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Epidemiology and Social: Concise Communication

Bone marrow macrophage iron grade and survival of HIV-seropositive patients

de Monyé, Cécile*‡; Karcher, Donald S.†; Boelaert, Johan R.§; Gordeuk, Victor R.*

Author Information

From the Departments of *Medicine and †Pathology, The George Washington University Medical Center, Washington, DC, USA; ‡University of Utrecht, Utrecht, The Netherlands; §Algemeen Ziekenhuis St Jan, B-8000 Brugge, Belgium.

Sponsorship: This work was supported in part by a grant from the Office of Minority Health to the Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, MD, USA.

Correspondence to: Victor R. Gordeuk, MD, Department of Medicine, The George Washington University Medical Center, Suite 3-428, 2150 Pennsylvania Avenue NW, Washington, DC 20037, USA. Tel: +1-202-994-3556; fax: +1-202-994-4524; e-mail: [email protected]

Request for reprints to: Dr Cécile de Monyé, Kerkstraat 21bis, 3581 RA, Utrecht, The Netherlands. Tel: +31-30-2333424; fax: +31-30-2317267.

Date of receipt: 16 September 1998; revised: 12 November 1998; accepted: 18 November 1998.

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Abstract

Introduction

Iron overload is associated with an increased risk of certain infections [1-6], possibly because of the increased availability of the essential nutrient, iron, and iron-induced dysfunction of macrophages [7-9] and neutrophils [10,11]. HIV infection is associated with profound impairment of the body‚s immune system [12]. Most patients in advanced stages of AIDS also have excessive iron deposits in the bone marrow, liver, and other organs [5,13-18] caused by one or a combination of several mechanisms [5,17,19-21]. These mechanisms include the sequestration of iron in the macrophages with chronic inflammation [19,20], a shift of iron from hemoglobin in red blood cells to macrophage stores during the development of hypoplastic anemia, the transfusion of blood [5,17], the upregulation of iron acquisition associated with antiretroviral agents causing ineffective erythropoiesis [21], a variety of inherited or acquired iron-loading conditions [22], and a large intake of dietary iron [23-25].

The literature examining whether high body iron stores affect the course of HIV infection is scanty. One postmortem study [5] found a significant association between increased hepatic iron levels and infection with Mycobacterium avium intracellulare. A few other studies [26-29] have provided indirect evidence for an association between high iron stores and high mortality. It was postulated that HIV-seropositive patients with high iron stores have shorter survival times than do HIV-seropositive patients with normal or low iron stores. Investigating the effect of high body iron on the course of HIV infection is difficult, because indirect measures of iron status such as serum ferritin concentration and transferrin saturation are profoundly influenced by inflammation and are therefore unreliable measures for these patients. In addition, direct measures of iron status, such as liver biopsy and bone marrow aspiration, are invasive and therefore are rarely performed for investigative purposes. For our study we retrospectively examined a group of patients who had had bone marrow aspirations performed for diagnostic purposes. The aspirates were stained for iron and graded to provide a direct estimate of macrophage iron stores.

Materials and methods

The study cohort consisted of 348 HIV-seropositive adults (persons >18 years of age) who had diagnostic bone marrow studies performed between January 1985 and June 1996 at The George Washington University Hospital in Washington, DC, USA, an urban academic hospital. These subjects had some clinical follow-up information available and their bone marrow aspirates were suitable for grading macrophage iron stores. The research was approved by the Committee on Human Research of The George Washington University.

The bone marrow aspirates were stained with Prussian blue, and iron stores in the macrophages were graded on a scale of 0 to 5 [30], using light microscopy by investigators who were blinded to the outcome of the patients. Grade 0 slides (n=9) had no iron granules indicating absent iron stores; grade 1 (n=27) had rare iron granules by oil immersion indicating low iron stores; grade 2 (n=94) had small iron granules by low power indicating normal iron stores; grade 3 (n=30) had numerous iron granules in all bone marrow spicules indicating borderline high to increased iron stores; grade 4 (n=178) had large granules in small clumps indicating markedly increased iron stores; grade 5 (n=10) had large clumps of granules indicating massively increased iron stores. For the analysis of survival, subjects with markedly or massively increased iron stores (grades 4-5) were compared with subjects with normal or low iron stores (grades 0-2).

Hospital records were reviewed for the dates of the bone marrow aspiration, determination of HIV seropositivity, latest recorded clinical follow-up, and death (if it occurred). The indications for and the histological findings of the bone marrow evaluation were noted. The complete blood count and the CD4 cell count at the time of the bone marrow aspiration were also noted, as was the number of units of red blood cells transfused before the aspiration, and the medical diagnoses up to and including the hospital admission during which the aspiration was performed. Because not all medical records were available for every patient, it was not possible to retrieve all of this information for each patient.

