Production of early and late pulmonary responses with inhaled leukotriene D4 in allergic sheep

Some allergic sheep respond to inhalation of antigen with both immediate and late increases in airflow resistance (late response). The mechanism of the late response is unknown but recent evidence suggests that the initial generation of slow-reacting substance of anaphylaxis (SRS-A) immediately after antigen challenge is a necessary pre-requisite for the physiologic expression of this late response. Based on this evidence we hypothesized that airway challenge with leukotriene D₄ (LTD₄), an active component of SRS-A would produce acute and late airway responses in allergenic sheep similar to those observed with antigen. In five allergic sheep with documented early and late pulmonary responses to antigen, inhalation of leukotriene D₄ aerosol (delivered dose {mean ±SE} 0.55±0.08 ug) resulted in significant early and late increases in specific lung resistance (SR_L). In three allergic sheep which only demonstrated acute responses to antigen, LTD₄ aerosol (delivered dose 0.59±0.09ug) only produced an acute increase in SR_L. In the late responders pretreatment with aerosol cromolyn sodium (1 mg/kg) did not affect the acute response but blunted the late increase in SR_L. Pretreatment with aerosol FPL-57231 (1% w/v solution) completely blocked both the acute and late responses. These data support the hypothesis that initial release of LTD₄ in the airways of sensitive animals is important for the physiologic expression of the late response.     

Immune retention: immunological requirements for maintaining an easily degradable antigen in vivo.

Radioactive human serum albumin (125I-HSA) was injected into the hind foot pads of unimmunized mice, actively immunized mice and mice passively immunized with mouse or rabbit anti-HSA serum. Eleven days later the unimmunized mice had cleared most of the 125I-HSA. In contrast, a high concentration of 125I-HSA was retained in the feet and draining lymph nodes and, to a lesser extent, in the spleen of the actively or passively immunized mice. Although immune retention required specific antibody, it appeared to be independent of T-cells or T-cell factors, since passively immunized nude mice retained antigen as well as actively or passively immunized normal mice. Depletion of the complement system with cobra venom factor (CVF) increased antigen retention in the feet but decreased retention in the spleen. Treatment with CVF did not decrease antigen retention in lymph nodes of actively immunized mice. Such treatment did, however, decrease retention in lymph nodes of passively immunized mice although not to the same extent as in the spleen. Retention of antigen in the feet was not only complement-independent but was also Fc independent, since F(ab')2 fragments of IgG could mediate immune retention. Antigen dose response studies indicated that immune retention in lymph nodes occurred optimally with minute amounts of antigen, whereas optimal retention in the feet required much higher concentrations of antigen. Foot pad injections of non-radiolabelled HSA eliminated 60% of the radioactivity retained in the foot pads of immunized mice. In contrast, non-radioactive egg albumin (EA) had almost no effect on retained HSA. However, if the mice were immunized to both EA and HSA, an injection of EA would displace a significant amount of retained HSA. Complexes of one specificity can apparently displace some retained antigen of a differing specificity.     

Production of early and late pulmonary responses with inhaled leukotriene D4 in allergic sheep

Some allergic sheep respond to inhalation of Ascaris suum antigen with both immediate and late increases in airflow resistance (late response). The mechanism of the late response is unknown but recent evidence suggests that the initial generation of slow-reacting substance of anaphylaxis (SRS-A) immediately after antigen challenge is a necessary pre-requisite for the physiologic expression of this late response. Based on this evidence we hypothesized that airway challenge with leukotriene D4 (LTD4), an active component of SRS-A would produce acute and late airway responses in allergic sheep similar to those observed with antigen. In five allergic sheep with documented early and late pulmonary responses to Ascaris suum antigen, inhalation of leukotriene D4 aerosol (delivered dose (mean +/- SE) 0.55 +/- 0.08 ug) resulted in significant early and late increases in specific lung resistance (SRL). In three allergic sheep which only demonstrated acute responses to antigen, LTD4 aerosol (delivered dose 0.59 +/- 0.09 ug) only produced an acute increase in SRL. In the late responders pretreatment with aerosol cromolyn sodium (1 mg/kg) did not affect the acute response but blunted the late increase in SRL. Pretreatment with aerosol FPL-57231 (1% w/v solution) completely blocked both the acute and late responses. These data support the hypothesis that initial release of LTD4 in the airways of sensitive animals is important for the physiologic expression of the late response.     

