mechanisms by which SP suppressed BPO-specific IgE AFC responses were induced in vitro, these responses were induced by culturing spleen cells from BPO-KLH sensitized mice for 5 days with BPO-KLH with or without whole SP, amino terminal SP (SP 1-4: Arg-Lys-Pro-Lys), or carboxy terminal SP (SP 8-11: Phe-Gly-Leu-Met). In some experiments, the SP receptor antagonist (D-Pro2, D-Phe7, D-Trp9)-SP (D-SP) was included in culture. In other experiments anti-interferon monoclonal antibody (anti-IFN gamma mAb) was in culture. Whole SP and SP 8-11, but not SP 1-4, suppressed BPO-specific IgE AFC responses induced in vitro. The suppression obtained was IgE isotype-specific and dose-dependent. Inclusion of SP receptor antagonist (D-Pro2, D-Phe7, D-Trp9)-SP inhibited suppression of BPO-specific memory IgE AFC responses by SP or SP 8-11. The SP-mediated suppression of BPO-specific memory IgE responses appeared to involve interferon gamma (IFN gamma).

The roles of Thy-1+ and AsGM1+ spleen cells and cytokines (IL-4, IL-5, IL-6, IFN-alpha, and IFN-gamma) in regulation of hapten-specific memory IgE antibody-forming cell (AFC) responses induced in vitro were examined. BALB/c mice, injected i.p. with benzylpenicilloyl-keyhole limpet hemocyanin (BPO-KLH) (10 micrograms) on days 0 and 21, were killed on day 60 or day 120. Numbers of BPO-specific IgGI, IgE, and IgA AFC in spleen were determined by enzyme-linked immunosorbent spot assay after 0 to 6 days of culture +/- BPO-KLH. BPO-specific AFC of all isotypes were detected in spleen on day 60, but not on day 120. Day 60 AFC responses did not persist in culture in that no AFC were detected by day 2 of culture +/- BPO-KLH. When either day 60 or day 120 cells were cultured for 3 days with BPO-KLH, BPO-specific AFC responses were induced, and peaked on day 5, with similar numbers of AFC of each isotype induced with day 60 and day 120 cells. On day 60, spleen contained two subsets of Thy-1+ cells: AsGM1- (approximately 32% of total cells) and AsGM1+ (approximately 4%). Depletion and reconstitution experiments established that both subsets were required for induction of BPO-specific IgE AFC responses. Cytokines could not substitute for the Thy-1(+)-depleted cells. However, when unfractionated day 60 cells were cultured with cytokines or anti-cytokine antibodies, BPO-specific IgE AFC responses induced were both IFN-alpha and IL-4 dependent; either increased or decreased by IFN-gamma, depending on its concentration, and unaffected by IL-5 or IL-6.

Muramyldipeptide (MDP) and murabutide (MB), a pyrogen free derivative of MDP, suppressed BPO specific IgE antibody forming cell (AFC) responses in vivo. To induce IgE responses, BALB/c mice were injected intraperitoneally (i.p.) with BPO-KLH (10 micrograms) in alum on days 0 and 21, or on days 0, 21 and 42. On day 44, mice were fed (gavage) or injected subcutaneously (s.c.) with MDP or MB (0.1-500 mg/kg). Mice were killed on days 45-70, and the numbers of BPO specific IgM, IgG1, IgE, and IgA antibody forming cells (AFC) in lymphoid organs determined in ELISPOT assay. With either immunization schedule, oral treatment with MDP or MB on day 44 suppressed BPO specific IgE AFC responses within 48 h (65-100%). With both molecules, the suppression was IgE isotype specific, dose dependent and transient. The suppression was also route specific since it was obtained only when MDP or MB was given by gavage, and not when injected s.c. These results show that peak antigen specific IgE responses can be suppressed in vivo, in isotype specific fashion, by a clearly defined class of molecules, one of which, MB, is a candidate for clinical studies in man. Pharmacologic agents of this type may be suitable for use in the therapeutic or prophylactic suppression of IgE and, hence, in the therapy of IgE mediated diseases such as allergic rhinitis, asthma, and other atopic diseases.

