Sarah Howie

A type 2 (Th2-like) pattern of immune response predominates in the pulmonary interstitium of patients with cryptogenic fibrosing alveolitis (CFA)

CFA is an inflammatory condition of the lungs resulting in scarring, pulmonary failure and death. The etiology of the disease is unknown, but the pathogenesis is believed to involve a persistent immunological reaction to unidentified antigen in the lung resulting in tissue damage. Recent advances in our understanding of the immune system have shown that different patterns of stimulatory cytokines are produced at sites of inflammation by a range of cell types. Patterns of cytokine production by inflammatory cells are recognized to be associated with different patterns of immunological response, and these have been described as type 1 (or Th1‐like) and type 2 (or Th2‐like) on this basis. We have studied cytokine expression in the intestinal inflammatory cell infiltrate in lung tissue from patients with CFA using mRNA in situ hybridization and immunohistochemistry. Our results show that while there is evidence for both a type 1 (characterized by interferon‐gamma (IFN‐γ)) and type 2 (characterized by IL‐4 and IL‐5) response present in CFA, the type 2 (or Th2) pattern of cytokines appears to predominate. This would be consistent with a possible role for the humoral immune response in the pathogenesis of this condition. In addition, recent evidence suggests that IL‐4 and IFN‐γ may be important regulatory factors for pulmonary fibroblasts. The relative paucity of IFN‐γ may contribute to the excessive fibroblast activation, deposition of collagen and scar formation that occurs in CFA.

Immunoreactive interleukin 4 and interferon-γ expression by type II alveolar epithelial cells in interstitial lung disease

Cryptogenic fibrosing alveolitis (CFA), extrinsic allergic alveolitis (EAA), and sarcoid are all immunologically mediated forms of interstitial lung disease. In contrast to most patients with EAA and sarcoid, patients with CFA show relentless pulmonary fibrosis which is unresponsive to immunosuppressive therapy. Previous studies have indicated a possible role for epithelial‐derived cytokines in the regulation of immunological and fibrotic responses in the lung. This study has examined lung biopsy specimens from patients with CFA, EAA, and sarcoid for immunoreactive interleukin 4 (IL4) and interferon‐γ (INFγ) expression by type II alveolar epithelial cells, as these cytokines have been suggested to have in vitro stimulatory and inhibitory effects on fibroblast function. In addition, mRNA in situ hybridization was performed on the CFA lung biopsies to confirm transcription of these cytokine genes within the cells. The results show that type II epithelial cells in EAA and sarcoid show up‐regulation of immunoreactivity for both IL4 and INF‐γ, but that in CFA only IL4 is detectable. The mRNA in situ hybridization results indicate that this may represent post‐transcriptional regulation of INFγ expression in CFA. These results are consistent with previous observations of a paucity of INFγ and a predominantly type II (Th2‐like) pattern of immune response in patients with CFA and suggest a possible imbalance of pro‐fibrogenic cytokines in the distal lung of patients with this condition, compared with those with EAA or sarcoid. Copyright

Peripheral T lymphocyte depletion by apoptosis after CD4 ligation in vivo: selective loss of CD44- and 'activating' memory T cells.

We have demonstrated that a single intravenous bolus of rat anti‐CD4 MoAb caused a small but prolonged increase in apoptosis in murine lymph nodes. We have quantified this process using the novel Highly Optimized Microscope Environment (HOME) interactive image analysis system and shown that the increase in apoptosis was sufficient to account for the observed depiction of the peripheral CD4+ T cell subset. This occurred in the absence of any other exogenous signal. Furthermore, there was no evidence of an inflammatory or necrotic response in the tissues, indicating that this was unlikely to be He or complement‐mediated antibody killing. The anti‐CD4‐induced depiction selectively removed CD44− T cells. Using mice previously immunized with yeast‐derived HIV‐1 p24 recombinant protein there was sparing of memory T cell function after in vivo anti‐CD4 treatment, except during a window of less than 24 h duration, when simultaneous exposure to antigen and anti‐CD4 antibody resulted in the depiction of specific memory T lymphocyte function. This indicated that a very minor alteration in the frequency of apoptosis had a marked effect on cell number over time, and suggested that opportunistic infection associated with CD4+ T cell depletion may be explained by loss of memory cells when there is antigenic stimulation at the same time as CD4 ligation. These results have implications for the pathology of HIV‐associated disease which is associated with ligation of CD4 molecules in vivo.