Late pulmonary allergic responses in actively but not passively IgE-sensitized rats

Previous studies suggested that although rats that were passively sensitized [monoclonal murine immunoglobulin E (IgE)] would respond to pulmonary antigen challenge with an immediate increase in resistance, they exhibited no late increases in resistance, unlike late changes in rats actively sensitized to preferentially produce IgE antibody. We hypothesized that passively sensitized rats also would not develop antigen-induced pulmonary inflammation. In a blinded protocol we compared immediate responses and pulmonary resistance and inflammation at 8, 19 and 24 h after challenge with placebo antigen, with dinitrophenol-bovine serum albumin (DNP-BSA) to elicit a passively sensitized response, or with ovalbumin (OA) to elicit an actively sensitized response. Despite similar immediate responses to OA and DNP-BSA, only the rats challenged with OA had marked inflammatory changes and a significant incidence of late elevations in resistance. Inflammation scores and lung resistance were significantly correlated only in the OA group. We also observed that anesthesia with fentanyl/droperidol significantly attenuated the immediate but not the late responses to antigen challenge, compared with rats anesthetized with ketamine. We conclude that IgE-mediated immediate responses to pulmonary antigen challenge are insufficient, and may be unnecessary, to initiate antigen-induced late inflammatory changes.     

Immunosuppression in experimental cryptococcosis in rats

Using a rat model, we have previously demonstrated that infection with Cryptococcus neoformans can trigger the production of a series of suppressor cells that specifically inhibit the cell-mediated immune response to a non-related antigen, human serum albumin (HSA), that has been injected 7 days after the infection. We previously determined that the cryptococcal infection induces afferent suppressor or suppressor induction cells (Ts₁) to HSA. The primary objective of the present study was to investigate the suppressor cells involved in the efferent phase of delayed-type hypersensitivity (DTH) response to HSA in rats infected with C. neoformans and immunized with the non-related antigen and determine the role that the Ts₁ cell plays in the induction of that cell. For this purpose, the spleen mononuclear (SpM) cells containing the Ts₁ or SpM cells from immunized non-infected rats (used as donor controls) were transferred to two groups of syngeneic naive recipients (first recipients). Later, the SpM cells from both groups of animals were transferred to rats immunized with HSA (second recipients). The efferent limb of the DTH response to HSA was suppressed in the recipients that received SpM cells from donors injected with Ts₁ cells. Additional HSA antigen was not required for induction of these efferent suppressor cells. Furthermore, we here show that these cells are resistant to treatment with cyclophosphamide (Cy), and that they can activate another suppressor population. The latter are Cy sensitive and are present in the immune recipient.     

Clinical hypersensitivity to specific aerosol challenge in parenterally immunized calves.

This paper reports an experiment designed to demonstrate that the calf lung can be sensitized to a specific respirable challenge following parenteral immunization with a nonliving antigen (human serum albumin). The possibility that immune-mediated injury could subsequently interfere with nonspecific mucosal defenses was also investigated by infecting calves with Pasteurella haemolytica after the antigen challenge and assessing pulmonary clearance of the organism. The results indicated that specific aerosol challenge produces reversible signs of respiratory hypersensitivity and that persistence of incidental infection in the upper respiratory tract is potentiated. Since the calves were sensitized by an immunization regime which imitated conventional vaccination, this study highlights the potential dangers of inactivated parenteral respiratory vaccines.     