To study the effects of cytokines on human IgE antibody forming cells (AFCs), log phase U266 myeloma cells (3 x 10(3)/ml), which secrete immunoglobulin E (IgE), were cultured for 0-24 h with and without cytokine or with or without antibodies against various cytokines. The numbers of IgE AFCs were determined in ELISPOT assay. We found that interleukin-6 (IL-6) suppressed (to 95%) whereas anti-IL-6 increased (to 148%) the numbers of IgE AFCs and that both worked in a dose-dependent fashion. IL-4 and interferon-gamma (IFN-gamma) also suppressed IgE AFC responses in a dose-dependent fashion. However, antibodies to these cytokines had no effect. In contrast, IFN-alpha increased (to fourfold) the numbers of IgE AFCs in a dose-dependent fashion. The data are the first to show a suppressive effect of IL-6 on human IgE responses and may also suggest a role for IL-6 in the treatment of atopic disease.

The ability of subcutaneously (s.c.) injected cytokines (IL-4, IL-5, IL-6, IFN alpha, IFN gamma, GMCSF) to regulate the induction of hapten-specific immediate hypersensitivity (IH) responses was studied in BPO-KLH (benzylpenicilloyl-keyhole limpet hemocyanin) sensitized BALB/c mice at the peak of a hapten-specific IgE antibody forming cell (AFC) response. To induce IH responses, mice were injected in the right pinna with either BPO-BSA (benzylpenicilloyl-bovine serum albumin), BPO-KLH (0.01-1.0 micrograms/ml) or mcAb anti-IgE (0.001 - 1.0 micrograms/ml); and in the left pinna with an equal volume of saline (0.05 ml). Pinnae were measured 5 min to 4 hr later using a micrometer caliper. Treatment of mice with IL-4 or IFN gamma dramatically suppressed the induction of IH responses in dose dependent fashion. In contrast, treatment of mice with IL-6 and IFN alpha increased these responses in dose dependent fashion, while GMCSF and IL-5 had no effect. The suppression obtained with IL-4 and IFN gamma, and the increases seen with IL-6 and IFN alpha, were transient since these cytokines, as well as GMCSF and IL-5, had no effect on IH responses elicited 21 days after the peak of BPO-specific IgE AFC responses. The data suggest that cytokine mediated effects on IH responses occur via changes in serum levels of BPO-specific IgG1 or IgE, through direct or indirect effects of cytokines on mast cells or other cell types, or by affecting the ability of BPO-specific homocytotropic antibodies to bind to mast cell surfaces.

The ability of cytokines (IL-4, IL-5, IL-6, IFN-alpha, IFN-gamma, TNF-alpha, GmCSF) to regulate peak benzylpenicilloyl (BPO)-specific IgE antibody-forming cell (AFC) responses was investigated. These responses were induced in BALB/c mice by ip injection of BPO-keyhole limpet hemocyanin (BPO-KLH; 10 micrograms) in aluminum hydroxide gel on Days 0, 21, and 42. On Day 44, or on Days 43, 44, and 45, mice were injected sc with varying doses of cytokine or anti-cytokine antibody. On Day 46, the numbers of BPO-specific AFC (IgM, IgG1, IgE and IgA) in spleen were determined ex vivo in enzyme-linked immunosorbent spot assay. Among the cytokines tested, only IL-6 suppressed BPO-specific IgE AFC responses in an isotype-specific fashion (60-90%). However, treatment of mice with anti-IL-6 also suppressed these responses, suggesting that IL-6 can either suppress or increase peak antigen specific IgE responses, depending upon its concentration. Among the cytokines tested, only IFN-alpha increased BPO-specific IgE AFC responses in an isotype-specific fashion. Since treatment with anti-IFN-alpha suppressed these responses, it appears that IFN-alpha is required to maintain peak antigen-specific IgE AFC responses. IL-4 or IFN-gamma nonspecifically suppressed responses of all isotypes. Treatment with anti-IL-4 also suppressed IgE responses, suggesting that this cytokine is required to maintain peak antigen specific IgE responses. Treatment with anti-IFN-gamma increased IgE responses, indicating that IFN-gamma suppresses peak antigen-specific IgE responses.

