Modulation of T-helper cell populations: potential mechanisms of respiratory hypersensitivity and immune suppression.

Information presented at this symposium indicates that modulation of Th cell responses is one means by which xenobiotics may cause immunotoxicity. A shift from Th1 to Th2 responses can enhance both infectious and allergic disease. Hence, in some cases, a common mechanism may be responsible for effects that are generally considered to be very different. Because cytokines produced in the inflammatory process play a role in modulation of Th cell responses, there is a mechanism by which agents that appear to have only local effects at the portal of entry may, in fact, affect immune responses systemically. An understanding of conditions which trigger certain cytokine responses may be useful not only in understanding inflammation but also in predicting certain kinds of immunosuppressive and allergic responses. Future studies in this area are likely to provide insights into many areas of immunotoxicology.

The regulation of pulmonary immunity.

No evidence has emerged which suggests that the principles of immunity derived from studies on cells from other body sites are contradicted in the lung and its associated lymphoid tissue. What is clear, however, is that the environment dictates the types of cells, their relationship to one another, and what perturbing events will set in motion either the development of an "active" immune response or tolerance. Investigating mechanisms for the development of lung immunity has increased our understanding of how human diseases develop and is continuing to suggest new ways to manipulate pulmonary immune responses. Demonstration that lung cells regulate both nonspecific inflammation and immunity through the expression of adhesion molecules and the secretion of cytokines offers hope for ways to design more effective vaccines, enhance microbial clearance in immunosuppressed hosts, and to suppress manifestations of immunologically mediated lung disease. Important lung diseases targeted for intensive research efforts in the immediate future are tuberculosis, asthma, and fibrotic lung disease. Perhaps even the common cold might be conquered. Considering the pace of current research on lung immunity, it may not be too ambitious to predict that these diseases may be conquered in the next decade.

Intrapulmonary antigen deposition in the human lung: local responses.

We hypothesized that, as in animal models, localized deposition of antigen into the human lung would induce local inflammatory and immune responses in antigen-exposed sites. To test this hypothesis, segmental instillation of a well-characterized, highly immunogenic, soluble antigen, keyhole limpet hemocyanin (KLH) was performed in 10 healthy, nonsmoking volunteers. Ten to fifteen days after instillation, bronchoalveolar lavage (BAL) was performed in immunized segments (IS) and contralateral control segments (CS) and local responses to antigen instillation were assessed by comparing IS and CS BAL. Greater albumin concentrations and cell recoveries were found in IS than in CS BAL, suggesting local inflammation. Although total numbers of each cell type were increased, relative proportions of alveolar macrophages, lymphocytes, and neutrophils were similar in IS and CS BAL. CD4/CD8 ratios in IS BAL samples were greater than those in CS samples, because of higher numbers of CD4+ lymphocytes in IS than in CS BAL but similar numbers of CD8+ lymphocytes. Anti-KLH IgG and IgA concentrations were greater in IS than in CS BAL. However, anti-KLH IgG/albumin ratios were similar in IS BAL and serum, suggesting that anti-KLH IgG had reached IS by passive transudation from the circulation. In contrast, anti-KLH IgA/albumin concentrations were greater in IS BAL than in serum, suggesting local production, and/or active transport of serum-derived anti-KLH IgA into the IS. Fractionation of serum and IS BAL on sucrose gradients demonstrated that anti-KLH IgA activity was largely associated with 11S polymeric IgA in both locations.(ABSTRACT TRUNCATED AT 250 WORDS)

Adoptive transfer of experimental hypersensitivity pneumonitis: CD4+ cells are memory and naive cells.