Comparison of simian virus 40 large T antigen recombinant protein and DNA immunization in the induction of protective immunity from experimental pulmonary metastasis

In this report we examine the ability of a recombinant tumor antigen preparation to prevent the establishment of experimental pulmonary metastasis. Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-Ag) was injected into BALB/c mice followed by challenge with an intravenous injection of syngeneic SV40-transformed tumorigenic cells. The experimental murine pulmonary metastasis model allows for the accurate measurement of metastatic lessions in the lungs at various times after the challenge, using computer-assisted video image analysis. Following challenge, lung metastasis and survival data for the groups of mice were obtained. Animals immunized with recombinant SV40 T-Ag showed no detectable sign of lung metastasis and survived for more than 120 days after challenge. Antibodies specific for SV40 T-Ag were detected in the serum of immunized mice by enzyme-linked immunosorbent assay. Splenocytes obtained from mice immunized with recombinant SV40 T-Ag did not lyse syngeneic tumor cells, indicating that no cytotoxic T lymphocyte response was induced. Control mice developed extensive lung metastasis and succumbed to lethal tumor within 4 weeks after challenge. These data indicate that immunization with the recombinant SV40␣T-Ag induces protective, T-Ag-specific immunity in an experimental pulmonary tumor metastasis model. Received: 20 August 1998 / Accepted: 25 November 1998     

The ovine nasal mucosa: an alternative tissue site for mucosal immunization

The ovine nasal mucosal environment has histological and ultrastructural features that resemble well-known inductive sites of mucosa-associated lymphoid tissue. In the present study, the nasal mucosa was assessed as a potential mucosal tissue site for delivering vaccines to sheep. Sheep were immunized by either injection with the model antigen, Keyhole Limpet Haemocyanin (KLH), and aluminium hydroxide gel (alum) or by aerosol spray with KLH with and without cholera toxin (CT). Sheep immunized by injection with KLH/alum and aerosol spray with KLH/CT induced strong anti-KLH IgG and IgA serum antibody responses as well as specific T cell memory. Anti-KLH IgG1 responses were significantly higher following immunization by injection and no significant differences in anti-KLH IgG2 responses were detected between groups. Sheep immunized with KLH by aerosol spray without CT did not produce serum antibody and T cell memory responses. Antibody-secreting cells were present in the parotid lymph nodes (draining lymph nodes) of sheep immunized with KLH/alum and KLH/CT, but secreted only Ag-specific IgG1, and not IgG2 or IgA. These results suggest that aerosolization of soluble antigen formulations with CT may provide an alternative method of delivering nasal vaccines to sheep and other large animal species, and that further improvements in antigen penetration of nasal tissues may dramatically improve the strength of the immune response.     

Studies on the mechanism of suppression by T cells in an adoptive secondary response.

Suppression of antibody synthesis by lymphocytes was studied using an adoptive secondary response model in which human serum albumin (HSA)-primed lymphocytes (memory cells) from the thoracic ducts of inbred rats were inhibited in irradiated recipients by nonimmune lymphocytes after mixed cell transfer. This investigation extended earlier work and formally showed that the suppressor cells were peripheral thymus-derived lymphocytes, which could rapidly recirculate from the blood to lymph, were present in spleen but not in bone marrow, and that primed T cells lacked this property to inhibit. The suppressive effect was independent of antigen dose but was markedly influenced by the form of antigen used for challenge in that suppression was significantly abrogated with aggregated HSA or with soluble HSA in the presence of specific antibody. Suppressor cells were found to exert their effect maximally at the time of antigen injection, but became ineffective by 40 hr following challenge. The results are considered within a larger framework of cellular regulation of antibody synthesis.     

In vitro evidence indicating a role for the Fc region of IgG in the mechanism for the long-term maintenance and regulation of antibody levels in vivo

The synthesis of anti-human serum albumin (HSA) antibody was induced spontaneously in cell cultures prepared from the draining lymph nodes of rabbits immunized months earlier with polymerized HSA. Serum from HSA-immunized rabbits suppressed this response. Removal of specific antibody from immune serum eliminated suppression and the addition of specific IgG restored suppression, indicating that the feedback phenomenon may be explained by an effect of specific IgG antibody. Fab and F(ab′)₂ fragments masked antigen as effectively as IgG; however, they were markedly inferior to IgG in mediating suppression. Furthermore, Fab competed with IgG and interfered with IgG mediated suppression. The addition of small amounts of antigen to antibody-suppressed cultures induced an antibody response. The level of induction was proportional to the antigen-antibody ratio. However, 80 to 100 times the antibody concentration needed to mask all antigenic determinants was needed in order to eliminate induction of antibody synthesis. High concentrations of antigen-antibody complexes at equivalence also suppressed the spontaneous response. This suppression was similar to antibody mediated suppression at the spontaneous response in that the Fc region of IgG was required.     