Muramyldipeptide (MDP) and murabutide (MB) suppressed hapten-specific IgE antibody-forming cell (AFC) responses in vivo. IgE responses were induced in BALB/c mice by intraperitoneal injection with benzylpenicilloyl-keyhole limpet hemocyanin (BPO-KLH) (10 micrograms) in aluminum hydroxide gel (Alum) on days 0, 21 and 42. On day 44, mice were fed (gavage) or injected subcutaneously with varying concentrations of MDP or MB (0.1-500 mg/kg). The mice were killed on days 45-70, and the numbers of BPO-specific IgM, IgG1, IgE, and IgA AFC in various lymphoid organs were determined in an enzyme-linked immunosorbent spot (ELISPOT) assay. In addition, levels of BPO-specific IgE in serum were determined by ELISA. Data are expressed as AFC/10(7) cells or as micrograms/ml. Feeding with MDP or MB on day 44 suppressed BPO-specific IgE AFC responses and serum levels of BPO-specific IgE within 48 h (day 46) (65-100% and approximately 50% decrease, respectively). With both molecules, the suppression was IgE isotype-specific, dose-dependent and transient. The suppression was also route-specific since it was obtained only when MDP or MB were given by gavage, and not when injected subcutaneously. These results show that peak antigen-specific IgE responses can be downregulated in vivo, in isotype-specific fashion, by a clearly defined class of molecules, MDP and MB, one of which, MB, is a candidate for clinical studies in man. The mechanism of suppression probably involves the modulation of gut-associated lymphoid tissue and mucosal immunity. The clinical implications are that pharmacologic agents of this type may be suitable for use in the therapeutic or prophylactic downregulation of IgE and, hence, in the therapy of IgE-mediated diseases in man such as allergic rhinitis, asthma, and other atopic diseases.

Pulmonary immunity has not been studied in children with acquired immunodeficiency syndrome (AIDS) or tuberculosis (TB), even though lungs of both children and adults infected with human immunodeficiency virus (HIV-1) or Mycobacterium tuberculosis are affected frequently and severely. In the present studies, the distributions of T (CD3+, CD4+, CD8+) and B (CD19+) lymphocytes in bronchoalveolar lavage fluid (BALF) and blood of children with AIDS (N = 28) and children with pulmonary TB (N = 18) were determined using direct immunofluorescence (flow microfluorimetry). The distributions of lymphocyte subsets in BALF differed dramatically from those in blood. In pediatric AIDS, reduction of CD4/CD8 ratio was much more pronounced in BALF than in peripheral blood (0.15 +/- 0.04 vs. 0.43 +/- 0.11). This difference was due to selective depletion of BALF CD4+ lymphocytes, rather than to a great influx of CD8+ cells into the lung. In childhood TB, the CD4/CD8 ratio in BALF also was significantly decreased, despite its elevation in blood (1.02 +/- 0.26 vs. 1.96 +/- 0.32). The results show that (1) examination of peripheral blood lymphocytes does not reflect the kind and extent of changes observed in the distribution of pulmonary lymphocyte subsets, and (2) the profound decrease of the CD4/CD8 ratios in BALF of children with AIDS or TB is due to decreased percentages and absolute numbers of BALF CD4+ lymphocytes. The data suggest that analysis of BALF provides a more accurate evaluation of the patient pulmonary immune status than monitoring peripheral blood.