We previously demonstrated that CD4+ cells are responsible for transfer of adoptive murine experimental hypersensitivity pneumonitis. To further characterize the CD4+ cells as naive or memory cells, we depleted Micropolyspora faeni-sensitized cultured C3H/HeJ spleen cells of surface IgM+ cells by panning and Ia+ cells by lysis. We measured the proportion of CD4+ cells that expressed CD45RB, CD44 or LECAM-1 (markers used to distinguish memory from naive CD4+ cells) in spleen cell populations able to transfer experimental hypersensitivity pneumonitis (M. faeni sensitized and cultured) and those unable to transfer experimental hypersensitivity pneumonitis (ovalbumin sensitized and M. faeni cultured). Planning reduced the proportion of B cells from 53% to 11% and of Ia+ cells from 54% to 11%. Further lysis of Ia+ cells from the SIgM(+)-depleted population reduced Ia+ cells to 6%. Thy1+ cells increased from 26% to 55% after panning and to 72% after Ia+ cell lysis. Cultured M. faeni-sensitized spleen cells could transfer experimental hypersensitivity pneumonitis. Depletion of SIgM+ cells enhanced and depletion of Ia+ cells did not affect the capacity to transfer experimental hypersensitivity pneumonitis. The CD4+ cells from M. faeni-sensitized animals were 47% CD45RBhi, 36% CD44+ and 0% LECAM-1hi before culture with M. faeni and 48% CD45RBhi. They were 34% CD44+ and 27% LECAM-1hi after culture. The origin of the cells (from M. faeni- or ovalbumin-sensitized animals) did not affect the phenotype of the CD4+ cells, either before or after culture with M. faeni. We conclude that the active cells in spleen cell cultures are SIgM-, Ia-, CD4+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

CD5-negative cell mediated experimental hypersensitivity pneumonitis.

Experimental hypersensitivity pneumonitis (EHP) can be transferred to Strain 2 guinea pigs by peripheral lymph node (PLN) cells cultured in vitro with antigen. The phenotype of the active cells has not been delineated. In addition, it is not known if cultured lung-associated lymph node (LALN) cells can transfer EHP. PLN and LALN cells from donor animals were cultured with a soluble extract of Micropolyspora faeni (10 micrograms/ml), and blast cells were isolated by centrifugation over Percoll gradients. Cultured PLN cells were passed through nylon wool columns, and the adherent and nonadherent fractions were tested for their ability to transfer EHP. PLN blast cell populations were depleted of CD5+ cells by treatment with monoclonal anti CD5 antibody (8BE6) and complement. Syngeneic recipients received media or LALN or PLN blast cells treated with antibody plus complement, media, or complement intravenously. Recipients were challenged intratracheally with M. faeni 48 h after the cell transfer, and they were killed 4 days later. The nonadherent PLN cell population was enriched for CD5+ (T) cells and depleted of surface immunoglobulin-positive (SIg+) cells. Treatment of the PLN blast cell population with 8BE6 and complement decreased CD5+ cells from 25 to 4% and increased SIg+ cells from 62 to 80%. All animals were maintained in HEPA-filtered air. Randomly selected microscopic fields of the lung (250/animal) were judged to be normal or abnormal without knowledge of treatment. There was a low level of pulmonary response to an intratracheal challenge of M. faeni in media recipients. There was a substantial increase (p less than 0.01) of the extent of pulmonary abnormalities in the animals receiving cultured PLN cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary immunization in the canine lung. Soluble antigen induces a localized response.

Primary immunization of the dog by intralobar instillation of particulate antigen induces an intense, localized pulmonary antibody response. In contrast, although soluble antigen can also induce local antibody responses after repeated deposition in the canine respiratory tract, its ability to induce local responses after primary immunization has not been well characterized. To document such responses, we immunized five beagle dogs using a bronchoscope to instill 10 mg keyhole limpet hemocyanin (KLH) into a single lung lobe (immunized) and saline into a contralateral lung lobe (control). Over the next 3 wk, we monitored specific immune responses in blood and bronchoalveolar lavage (BAL) fluids obtained from the immunized and control lung lobes. Primary intrapulmonary immunization of dogs with KLH resulted in anti-KLH antibody responses both in blood and in immunized and control BAL fluids. However, immunoglobulin class-specific expression of response differed between the immunized and control lung lobes. Specific IgM and IgA responses were significantly greater in the immunized lobes. In contrast, specific IgG, and cells producing specific IgG, were quantitatively similar in lavage fluids derived from immunized and control lung lobes. These studies demonstrate that primary immunization of the dog by intralobar instillation of soluble antigen stimulates a local IgM and IgA response and an IgG response that distributes to both immunized and unimmunized lung. This pattern of immunoglobulin class-specific pulmonary antibody response has the potential to importantly influence regional responses to intrapulmonary antigen.

