Background: HIV-infected adults are highly susceptible to pneumococcal disease.
Objective: To examine if alveolar macrophages from HIV-infected subjects exhibited a failure of cytokine production in response to Streptococcus pneumoniae in vitro.
Design: Case–control comparison of alveolar macrophages from 11 HIV-infected and 13 non-infected adults.
Methods: Type 1 opsonized S. pneumoniae were used to challenge the alveolar macrophages in vitro. Cell supernatant fluid was collected from unstimulated cells, and cells challenged with bacteria for 0, 6, 12 and 24 h. Cytokine production (interleukins 1β, 6 and 8) was measured in all fluids using an enzyme-linked immunosorbent assay.
Results: All the cytokines tested increased over time in both HIV-infected and uninfected subjects. Interleukin-8 release was significantly lower in HIV-infected than in non-HIV-infected subjects (P = 0.02).
Conclusion: Reduced interleukin-8 production may result in decreased neutrophil recruitment, and hence increased susceptibility to pneumococcal infection in HIV-infected subjects.
From the aMalawi–Liverpool–Wellcome Trust Research Programme, Universities of Malawi and Liverpool
bLiverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK
cDivision of Genomic Medicine, University of Sheffield Medical School, Royal Hallamshire Hospital, Sheffield, UK
dDepartment of Medicine, University of Malawi College of Medicine, Blantyre, Malawi.
Received 14 February, 2005
Revised 19 April, 2005
Accepted 17 May, 2005
Correspondence to Dr S. Gordon, Malawi–Liverpool–Wellcome Programme of Clinical Tropical Research, PO Box 30096, Blantyre, Malawi. E-mail: firstname.lastname@example.org; email@example.com.
Pneumococcal infections in HIV-infected Africans are common, severe and not yet preventable by vaccination [1,2]. The rate of invasive pneumococcal disease in HIV-infected adults is 20- to 100-fold higher than that measured in adults uninfected with HIV, with the higher rates occurring late in HIV disease. The immune defect responsible for this susceptibility has not yet been identified but it is now known to be critically dependent on CD4 T cell-mediated immunity . Increased pulmonary epithelial binding of pneumococci and decreased pulmonary phagocytic defence are also potential mechanisms . We have reported that alveolar macrophage phagocytosis of pneumococci is dependent on opsonins  and is intact in HIV-infected adults . Further, pulmonary levels of pneumococcus-specific immunoglobulin are normal or increased in HIV-infected adults . Alveolar macrophages produce proinflammatory cytokines, including the interleukins (IL) IL-1β, IL-6 and IL-8, both in response to HIV infection and when challenged with bacteria . In the lung, the neutrophil concentration within pulmonary capillaries is three times greater than that measured in peripheral veins as a result of the relatively small pulmonary capillary diameter and local endothelial adhesion factors . Neutrophils are, therefore, available to respond promptly to alveolar inflammatory stimuli derived from cells within the alveolar unit, and IL-8 is the cytokine that is chiefly responsible for the initial recruitment of neutrophils to the alveolar space .
The present study compares cytokine release by unstimulated alveolar macrophages from HIV-infected and non-infected subjects, and cytokine release after in vitro challenge with opsonized Streptococcus pneumoniae.
Subjects recruited by advertisement gave written informed consent after a culturally appropriate written and oral information process for the study, which was approved by the University of Malawi College of Medicine Research Ethics Committee and the Research Committee of the Liverpool School of Tropical Medicine. HIV testing was by two rapid tests with results conveyed to the subject. All subjects were well and had a normal chest radiograph at bronchoscopy. In particular, no subject had an acute respiratory illness or any history of asthma. A 200 ml lavage of the right middle lobe was carried out and the bronchoalveolar lavage collected into glass containers on ice.
The bronchoalveolar lavage was filtered through gauze, centrifuged and the cell pellet resuspended at 1 × 106 cells/ml in RPMI 1640 medium (GibcoBRL, Paisley, Scotland) supplemented with antibiotics and 10% fetal calf serum. Samples (1 ml) of this cell suspension were incubated for 24 h in tissue culture wells, after which the cells were washed and given fresh medium without antibiotics. Greater than 95% viability of adherent cells was demonstrated using Trypan blue; these cells have been demonstrated previously to be predominantly (> 98%) macrophages [5,6]. Stocks of type 1 S. pneumoniae were grown to mid-log phase and stored in portions at −80°C.