The organs in which B cells bearing membrane-bound IgE (sIgE+) and benzylpenicilloyl (BPO)-specific IgE antibody-forming cells (AFC) first appeared were determined in BALB/c mice given BPO-keyhole limpet hemocyanin (10 micrograms) in aluminum hydroxide by various routes (i.p, gavage, s.c., i.v., or i.m.). In mice immunized by the i.p. route, the numbers and location of sIgE+ B cells and asialo GM1 ganglioside (AsGm1+) cells, the location of IgE/CD23 immune complexes, and the numbers of BPO-specific IgE AFC in lymphoid organs were determined. With all routes of immunization, no sIgE+ B cells or BPO-specific IgE AFC were ever detected in any organ before day 8. On day 8, with the s.c., i.v., or i.m. routes, sIgE+ B cells and IgE AFC appeared exclusively in Peyer's patches (PP); with the i.p. or gavage routes, sIgE+ B cells simultaneously appeared in both PP and mesenteric lymph nodes, whereas IgE AFC appeared only in PP. In mice immunized by the i.p. route, IgE/CD23 immune complexes and strikingly increased numbers of AsGm1+ cells transiently appeared only in PP after the appearance and preceding the "disappearance" of the sIgE+ B cells and IgE AFC. The data suggest that specific IgE responses originate in gut-associated lymphoid tissue and appear later in spleen. The data also associate the appearance of IgE/CD23 immune complexes and AsGm1+ cells with the "disappearance" of sIgE+ B cells and IgE AFC from PP.

To characterize the activation state of monocytes during human immunodeficiency virus (HIV) infection, peripheral blood monocytes (PBMs) from patients with acquired immunodeficiency syndrome (n = 10) and from healthy controls (n = 10) were cultured for 4 days. Monocyte culture supernatant (MCS) was collected daily, and levels of urokinase (UK) inhibitor PAI-II, a product of activated monocytes, released into MCS were determined (fibrin plate assay). To examine the activation state of PBMs independently, expression of GM1 ganglioside on PBMs from patients with AIDS (n = 9), patients with AIDS-related complex (ARC) (n = 8), HIV+ asymptomatic patients (n = 6), and HIV- healthy controls (n = 11) was determined (flow cytometry; living cells in suspension). Data are expressed as percent inhibition of UK, or as percent total cells. Patients' MCS collected on days 1-4 of culture contained similar levels of PAI-II because it inhibited UK in similar fashion (70-90%). In contrast, MCS from healthy controls, collected after 2 days, had decreased ability to inhibit UK (15-50%) and thus contained lower levels of PAI-II. Monocyte activation, measured by increased expression of GM1 ganglioside on PBM surfaces, directly correlated with the progression of HIV infection into the development of AIDS, since the order of magnitude of GM1 ganglioside expression on PBMs was AIDS greater than ARC greater than HIV+ asymptomatic = healthy controls. Our data indicate that PBMs from patients with AIDS are constitutively activated and suggest that activation directly correlates with disease progression.