Antigen sensitization and methacholine responses in dogs with hyperreactive airways

Antigen sensitization was induced in six Basenji-Greyhound (BG) dogs by weekly aerosol exposure to Ascaris suum. The effects on airway responsiveness to inhaled methacholine were studied before and at least 2 wk following Ascaris sensitization. All dogs developed detectable serum levels of Ascaris-specific immunoglobulin E (IgE), and five out of six dogs developed airway responsiveness to antigen over the 4- to 6-mo period. This was accompanied by a decrease rather than an increase in airway responsiveness to inhaled methacholine. When dogs were challenged with methacholine 30 min after Ascaris antigen aerosol challenge, however, dogs reactive to Ascaris became hyperresponsive to methacholine. The magnitude of the response to antigen correlated (r = 0.85) inversely with the dose of methacholine increasing pulmonary resistance 200%. These data show that in BG dogs airway responsiveness to methacholine is increased by acute antigen exposure but that sensitization of previously unsensitized dogs does not increase nonspecific airway responsiveness.     

The effect of arachidonic acid on airway responses of rhesus monkeys.

Rhesus monkeys with immediate-type airway responses to ascaris antigen were used to study the effect of arachidonic acid (Ar). Arachidonic acid produced no changes in pulmonary function parameters when delivered by aerosol at varying doses. However, when Ar was aerosolized prior to aerosol antigen challenge this resulted in a selective increase in pulmonary resistance (PR). This effect in PR was variable with intravenous Ar. Indomethacin (I) by aerosol was evaluated to determine whether it would block the post Ar increase in PR. This could not be accomplished because both aerosolized and intravenous I had a similar effect on the PR following antigen challenge. These effects of Ar and I may be due to production of airway reactive prostaglandins, their precursers or substances such as slow reacting substance.     

Interaction between granulocytes and antibodies in the enhancement of lung defenses against Staphylococcus aureus after intranasal immunization of mice with live-attenuated bacteria

Abstract Immunization with live-attenuated Staphylococcus aureus induced measurable levels of specific IgG and IgA in the lungs, but the pulmonary clearance of S. aureus in immunized mice did not differ from that of control mice. Aerosol exposure of mice to Pseudomonas aeruginosa induced a significant recruitment of polymorphonuclear leukocytes (PMNL) to the lungs in both immunized and control mice, whereas S. aureus challenge did not. However, challenge with a mixture of P. aeruginosa-S. aureus or exposure to an aerosol of Escherichia coli lipopolysaccharide (LPS) before S. aureus challenge induced PMNL migration and a significant enhancement of pulmonary clearance of S. aureus in immunized mice. The presence of both antibodies and PMNL was required for enhancement of S. aureus pulmonary clearance.     

Influence of active and passive immunity on the disposition of dihydromorphine-H3

The effect of circulating antibodies on the plasma and brain concentration of dihydromorphine-³H was examined. Rabbits immunized with the morphine antigen 3-0-carboxymethyl morphine coupled to bovine serum albumin served as the source of the antibodies which were used for passive immunization of mice and rats. The 45 minute plasma concentration of the narcotic was increased 90–100 fold in passively immunized animals whereas the brain concentration decreased by at least 75 percent. Mice were also actively immunized with the morphine antigen. Compared to non-immunized mice, plasma levels of dihydromorphine-³H were increased in the immunized mice from 7–30 fold at all intervals measured for at least 4 days. The plasma half life was markedly slowed in immunized mice. The narcotic bound to the antibody in the plasma $\text{in vivo}$ could be displaced by the administration of morphine. The consequence of active immunization on brain narcotic content varied with the dose of the drug and time interval studied. We suggest the possibility that the antibodies may initially act to sequester the narcotic but with time as the narcotic is slowly released from the antibodies that they may also act as a circulating source of the drug. It is apparent that the presence of circulating antibodies can have marked effects on the disposition of narcotics.     