Experimental hypersensitivity pneumonitis: suppressor cell influences.

Experimental hypersensitivity pneumonitis (EHP) can be transferred to strain 2 guinea pigs by lymph node cells (LNC) cultured in vitro with antigen. Using mixtures of cell populations, we sought to determine if functional suppressor cells were present in our system. We also characterized the composition of cell populations that were capable (blast 10 micrograms/mL Micropolyspora faeni from 2-week donor animals) and incapable (blast 0 micrograms/mL M. faeni from 2-week donor animals; blast 10 micrograms/mL from 8-week donor animals) using flow cytometry, anti-Ig and monoclonal antibody 8BE6 (T cell marker) of transferring EHP. Two groups of donors were used: animals sensitized with Freund's adjuvant and M. faeni and challenged with either two or eight weekly intratracheal (IT) injections of M. faeni (2- and 8-week groups). LNC from donor animals were cultured with a soluble extract of M. faeni (10 or 0 micrograms/mL) blasts isolated and transferred IV to syngeneic recipients. Control animals received media IV. Recipients were challenged IT with M. faeni 48 h after the cell transfer and sacrificed 4 days thereafter. All animals were maintained in HEPA filtered air. Randomly selected microscopic fields of the lung (250/animal) were judged to be normal or abnormal without knowledge of treatment. There was a low level of pulmonary response to an IT challenge of M. faeni in media recipients. There was a substantial increase (P less than .01) in pulmonary abnormalities in the animals receiving blasts from the 10-micrograms/mL M. faeni 2-week group. Addition of cells from incompetent cell populations (0 micrograms/mL M. faeni 2-week donors or 10 micrograms/mL M. faeni 8-week donors) did not alter the ability of competent populations to transfer EHP. Cells cultured with antigen had a decreased proportion of T cells and an increased proportion of SIg+ and large cells. Competent and incompetent cell populations did not differ in regard to proportion of large cells, surface Ig+, or T cells. We conclude that the inability of certain cell populations to transfer EHP is not associated with the appearance of functional suppressor cells. Differences of ability to transfer EHP do not correlate with differences of size distribution or T and B cell composition.

Dynamic T-cell changes in peripheral blood and bronchoalveolar lavage after antigen bronchoprovocation in asthmatics.

We previously demonstrated decrease of peripheral blood CD4+ cells 48 to 72 h after antigen-induced bronchoprovocation in asthmatic subjects. To determine if this was accompanied by reciprocal changes in bronchoalveolar lavage (BAL) fluid, we sampled BAL before and 48 h after antigen administration. Peripheral blood lymphocytes (PBL) and BAL T-cell subset composition were examined in eight extrinsic asthmatics during three different weeks. During control week (CW) BAL was preformed at baseline, and PBL subsets were followed for 3 days. During placebo week (PW) a placebo inhalation was followed by a BAL at 48 h and daily PBL for 3 days. Asthmatic attacks were induced by inhalation of a relevant antigen at the start of antigen week (AW) and followed by examining the BAL at 48 h and PBL daily for 72 h. We found that BAL as a stimulus had no effect on T-cell composition of the PBL after the procedure. After induction of an asthmatic attack there was a reduction of PBL CD4+ cells from a baseline mean of 52.6% to 38.8% at 48 h (p less than 0.05). At the same time BAL CD4+ cells increased from 35.6% at PW to 47.3% in AW (p less than 0.05). These data are compatible with specific recruitment of PBL CD4+ cells into the respiratory system in atopic subjects undergoing antigenic bronchoprovocation.

Murine lung immunity to a soluble antigen.