THE IMMUNOLOGICAL ARCHITECTURE OF B-LYMPHOCYTE AGGREGATES IN CRYPTOGENIC FIBROSING ALVEOLITIS

Cryptogenic fibrosing alveolitis (CFA) is believed to have a pathogenesis mediated by the cellular arm of the immune system. Previous studies have, however, indicated the presence of B‐lymphocyte aggregates, as well as evidence of local immunoglobulin production and increased levels of B‐cell growth factors. It has recently been shown that CFA is associated with the production of circulating IgG autoantibodies to antigen(s) associated with alveolar lining cells. This prompted an examination of the immunological architecture of the B‐lymphocyte aggregates, in order to assess whether they might provide histological confirmation of a local humoral immune response in these patients. Thirty‐eight consecutive open lung biopsy specimens were examined from patients with CFA and aggregates of B lymphocytes were identified in 37/38. In only five cases were germinal centres seen. The morphological appearances of the aggregates were reminiscent of those observed in mucosal associated lymphoid tissue (MALT). Using immunohistochemistry, despite the low frequency of true germinal centre formation, the B‐lymphocyte aggregates were shown to contain the cellular micro‐environment necessary for a humoral immune response. In addition, there was evidence of lymphocyte proliferation and activation within these aggregates. These results provide evidence of a local humoral immune response associated with B‐lymphocyte aggregates in the lungs of patients with CFA.

Prostaglandin E2 constrains systemic inflammation through an innate lymphoid cell–IL-22 axis

A prostaglandin barrier to inflammation Blood-borne bacterial infections and severe trauma can send the immune system into overdrive, causing it to pump out inflammatory mediators, sometimes at lethal doses. Duffin et al. now report on a role for prostaglandins in keeping systemic inflammation in check. Systemic inflammation correlates with decreased production of the prostaglandin E2 (PGE2). Blocking PGE2 signaling in mice led to severe inflammation associated with the translocation of gut bacteria. PGE2 acts on innate lymphoid cells, which produce interleukin-22, a secreted protein that helps promote intestinal integrity. Science, this issue p. 1333 Prostaglandin E2 prevents systemic inflammation by maintaining gut barrier integrity. Systemic inflammation, which results from the massive release of proinflammatory molecules into the circulatory system, is a major risk factor for severe illness, but the precise mechanisms underlying its control are not fully understood. We observed that prostaglandin E2 (PGE2), through its receptor EP4, is down-regulated in human systemic inflammatory disease. Mice with reduced PGE2 synthesis develop systemic inflammation, associated with translocation of gut bacteria, which can be prevented by treatment with EP4 agonists. Mechanistically, we demonstrate that PGE2-EP4 signaling acts directly on type 3 innate lymphoid cells (ILCs), promoting their homeostasis and driving them to produce interleukin-22 (IL-22). Disruption of the ILC–IL-22 axis impairs PGE2-mediated inhibition of systemic inflammation. Hence, the ILC–IL-22 axis is essential in protecting against gut barrier dysfunction, enabling PGE2-EP4 signaling to impede systemic inflammation.

Theileria annulata induces abberrant T cell activation in vitro and in vivo

The protozoan parasite of cattle, Theileria annulata, causes a severe lymphoproliferative disease, developing initially in the draining lymph node, which is often fatal in naive animals. Infection of macrophages with T. annulata leads to an augmentation of their antigen‐presenting capability in vitro and infected cells can induce proliferation of autologous resting T cells from naive animals. This inappropriate activation of T cells may play an important role in the failure of the host to mount an effective immune response in vivo. To investigate this hypothesis we characterized further the response of T cells from naive cattle to infected cells in vitro, and also examined the development of the immune response in lymph nodes draining the sites of T. annulata infection. Both CD4+ and CD8+ T cells from naive peripheral blood mononuclear cells (PBMC) were induced to proliferate and express the activation markers IL‐2R and MHC class II when cultured with infected cells. This effect was seen in both ‘naive’ and ‘memory’ T cells, and was dependent upon contact with infected cells. In vitro, infected cells are therefore capable of activating T cells irrespective of their antigen specificity or memory status. In draining lymph nodes, although large numbers of IL‐2R+ cells developed following infection, these activated cells were only associated with areas of parasite‐induced proliferating cells, and subsequently disappeared from the node. Cells expressing IL‐2R were not present in recognized sites for T cell development. Germinal centres were severely affected, losing T cell‐dependent zones followed by a total destruction of morphology. T cell function is therefore severely disrupted within draining nodes. This study has shown that parasitized cells supply sufficient signals in vitro to activate T cells irrespective of specificity. T cells also are not stimulated in a conventional manner in vivo, and this may play an important role in preventing an effective immune response from being generated.