Antigen specific IgE responses originate in gut associated lymphoid tissue (GALT) of mice sensitized with benzylpenicilloyl-keyhole limpet hemocyanin (BPO-KLH) in alum, regardless of the route (intraperitoneal [i.p.], oral [gavage], subcutaneous [s.c.], intramuscular [i.m.] or intravenous [i.v.]) used for immunization. When BALB/c mice were injected i.p. with BPO-KLH (10 micrograms) in alum, B lymphocytes bearing membrane bound IgE (sIgE+B cells) first appeared simultaneously in Peyer's patches (PP) and mesenteric lymph node (MLN) on day 8. BPO specific IgE antibody forming cells (AFC) also appeared in PP on day 8, but were not found in MLN until day 10. On day 8, no sIgE+B cells or IgE AFC were found in bone marrow (BM) or other lymphoid organs. The appearance of sIgE+B cells and IgE AFC in PP and MLN was transient; these cells were no longer detected in PP on days 14 and 24, respectively, or in MLN on days 14 and 36, respectively. sIgE+B cells and IgE AFC did not appear in spleen until day 12, where they were detected through day 70. Although sIgE+B cells were never found in BM, IgE AFC appeared in BM on day 18, where they were detected through day 70. No sIgE+B cells or IgE AFC were found in other lymph nodes (OLN) on days 0-70. Boosting did not induce the reappearance of sIgE+B cells or IgE AFC in PP, the reappearance of sIgE+B cells in MLN, the appearance of sIgE+B cells in BM, or the appearance of sIgE+B cells or IgE AFC in OLN.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmin activity induced by different concentrations of added urokinase (UK) in serum from 20 patients with AIDS (P) and 10 healthy control (HC) subjects was measured using the fibrin plate assay. 125I-fibrin coated 24 well plates were exposed to UK (0.34-6.8 ng/ml) or to trypsin (T) (2.5 micrograms/ml) in the presence of serum (10%) from P or HC. Control wells were exposed to either T or tris buffer (pH 8.1) alone. Volumes were adjusted to 1 ml with buffer and after 1 hr at 37 degrees C, radioactivity (cpm) released into the medium was determined using a gamma counter. Data are expressed as % plasmin activity or as % inhibition of plasmin activity. All sera from HC totally abrogated (greater than 98%) the plasmin activating ability of UK (1.7 ng/ml). In contrast, sera from approximately 50% of P were less able to inhibit plasmin activation (to 26%). The inability of P sera to inhibit plasmin activation was specific since P and HC sera were equally capable of inhibiting T. Mixing experiments using P and HC sera demonstrated that P sera did not block the ability of HC sera to inhibit plasmin activation. The inhibitory activity of HC and active P sera eluted in the void volume of a spehadex G100 column (MW greater than 100,000 daltons) and is acid sensitive, however HC sera also contains an acid stable inhibitor(s) of plasmin activation not detected in P sera. These data suggest dysregulation of UK dependent proteolysis may be associated with AIDS.

A new murine model system was developed to study hapten specific IgE anamnestic antibody forming cell (AFC) responses induced in vitro. BALB/c mice injected intraperitoneally with BPO-KLH (10 micrograms) in aluminum hydroxide gel (alum) on day 0 and 21 were killed on day 42. Spleen cells (4 X 10(7] were cultured for 0-8 days in the presence of BPO-KLH (0.25-2500 ng/ml), after which the numbers of BPO specific IgE AFC were enumerated in an ELISPOT assay at 37 degrees C using BPO-BSA coated plates. Peak BPO specific IgE anamnestic AFC responses (approximately 40 AFC/10(7) cultured cells) occurred when cells were cultured with 100 ng/ml BPO-KLH for 5 days; peak BPO specific IgG1 and IgA responses (approximately 3000 and approximately 280 AFC/10(7) cultured cells) were detected one day earlier with the same antigen concentration. No ELISPOTs of any isotype were detected if: cells were cultured in the absence of specific antigen; cells from alum treated or unsensitized mice were used; assay wells were coated with BSA alone; isotype specific antibodies were omitted; or assays were performed in the presence of cycloheximide or at 4 degrees C. Cellular interactions in IgE anamnestic AFC responses were studied using the newly developed model system. We found that these responses were partially T cell independent, and that spleens from sensitized and unsensitized adult mice, as well as neonatal mice, contained cells which suppressed these responses.

Sensitized T cells in inguinal lymph nodes (LN) of rats immunized with ovalbumin and adjuvant can be separated on BSA density gradients from T cells responding to Con A and PHA. Further, these OA-sensitized T cells are separable from B memory cells responsive to OA. On days 9 and 17 after sensitization, OA-reactive cells peak and are found in enriched numbers in the lighter bands A + B (B-band), and C, respectively whereas Con A and PHA reactive cells, on both days, are concentrated in bands C and D, respectively. On day 9 at least 20 to 30% of B-band cells can reinitiate DNA synthesis and produce high concentrations of lymphotoxin in response to OA in culture, but show only a marginal blastogenic response to Con A or PHA and cannot produce lymphotoxin in response to PHA. These cells are defined as OA-sensitized T cells because a) treatment with rat isoantiserum specific for T lymphocytes plus complement abolished their ability to incorporate 3H-thymidine in response to OA and Con A or PHA; b) membrane-bound immunoglobulin could not detected with indirect immunofluorescence staining; c) histologic studies demonstrate massive hypertrophy of thymus-dependent paracortical areas and absence of germinal centers in inguinal LN at this time, and d) they produce lymphotoxin after challenge with OA, but not with PHA, whereas cells of the other bands produce lymphotoxin in response to PHA, but not to OA. Addition of purified macrophages to the various cultures did not affect the levels of response observed. Day 17 C-band cells are regarded as B-memory cells because treatment with T-specific antiserum plus complement enhanced their response to OA, many germinal centers now are present in inguinal LN, and a significant proportion of cells of bands B and C, negative on day 9, show positive immunofluorescence staining at this time.