Although it is known that soluble antigen is immunogenic when deposited in the respiratory tract, less is known about lung immunity to soluble antigen than is known about lung immunity to particulate antigen. To test the hypothesis that soluble antigen triggers antigen-specific immunity in the respiratory tract in a fashion similar to that reported for particulate antigen, we examined the development of local and systemic immunity in C57BL/6 mice after intratracheal (i.t.) instillation of a soluble, large molecular weight protein neoantigen, keyhole limpet hemocyanin (KLH). Specific anti-KLH IgG and IgM first appeared in the sera of mice on day 7 after primary immunization by i.t. instillation of KLH, with specific serum antibody concentrations remaining elevated at day 11. Cell populations prepared from lung-associated lymph nodes of immunized mice released specific anti-KLH IgG and IgM in vitro; peak levels were obtained from cells isolated 7 days after antigen instillation, with levels of specific antibody released by cells isolated on days 9 and 11 decreasing markedly. Cultured spleen cells obtained from mice after primary immunization released only low levels of specific IgM, and no specific IgG. No specific antibody was released by cell populations derived from the lungs of animals undergoing primary immunization. When presensitized mice were given an i.t. challenge with KLH, responses differed markedly from those following primary immunization. Lung-associated lymph node cell populations from challenged mice released greater amounts of specific antibody earlier than did cell populations from mice undergoing primary immunization.(ABSTRACT TRUNCATED AT 250 WORDS)

Guidelines for the clinical evaluation of hypersensitivity pneumonitis. Report of the Subcommittee on Hypersensitivity Pneumonitis.

In general, a history of exposure to "moldy" hay, birds, or other incriminated occupational or environmental inhalants in a patient with clinical and radiologic features consistent with HSP should lead to the demonstration of serum precipitins to the suspected antigen and an established diagnosis, confirmed by avoidance of the agent involved. Occasionally, other diagnostic procedures are required. The diagnosis is often difficult in domestic exposures, such as humidification and air conditioning systems. A careful environmental history is essential, and at times the physician must inspect the patient's environment personally. In most cases, the diagnosis is established if (1) the history and physical findings and pulmonary function tests indicate an interstitial lung disease, (2) the x-ray film is consistent, (3) there is exposure to a recognized cause, and (4) there is antibody to that antigen. In other exceptional circumstances, bronchoalveolar lavage may help. Biopsy is rarely needed. Special environmental studies and identification of new antigens require research facilities. Provocation tests are research procedures, not necessary for the diagnosis, and not needed in contested workmen's compensation adjudications.

Macrophage activation by Micropolyspora faeni does not suppress anamnestic pulmonary immunologic reactivity.

Determinants of lung immunologic response to antigens are not known, but could include alveolar macrophage (AM) activation. We tested the ability of AM activation to modify the anamnestic response by administering bovine serum albumin (BSA) intratracheally, activating AM (by intratracheal Micropolyspora faeni), and then exposing rabbits again to intratracheal BSA. We compared the results from 4 groups of animals: intratracheal administration of either 50 mg M. faeni or normal saline and later administration of either intratracheal or intramuscular BSA. M. faeni administered intratracheally increased the number of AM. These AM were activated (increased phagocytosis of IgG-coated particles). We found no difference in the amount of antibody in either lavage fluid or serum or in antigen-induced pulmonary parenchymal and hilar node lymphocyte proliferation among these 4 groups.

Alveolar macrophage catabolism of Micropolyspora faeni.

Pulmonary histologic abnormalities resolve despite continuing intratracheal injections of Micropolyspora faeni in a rabbit model of hypersensitivity pneumonitis. We examined in vitro alveolar macrophage (AM) metabolism to determine if increased efficiency of M. faeni degradation by AMs was associated with resolution of pulmonary abnormalities. Rabbits were exposed to M. faeni with three sensitizing and two, four, or eight weekly intratracheal challenge injections. Bronchoalveolar cells (BAC) were obtained by lavage 4 to 6 days after the last intratracheal injection. We determined the fate of 125I-labeled M. faeni added to 48-hour cultures of BAC derived from naive and M. faeni-exposed animals. Label was transported from the pellet to the supernatant fraction of BAC cultures, and the proportion of supernatant label that was precipitated by trichloroacetic acid decreased. These phenomena were dependent on time, viable cells, and temperature. They were not altered by puromycin and were caused by AM. BAC from M. faeni-treated rabbits were slightly more effective in transport of label from pellet to supernatant than BAC from naive rabbits during the first 4 hours of culture but not thereafter. There was no difference between BAC from rabbits challenged two, four and eight times. We conclude that resolution of pulmonary histologic abnormalities in this model of hypersensitivity pneumonitis is not associated with evidence of enhanced AM particulate M. faeni catabolism.