A non-protective T helper 1 response against the intra-macrophage protozoan Theileria annulata

Theileria annulata is a protozoan parasite which infects and transforms bovine macrophages. Infected macrophages possess augmented antigen presentation capabilities, as they are able to activate the majority of T cells from unexposed animals. In vivo, T cells in the draining lymph node (principal site of parasite development) are activated ‘non‐specifically’ by the parasite. This event is followed by failure of the immune response to control the infection. Protective immune responses against intra‐macrophage protozoa are usually mediated by T helper 1 (Th1) T cell responses. Here we examine the cytokine responses made by T. annulata‐activated T cells. We show that the outcome of in vitro activation of T cells by parasitized macrophages is a skewing of their cytokine responses towards preferential expression of interferon‐gamma (IFN‐γ) mRNA. The in vitro response is mirrored during in vivo infection, as greatly elevated amounts of IFN‐γ protein are found in lymph efferent from infected lymph nodes, while expression of IL‐4 mRNA within the node stops. IFN‐γ production does not correlate with protection against the parasite, as infected cells flourish during peak IFN‐γ production, and only very small amounts of IFN‐γ are produced during the effective immune response of an immunized animal. Overproduction of IFN‐γ and loss of IL‐4 expression are also likely to account for the failure of B cells to reach the light zone of germinal centres, a developmental step which is tightly regulated by cytokines.

Circulating antibodies to lung protein(s) in patients with cryptogenic fibrosing alveolitis.

BACKGROUND--It has been hypothesised that cryptogenic fibrosing alveolitis has an immunological pathogenesis mediated by T lymphocytes. It is, however, recognised that patients may show dysregulation of the humoral immune system and that the presence of large numbers of B lymphocytes in open lung biopsies may be associated with a poor prognosis. Evidence of a role for the humoral immune system in the pathogenesis of cryptogenic fibrosing alveolitis has been suggested, but attempts to demonstrate circulating immunoglobulin to antigen within the lung have been inconclusive. METHODS--Plasma samples from 22 patients with cryptogenic fibrosing alveolitis, 22 patients with sarcoidosis, and 17 healthy controls were screened by SDS-PAGE and Western blotting for the presence of autoantibodies to lung proteins derived from cryptogenic fibrosing alveolitis, sarcoid and control lung tissue, as well as four normal non-pulmonary tissues. Possible site(s) of target protein(s) within the lung tissue were identified by immunohistochemical examination using IgG purified from the plasma of six patients and two controls. RESULTS--Eighteen of the plasma samples from patients with cryptogenic fibrosing alveolitis had reactive IgG to lung protein(s) in the 70-90 kDa molecular weight range compared with five of 18 plasma samples from patients with sarcoidosis and one of 17 controls. Plasma from patients with cryptogenic fibrosing alveolitis recognised antigen(s) of the same molecular weight in control and sarcoid lung tissue, but not non-pulmonary tissues, with a similar frequency. Immunohistochemical staining of cryptogenic fibrosing alveolitis biopsy material using IgG purified from plasma samples from patients with cryptogenic fibrosing alveolitis, but not control samples, revealed fine linear positivity in the lung parenchyma in a pattern suggestive of reaction with alveolar lining cells. The pattern was cytoplasmic/membranous and not nuclear. CONCLUSIONS--Patients with cryptogenic fibrosing alveolitis have a high frequency of plasma IgG autoantibodies to protein(s) within lung tissue associated with alveolar lining cells. This is believed to be the site where immunological injury occurs in cryptogenic fibrosing alveolitis, but the significance of these antibodies to the aetiology and pathogenesis is as yet unclear.

Role of inflammation in nocturnal asthma.