Lymph node cells of ovalbumin-sensitized rats were separated on the basis of buoyant density into fractions reciprocally enriched in cells responsive to ovalbumin or phytohemagglutinin (PHA). Recombination of high density and low density fractions in varying proportions resulted in potentiation or suppression of the DNA synthetic response to PHA in culture. The response of cultures containing equal numbers of high and low density cells was markedly greater than the sum of the two populations stimulated separately. However, when decreasing numbers of low density cells were cultured with a constant number of high density cells, profound suppression was observed.

Peyer's patches (PP) from adult conventionally raised (C) and germ-free (GF) rats of the same age differed strikingly in the distributions of cells bearing membrane-bound immunoglobulin of the different isotypes. High concentrations of cells with membrane-bound IgE (approximately 20% of total cells determined by indirect immunofluorescence) were found in PP of rats, whereas IgE+ cells were absent from PP of C rats. The numbers of IgA+ cells were almost threefold higher in PP of GF rats when compared with C rats. In contrast, PP of GF rats had greatly reduced numbers of IgM-bearing cells (4%) when compared with C rats (23%); in some experiments virtually no IgM-bearing cells were detected. The levels of IgA- and IgE-bearing cells in spleen of GF rats were increased (to 11% and 7%, respectively). Of the IgE-bearing cells in PP and spleen of GF rats, approximately one-half were simultaneously positive for IgA. When these PP cells were treated with pronase to remove membrane bound immunoglobulins and maintained in culture, both IgE and IgA reappeared within 12 h. The proportion of doubly labeled cells was similar to that of the untreated population. No IgE+ cells were detected in bone marrow of C of GF rats at any time, in agreement with the findings of Ishizaka et al., although up to 20% of bone marrow cells bore other immunoglobulin isotypes, suggesting that IgE-bearing cells arise in the PP either de novo or by switching from precursors carrying IgM or IgA.

Mechanisms regulating the appearance of sIgE+ B lymphocytes appear to be lacking in adult germfree (GF) rats in that their Peyer's patches (PP) contain high numbers of cells with sIgE (approximately 15% of total cells), one-half of which simultaneously express sIgA, whereas sIgE+ cells are absent from PP of conventional rats (less than 1%). GF rat PP also contain elevated numbers of sIgA+ cells and decreased numbers of sIgM+ cells, with elevated numbers of sThy-1+ RT 7.1+ Ig- T cells, and reduced numbers of sThy-1- RT 7.1+ Ig- T cells. The cellular composition of PP of GF rats was converted to that resembling a conventional rat within 18 h after either 1) use of standard (unautoclaved) food; 2) feeding with certain bacteria (Clostridium difficile, Corynebacterium pseudodiphtheriticum, Mycobacterium tuberculosis, and Klebsiella pneumoniae), in either live or heat-killed, but not autoclaved form; or with certain bacterial cell wall components: murein (peptidoglycan), and its synthetic derivatives, muramyltripeptide phosphatidylethanolamine and desmethyl-muramyldipeptide, but not with LPS, core lipid A or lipoprotein; there was no effect if any bacterial cell wall component was injected i.v.; or 3) thymectomy. Each procedure resulted in elimination of sIgE+ B cells and normalization of the other surface isotypes, and loss of sThy-1+ RT 7.1+ Ig- T cells and normalization of sThy-1- RT 7.1+ Ig- T cells. Irrespective of treatment, no sIgE+ cells were detected in bone marrow, thymus, other lymphoid organs or blood, excluding the possibility that the elimination of these cells from PP was associated with their redistribution to other sites. Thus, exposure to gut flora and bacterial peptidoglycan components may have resulted in IgE isotype switching, either directly or through the mediation of accessory and/or sThy-1+ RT 7.1+ regulatory T cells. The sites in which sIgE+ B cells are down-regulated appear to be PP.