Experimental hypersensitivity pneumonitis: lack of tolerance.

Most subjects repetitively exposed to agents responsible for hypersensitivity pneumonitis (HP) do not develop persistent or progressive pulmonary inflammation. To determine if immunologic tolerance is associated with resolution of pulmonary abnormalities despite continuing exposure, we examined markers of local immunologic reactivity in a model of HP in rabbits. Rabbits were exposed to Micropolyspora faeni (M. faeni), the agent responsible for farmer's lung disease, with 3 sensitizing and 2, 4, or 8 challenge intratracheal injections. We determined bronchoalveolar macrophage migration inhibition (MMI) induced by M. faeni antigen, mitogen, and antigen-induced lymphocyte proliferation of lymphocytes derived from the lungs and hilar lymph nodes, and the amount of IgG antibody to M. faeni in serum and bronchoalveolar lavage fluid. We found MMI of lavage cells from rabbits exposed to M. faeni. Migration inhibition was dependent on ongoing protein synthesis. Hilar node and pulmonary lymphocytes proliferated upon exposure to M. faeni antigen, and anti-M. faeni antibody was found in serum and lavage fluid from M. faeni-treated rabbits. There were no differences between rabbits challenged 2, 4, and 8 times. We conclude that resolution of pulmonary histologic abnormalities in this model of hypersensitivity pneumonitis is not associated with evidence of immunologic tolerance.

Prednisone and T-cell subpopulations.

Alteration of T-cell subset relationships may cause many of the anti-inflammatory and immunoregulatory effects of glucocorticosteroids. The effect of oral administration of lactose or 60 mg of prednisone on peripheral blood T-lymphocyte subset profile and total eosinophil count (TEC) was examined. A purified T-cell peripheral blood population was obtained and the proportion of T cells with T3, T4, T8, M1, and la surface antigens was determined before and five hours after ingestion of lactose or prednisone. Lactose caused no change of any of the measured values. Prednisone caused a large (72%) decrease of the total lymphocyte number and the TEC (97%) but no change of the proportion of T cells with the previously mentioned antigens. Administration of 60 mg of prednisone does not acutely selectively deplete subclasses of T lymphocytes from peripheral blood.

Changes in T-lymphocyte subpopulations after antigenic bronchial provocation in asthmatics.

To determine whether inhaled agents can alter T-cell subsets in the peripheral blood of patients with bronchial asthma, we tested six asymptomatic asthmatics who were sensitive to mixed grass (positive skin test) with mixed grass extract, methacholine, and an antigen to which they were not sensitized (negative skin test). Levels of OKT4 cells (helper T lymphocytes) were reduced in the peripheral blood immediately after the challenge with mixed grass extract, and remained low for at least 72 hours. Levels of Ia-positive (activated) T cells were increased 48 hours after the challenge. No changes were observed in any of these T-cell subpopulations after challenge with methacholine or after the inhalation of an equal amount of an antigen to which the subjects were not sensitized. These results suggest that the selective loss of circulating helper T cells and an increase in activated T cells after an asthmatic attack induced by antigenic inhalation may serve as an indicator of immune-mediated bronchoconstriction.

Pulmonary response to repeated exposure to Micropolyspora faeni.

Most human exposure to agents that cause hypersensitivity pneumonitis (HP) result in transient episodes of HP that resolve quickly. We repeatedly injected Micropolyspora faeni, which is responsible for farmer's lung disease, into rabbits in an attempt to elucidate mechanisms for this phenomenom (i.e., resolution of abnormalities). The character and the extent of lung disease, the amount of anti-M. faeni serum antibody, and skin reactivity to M. faeni were evaluated after 3 sensitizing and 2, 4, or 8 challenge injections. We also determined the fate of 125I labeled M. faeni injected intratracheally into both normal and previously exposed rabbits. Increased numbers of lymphocytes, macrophages, and few polymorphonuclear leukocytes were present in interstitial and intraalveolar regions and bronchial walls. Interstitial fibrosis was not observed. The extent of cellular abnormalities was maximal after 2 challenges and regressed thereafter, despite continuing intratracheal injections. Serum anti-M. faeni antibody peaked after 4 intratracheal challenges. Anti-M. faeni antibody level at the time of death appeared to be proportional to the extent of inflammatory reaction within the lung. Previous exposure of rabbits to M. faeni was associated with more rapid appearance of 125I in blood in the first 2 h after intratracheal injection of 125I M. faeni. However, 24 h after injection, there was less 125I in the lungs and more in the urine of immunized rabbits than in normal rabbits. Repeated intratracheal injections of M. faeni into rabbits produces transient interstitial, intraalveolar, and peribronchial inflammatory infiltration that regresses without fibrosis despite continued antigenic challenge. Immunization appears to markedly decrease pulmonary exposure to antigen that results from an intratracheal injection of M. faeni.