Intraocular Ia expression in vivo induced in the absence of immune T cells

The expression of Ia antigen on the ciliary body, retinal pigment epithelium, and retinal vascular endothelium was investigated using two models of ocular inflammation. Active systemic immunization with bovine serum albumin with subsequent ocular challenge resulted in increased Ia expression in the ciliary body and retinal pigment epithelium. This was compared with passive transfer of hyperimmune serum to bovine serum albumin followed by ocular challenge when Ia expression was found to occur only in the ciliary body. An intraocular T-cell infiltrate occurred only in the actively immunized animals and was not present after passive transfer, suggesting that the Ia induction in this latter situation occurred in the absence of T cells.     

Immunocytochemical staining of Histoplasma capsulatum at the electron microscopic level

Summary Rabbits were immunized with histoplasmin emulsified in Freund's complete adjuvant. Antibody raised in these rabbits was exposed to Histoplasma capsulatum yeast cells, either in tissue culture medium, or after in vitro or in vivo phagocytosis by mouse macrophages. The sites of antibody binding were identified using an immunoperoxidase technique. At least two sites of antibody binding were identified, one to the fungal cell wall and the other to the outer cell membrane. Within 6 h after phagocytosis by macrophages, fungal cell walls appeared roughened, with what appeared to be cell wall antigen released into the phagolysosome, appearing associated with the phagolysosome membrane, and possibly adjacent macrophage cytoplasm. Similar staining of fungal antigen was noted in alveolar macrophages which had ingested Histoplasma capsulatum after a respiratory challenge. This method may be useful in detailing the host/pathogen interactions which occur in human pulmonary histoplasmosis.     

Passively acquired antibodies suppress humoral but not cell-mediated immunity in mice immunized with live attenuated respiratory syncytial virus vaccines

A respiratory syncytial virus (RSV) vaccine will need to be administered by 1 mo of age to protect young infants; therefore, it will need to be effective in the presence of maternally acquired RSV Abs. In the present study, the immunogenicity and efficacy of two live attenuated RSV vaccine candidates of different level of attenuation were evaluated in mice passively immunized with varying quantities of RSV Abs. The replication of the RSV vaccines was suppressed in the lower, but not the upper, respiratory tract of the passively immunized mice. Immunization with either vaccine candidate was highly efficacious against challenge with wild-type RSV in both passively immunized and control mice. Nonetheless, a high level of immunity was seen even in passively/actively immunized animals that failed to develop a humoral immune response, suggesting that T cells mediated the immunity. Depletion of CD4+ and CD8+ T cells in passively/actively immunized and control animals at the time of challenge with wild-type RSV demonstrated that CD4+ and CD8+ T cells made significant independent contributions to the restriction of replication of RSV challenge virus in both the upper and lower respiratory tracts. Although passively acquired serum RSV Abs suppressed the primary systemic and mucosal Ab responses of IgM, IgG, and IgA isotypes, B lymphocytes were nevertheless primed for robust secondary Ab responses. Thus, immunity mediated by CD4+ and CD8+ T cells and Abs can be readily induced in mice by live RSV vaccine candidates in the presence of physiologic levels of RSV neutralizing Abs.     

Preparation and immunochemical properties of methoxypolyethylene glycol-coupled and N-carboxymethylated derivatives of ragweed pollen allergen, antigen E.