T memory cells specifically responsive to ovalbumin and performing the diverse functions of DNA synthesis, lymphotoxin release, and regulation can be isolated in enriched numbers in the most buoyant fractions (A+B) of bovine serum albumin gradients on day 9 after sensitization. At least 20-30% of these cells are capable of mounting a blastogenic response to ovalbumin. A+B cells responding to ovalbumin with DNA synthesis have adherent properties and are further enriched on passage through glass wool. The subpopulations capable of entering into blastogenesis and DNA synthesis and of lymphotoxin release are unresponsive to T mitogens. A+B cells are capable of either potentiating or suppressing DNA synthetic responses to both phytohemagglutinin and antigen when added to 5 X 10(5) D cells in different proportions. Potentiation or suppression of phytohemagglutinin responses were observed with 1 X 10(5) A+B cells, and total suppression was observed with A+B in the range of 4 X 10(3) to 2 X 10(4). The response to antigen was sometimes inhibited in the same cell combinations that gave a potentiated response to phytohemagglutinin and vice versa. Regulatory cells in this system were not macrophages since their effect was not mimicked by addition of peritoneal macrophages, and ablation of macrophages by carrageenan affected neither the potentiation nor suppression.

Peyer's patches (PP) in germ-free rats (GF) and in the hyper-IgE syndrome patient (HIES) differ from their conventional rat (C) and healthy human (HH) counterparts in that GF rats contained fewer (two-fold) PP and none was detected in HIES. Existing PP in GF rats had reduced cellularity (three-fold) and different B and T cell subsets: high numbers of IgE-bearing (sIgE+) B cells (approximately 15% of total cells), one-half of which also expressed sIgA, were present in GF rat PP while none was detected in C rat PP (less than 1%). GF rat PP also contained elevated numbers of sIgA+ cells and decreased sIgM+ cells, with elevated numbers of sThy 1+ RT 7.1+ Ig- T cells (suppressor phenotype) and reduced sThy 1- RT 7.1+ Ig- T cells (helper phenotype). The cellular composition of GF rat PP was converted to that resembling a C rat within 18 hr after (a) use of standard (unautoclaved) chow; (b) feeding with certain bacteria or "working" bacterial cell wall components (BCWC) and synthetic derivatives, murein, MTP-PE, and norMDP, but not with LPS, core lipid A, or lipoprotein; BCWC had no effect if injected intravenously; or (c) thymectomy. Each procedure resulted in (i) elimination of sIgE+ B cells and normalization of the other isotypes, and (ii) loss of T suppressor cells and normalization of T helper cells. After treatments, no sIgE+ cells were detected in bone marrow (BM), thymus, other lymphoid organs, or blood. PP were not detected in HIES, although they were present in HH (approximately 10/individual). P blood contained two distinct sIgE+ B cell subpopulations, the apparent source of which was mesenteric lymph node (MLN), the only organ in which high numbers of these cells (35%) (five nodes examined) were detected; far fewer IgE+ cells were found in spleen (less than 5%), and none was detected in BM, thymus, other LN, or appendix, which was virtually acellular. Virtually no IgE secreting plasma cells were detected in MLN, spleen, appendix, other lymphoid organs, or in gut lamina propria. IgE+ B cells in MLN were not detected in follicles (classical B cell areas); instead, they were found in high numbers in the thymus-dependent area and in medulla. Most follicles (greater than 98%) in MLN and spleen contained intercellular IgE complexed to bacterial antigen and/or CD23 (IgE-binding factor? antigen?), but contained no germinal centers.(ABSTRACT TRUNCATED AT 400 WORDS)