Activated alveolar macrophages: IgG and complement receptors.

Receptors for IgG and C on PM populations can be modulated by activation. AM populations constantly exposed to environmental particulates may not be subject to changes of IgG and C receptors after activation. We compared normal and activated rabbit AMs and PMs. Activation was accomplished by treatment with intravenous BCG or intratracheal Micropolyspora faeni. We examined the ability of these AM populations to attach to (form rosettes) and phagocytize SRBC coated with varying concentration of IgG or C. Compared to normal AMs, activated AM populations formed more rosettes with SRBC coated with IgG or C and phagocytized more via the IgG receptor. PMs from BCG-treated rabbits did not form more rosettes with IgG- or C-coated SRBC but demonstrated more IgG-mediated phagocytosis than did normal PMs. C-mediated phagocytosis occurred in AM populations from only five of 25 BCG-treated rabbits and two of 20 normal rabbits. The TG broth-induced rabbit PM also exhibited a low level of C and IgG-mediated phagocytosis compared to the mouse TG broth-induced PM. We conclude that rabbit AM IgG and C receptors can be altered by events that activate AMs. C-mediated phagocytosis is much lower in rabbit macrophages (both AMs and PMs) than in activated mouse PM populations. IgG-mediated phagocytosis can be used as an index of rabbit AM activation.

Corticosteroid-sensitive lymphocytes are normal in atopic asthma.

Corticosteroids, well known to increase susceptibility to infection, are often administered to atopic patients. Atopy may be associated with lymphocyte abnormalities and increased susceptibility to infections caused by intracellular organisms. We sought to determine whether atopic and nonatopic subjects respond in a similar manner to corticosteroids administered both systemically and locally. We compared the response of peripheral blood leukocytes of 15 atopic asthmatics and 10 nonatopic control subjects to prednisone or beclomethasone dipropionate. We determined leukocyte number, total eosinophil count, T-cell number, complement receptor lymphocyte number, and concanavalin A (Con A)- and phytohemagglutinin (PHA)-induced lymphocyte proliferation before and 5 hr after administration of 20 mg of prednisone orally or 336 micrograms of beclomethasone dipropionate by aerosol inhalation. Baseline values of the groups differed. The atopic asthmatic group had higher total eosinophil count, lower percent lymphocyte count, and slightly lower Con A- and PHA (high concentration)-induced lymphocyte proliferation. T-cell and complement receptor lymphocyte number were equivalent in both groups. Prednisone caused a profound eosinopenia, monocytopenia, T lymphopenia, depression of mitogen-induced lymphocyte proliferation, and increase in leukocyte number and complement receptor lymphocyte percent. Beclomethasone dipropionate was associated with little or no change in these parameters. We conclude that atopic asthma is not associated with a defect in corticosteroid-sensitive leukocyte populations and that beclomethasone dipropionate aerosol, as opposed to prednisone, does not alter peripheral blood mononuclear cell populations.

The effect of complement depletion on hypersensitivity pneumonitis lesions induced by Micropolyspora faeni antigen.

Animals sensitized by intratracheal administration of particulate Micropolyspora faeni antigen and subsequently challenged with the antigen intratracheally developed lesions of hypersensitivity pneumonitis histologically similar to those observed in man wih this disease. Animals sensitized with antigen but depleted of complement with cobra venom factor prior to challenge with the antigen manifested a significant reduction in mean lesion indices when compared to a group of control animals that were not complement-depleted. These data indicate that complement is necessary for the development of pulmonary lesions of experimental hypersensitivity pneumonitis in the rabbit.