On each experimental day, bacteria were opsonized with pooled immune serum  from volunteers vaccinated with 23-valent pneumococcal polysaccharide vaccine (Pneumovax II; Merck, Sharp and Dome, Philadelphia, Pennsylvania, USA). Supernatant fluid was removed from alveolar macrophages at day 2 for baseline cytokine measurement and the cells washed before opsonized bacteria were added at a multiplicity of infection of 10. After a 30 min incubation at 37°C, unbound bacteria were washed off and the media replaced. Supernatant fluid was harvested from two wells at this point and from further pairs of wells after 6, 12 and 24 h. In order to avoid bacterial overgrowth and consequent cellular damage, the culture medium was replaced with enriched RPMI plus antibiotics at 6 h.
Cytokines were measured by enzyme-linked immunosorbent assay using standard kits for IL-1β, IL-6 and IL-8, (BD-Pharmingen, Oxford, UK) according to the manufacturer's instructions. Standards and samples were serially 1:2 diluted in triplicate and duplicate, respectively, on each plate and results with < 15% variation between duplicates were accepted.
The study recruited 24 subjects with a mean age of 32 years (range, 19–53); demographic details and risk factors for pneumococcal infection are given in Table 1. There were no significant differences between the groups except in CD4 cell count and HIV viral load. No subject was taking antiretroviral medication, prophylactic antibiotics or other medication and none had received pneumococcal vaccination. Bronchoalveolar lavage samples obtained had a mean volume of 110 ml and a cell yield of 9 × 106 to 1.7 × 107 cells. Bronchoalveolar lavage samples from HIV-negative subjects contained 10% lymphocytes and those from HIV-positive subjects contained 20%, consistent with the known pulmonary CD8 cell lymphocytosis of HIV infection.
Baseline cell supernatants from HIV-infected subjects compared with those from HIV-uninfected subjects contained higher concentrations of IL-1β (70.8 versus 17.5 pg/ml; rank sum P = 0.01) and IL-6 (4234 versus 1116 pg/ml; rank sum P = 0.02), whereas IL-8 levels were not significantly different (89044 versus 22920 pg/ml; rank sum P = 0.09).
In response to bacterial challenge, IL-1β, IL-6 and IL-8 increased appropriately over the experimental period in all experiments (Fig. 1). There was a higher mean level of IL-1β and IL-6 in supernatants from HIV-infected subjects compared with uninfected subjects at all time points, but the area under the curve was not statistically significantly different between the groups. There was significantly less IL-8 measured in supernatants from HIV-infected subjects than in uninfected subjects at all time points except baseline, and the area under the curve was significantly less (98 versus 202; rank sum P = 0.02). Comparisons of groups stratified by CD4 cell count yielded no significant differences.
We have confirmed that alveolar macrophages from patients infected with HIV produce increased resting levels of proinflammatory cytokines and have shown that these cells can produce a further increase when subsequently challenged with opsonized pneumococci in vitro. The pattern and kinetics of cytokine production measured in this study are consistent with the known regulation of these cytokines by protein kinase C and transcription factor NFκB . The production of IL-8 in response to pneumolysin has been described in human monocytes . The relative deficiency of IL-8 production in macrophages from HIV-infected subjects in this study is an interesting and novel observation, however, and suggests a potential mechanism in which there is failure of early neutrophil recruitment, leading to an increased risk of disease following aspiration of pneumococci to the alveolar space. Other studies have also suggested that IL-8 regulation may occur independently of other NFκB-regulated cytokines, and that it can be inhibited in patients with severe infections [13,14]. However, the subjects in our study were well and had increased levels of IL-1 and IL-6 at baseline and in response to pneumococcal challenge. In contrast, IL-8 levels were not increased at baseline and showed a specific IL-8 deficit in response to challenge. This study was conducted using opsonized pneumococci and shows that there may be deficiency of chemotactic signal within the alveolus even following efficient uptake of pneumococci in an alveolar environment containing specific antibody. It suggests that even optimal antibody responses to vaccine may not be protective against pneumococcal disease in HIV-infected adults  and supports the use of antiretroviral therapy for prevention of pneumococcal infection.
The authors would like to thank their volunteers and the staff of the Queen Elizabeth Central Hospital, Blantyre, Malawi for their willing cooperation with this study. We also thank the bronchoscopy suite, research clinic and laboratory staff of the Wellcome Trust Research Laboratories, Blantyre, Malawi for technical assistance.
Sponsorship: This work received financial support from the Wellcome Trust of Great Britain. SG and NF hold Wellcome Trust Career Development Fellowships; MEM is a Director of the Liverpool Wellcome Trust Centre for Clinical Tropical Research and this work forms part of the Malawi–Liverpool–Wellcome Trust Programme of Research in Clinical Tropical Medicine.
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