A methoxypolyethylene glycol (PEG)-coupled and several N-carboxymethylated (N-CM) derivatives of antigen E, the major allergenic protein of ragweed pollen, were prepared. The PEG derivative contained seven residues of PEG groups (residue weight about 2100) per molecule of protein and the groups were linked to the lysyl residues of antigen via the 2,6-positions of 4-hydroxy-triazine nucleus. The maximally N-CM derivative contained, respectively, 10, 6, and 2 residues of mono-CM, di-CM, and unmodified lysyl residues per molecule of protein. The CM groups were introduced reductively on reaction with glyoxylic and sodium cyanoborohydride and the extent of mono- and dicarboxymethylation was controlled more by the concentration of cyanoborohydride than by that of glyoxylic acid. The molar allergenic activities of the PEG and the N-CM derivatives in man were, respectively, 0.02 and 0.5 of that of the native antigen. Rabbits immunized with the PEG derivative gave antibody titers about $\text{1}\text{8}$th of those obtained with animals immunized with the native antigen. However, the rabbits preimmunized with the PEG derivative gave a vigorous secondary response on challenge with the native antigen and their titers approached those of rabbits preimmunized with the native antigen. The immunogenicity of the reduced and S-carboxymethylated derivative of antigen E which has the denatured conformation was studied as a control. Rabbits immunized with the S-CM derivative gave antibody titers $\text{1}\text{34}$th of those obtained with animals immunized with the native antigen; on secondary challenge with the native antigen, these rabbits gave antibody titers about $\text{1}\text{6}$th of those of animals preimmunized with the native antigen.     

The Basenji-Greyhound dog model of asthma: leukocyte histamine release, serum IgE, and airway response to inhaled antigen

To understand the immunologic mechanisms underlying the variation in airway response to inhaled Ascaris antigen (AA) in Basenji-Greyhound (BG) dogs having hyperreactive airways, we examined the relationship between leukocyte histamine release, Ascaris-specific serum IgE, changes in pulmonary resistance (RL), and decreases in dynamic compliance (Cdyn). All Ascaris-sensitive BG dogs showing airway responses to AA aerosol challenge exhibited an antigen dose-dependent release of leukocyte histamine, with total leukocyte histamine ranging from 68 to 123 ng/10(7) cells. Airway response to inhaled antigen more closely correlated with antigen dose releasing 50% total leukocyte histamine (RL, r = 0.94); Cdyn, r = 0.82), than with circulating levels of antigen-specific IgE (RL, r = 0.68; Cdyn, r = 0.69). We conclude that the airway response of sensitized BG dogs to AA inhalations is more dependent on factors affecting mediator release from pulmonary sources than circulating specific reaginic antibody.     

Vaccine-induced serum immunoglobin contributes to protection from herpes simplex virus type 2 genital infection in the presence of immune T cells

Herpes simplex type virus 2 (HSV-2) is a sexually transmitted pathogen that causes genital lesions and spreads to the nervous system to establish acute and latent infections. Systemic but not mucosal cellular and humoral immune responses are elicited by immunization of mice with a replication-defective mutant of HSV-2, yet the mice are protected against disease caused by subsequent challenge of the genital mucosa with virulent HSV-2. In this study, we investigated the role of immune serum antibody generated by immunization with a replication-defective HSV-2 vaccine prototype strain in protection of the genital mucosa and the nervous system from HSV-2 infection. Passive transfer of replication-defective virus-immune serum at physiologic concentrations to SCID or B-cell-deficient mice had no effect on replication of challenge virus in the genital mucosa but did significantly reduce the incidence and severity of genital and neurologic disease. In contrast, B-cell-deficient mice immunized with replication-defective HSV-2 were able to control replication of challenge virus in the genital mucosa, but not until 3 days postchallenge, and were not completely protected against genital and neurologic disease. Passive transfer of physiologic amounts of immune serum to immunized, B-cell-deficient mice completely restored their capacity to limit replication of challenge virus in the genital mucosa and prevented signs of genital and systemic disease. In addition, the numbers of viral genomes in the lumbosacral dorsal root ganglia of immunized, B-cell-deficient mice were dramatically reduced by transfer of immune serum prior to challenge. These results suggest that there is an apparent synergism between immune serum antibody and immune T cells in achieving protection and that serum antibody induced by vaccination with replication-defective virus aids in reducing establishment of latent infection after genital infection with HSV-2.