BACKGROUND--Nocturnal airway narrowing is a common problem for patients with asthma but the role of inflammation in its pathogenesis is unclear. Overnight changes in airway inflammatory cell populations were studied in patients with nocturnal asthma and in control normal subjects. METHODS--Bronchoscopies were performed at 0400 hours and 1600 hours in eight healthy subjects and in 10 patients with nocturnal asthma (> 15% overnight fall in peak flow plus at least one awakening/week with asthma). The two bronchoscopies were separated by at least five days, and both the order of bronchoscopies and site of bronchoalveolar lavage (middle lobe or lingula with contralateral lower lobe bronchial biopsy) were randomised. RESULTS--In the normal subjects there was no difference in cell numbers and differential cell counts in bronchoalveolar lavage fluid between 0400 and 1600 hours, but in the nocturnal asthmatic subjects both eosinophil counts (median 0.11 x 10(5) cells/ml at 0400 hours, 0.05 x 10(5) cells/ml at 1600 hours) and lymphocyte numbers (0.06 x 10(5) cells/ml at 0400 hours, 0.03 x 10(5) cells/ml at 1600 hours) increased at 0400 hours, along with an increase in eosinophil cationic protein levels in bronchoalveolar lavage fluid (3.0 micrograms/ml at 0400 hours, 2.0 micrograms/l at 1600 hours). There were no changes in cell populations in the bronchial biopsies or in alveolar macrophage production of hydrogen peroxide, GM-CSF, or TNF alpha in either normal or asthmatic subjects at 0400 and 1600 hours. There was no correlation between changes in overnight airway function and changes in cell populations in the bronchoalveolar lavage fluid. CONCLUSIONS--This study confirms that there are increases in inflammatory cell populations in the airway fluid at night in asthmatic but not in normal subjects. The results have also shown a nocturnal increase in eosinophil cationic protein levels in bronchoalveolar lavage fluid, but these findings do not prove that these inflammatory changes cause nocturnal airway narrowing.

Localisation of a pulmonary autoantigen in cryptogenic fibrosing alveolitis.

BACKGROUND--Cryptogenic fibrosing alveolitis (CFA) is believed to have an immunological pathogenesis with a persisting inflammatory reaction to an as yet unidentified pulmonary antigen(s). A high frequency of IgG autoantibodies has previously been found in the plasma of patients with CFA to an extractable 70-90 kDa lung antigen by Western blotting. Preliminary immunohistochemical studies with patient IgG had indicated that the target protein(s) might be associated with alveolar epithelial lining cells which have previously been suggested as the site of immunological attack in CFA. METHODS--In order to confirm this finding immunohistochemical analysis and Western blotting were performed on a human type II alveolar cell line (A549) using CFA patient plasma. In order to study further the distribution of the antigen, antibodies were raised in a rabbit to the partially purified 70-90 kDa CFA lung protein. RESULTS--The results showed that the human CFA autoantibody recognised a 70-90 kDa protein with a cytoplasmic distribution present in the A549 cells, confirming previous observations. The immune rabbit IgG recognised a protein of similar molecular weight by Western blotting of protein derived from lung biopsy samples of patients with CFA and A549 cells. In addition it immunoprecipitated protein(s) of this molecular weight from lung biopsy protein extracts from patients with CFA. The precipitated protein(s) were found to cross-react with the autoantibody found in the plasma of patients with CFA. Immunohistochemical analysis with immunised rabbit antibody revealed positive staining of type I and II alveolar epithelial lining cells in CFA. A similar pattern of epithelial staining was also observed with the rabbit IgG on biopsy specimens of lung from patients with sarcoidosis and control lung tissue, although this was more focal and less intense. No positive staining was seen on sections from a number of non-pulmonary tissues (colon, liver, kidney, tonsil, lymph node, skin, cervix). Cytoplasmic staining of the A549 cell line was also detected. CONCLUSIONS--The 70-90 kDa protein recognised by autoantibodies in patients with CFA is associated with pulmonary epithelial lining cells. The immune rabbit IgG produced appears to recognise antigen by Western blotting and immunohistochemical staining of lung tissue in a similar pattern to the patient autoantibodies. Immunohistochemical data obtained with this antibody suggest that the putative autoantigen against which patients with CFA mount a humoral immune response may be endogenous and specific to the lung.