Proportions were compared by using Pearson‚s chi-square test or the Fisher exact test, normally distributed continuous variables by using the Student t-test, and skewed continuous variables by using the Kruskall-Wallis test [31]. The survival times from the bone marrow aspiration and from the determination of HIV seropositivity were analysed, according to bone marrow macrophage iron grade, with Kaplan-Meier estimates [32] and log-rank tests [33] (Fig. 1), and with Cox proportional hazards analyses [34] adjusted for several variables that might be expected to influence the duration of survival. Although we wished to adjust these analyses for the CD4 count at the time of the bone marrow study, they were adjusted for the absolute lymphocyte count instead, because the CD4 count was available for less than one-third of the research subjects. The use of the absolute lymphocyte count as a surrogate for the CD4 count is supported by the fact that the CD4 count correlated well with the absolute lymphocyte count in the patients in the study for whom it was available, (r=0.62; P<0.001), and that the World Health Organization has proposed using the absolute lymphocyte count as a surrogate for the CD4 count in parts of the world where it is difficult to obtain CD4 counts [35]. The Cox proportional hazards analyses were also adjusted for the number of units of red blood cells transfused [36,37], hemoglobin concentration [38], absolute neutrophil count, year of the bone marrow aspiration, and histories of infection with cryptococcus, M. avium intracellulare, cytomegalovirus and Candida spp. [39].

F1-10
Fig. 1:
Kaplan-Meier plot of survival times by bone marrow macrophage iron grade. Grades 0-2, absent, low or normal iron stores; grades 4-5, markedly or massively increased iron stores. The P-value gives the significance level of the log-rank test. A. Survival after the bone marrow study. B. Survival after the diagnosis of HIV seropositivity.

Results

Compared with subjects with bone marrow macrophage iron grades of 0-2, patients with iron grades of 4-5 were less likely to have a history of hemophilia (P=0.013) and were more likely to have histories of blood transfusions (P=0.002) and of infections with Candida spp., Pneumocystis carinii, and Mycobacterium spp. (P≤0.006). They also had lower CD4 counts (P=0.027), absolute lymphocyte counts (P≤0.001), and hemoglobin concentrations (P<0.001) (Table 1). No indications for the bone marrow study were significantly associated with iron grade, and the only morphological finding of the bone marrow study that was significantly associated with iron grade was dysplasia, which was more common in patients with iron grades of 4-5 (P=0.009; data not shown).

T1-10
Table 1:
Demographic and clinical characteristics of study cohort by bone marrow macrophage iron grade

Follow-up information was obtained for the 348 subjects for a median of 3 months (range: <1 to 84 months) after the bone marrow aspiration and a median of 32 months (range: <1 to 164 months) after the determination of HIV seropositivity. By February, 1997, 118 (33.9%) of the patients had died. Unadjusted Kaplan-Meier estimates of survival times from the bone marrow aspiration or from the determination of HIV seropositivity according to bone marrow macrophage iron grade are shown in Fig. 1. In both cases, elevated iron grades were associated with shorter survival times (P≤0.006). Cox proportional hazards models confirmed that iron grades of 4-5 were associated with higher mortality rates than iron grades of 0-2, both from the time of the bone marrow aspiration and from the time of the determination of HIV seropositivity. The estimated rate of death (hazards ratio) after the bone marrow aspirate in subjects with bone marrow iron grades of 4-5 was 2.1-fold higher than the rate in patients with iron grades of 0-2 (95% confidence interval of 1.3 to 3.5; P=0.003), after adjustment for the absolute lymphocyte count, absolute neutrophil count, hemoglobin concentration, number of blood transfusions, year of the bone marrow study, and histories of infection with cryptococcus, cytomegalovirus, M. avium intracellulare, or Candida spp. The estimated rate of death (hazards ratio) after the diagnosis of HIV seropositivity in subjects with bone marrow iron grades of 4-5 was 2.8-fold higher than the rate in patients with iron grades of 0-2 (95% confidence interval of 1.5 to 4.9; P=0.001), after adjustment for the year of the bone marrow study, time from the diagnosis of HIV seropositivity to the bone marrow aspirate, absolute lymphocyte count, absolute neutrophil count, hemoglobin concentration, number of blood transfusions, and histories of infection with cryptococcus, cytomegalovirus, M. avium intracellulare, or Candida spp.

Discussion

In this retrospective study, high bone marrow macrophage iron grades in HIV-seropositive patients were found to be significantly associated with the occurrence of certain infections and with shortened survival times.

Although infections in general cause a shift of iron into stores that is secondary to the associated inflammatory process, most of the infections listed in Table 1 were not associated with macrophage iron grade. On the other hand, infections with Candida spp., P. carinii, and Mycobacterium spp. were associated with higher macrophage iron grades, and each of these infections has been reported in other studies to be influenced by iron status. For example, iron loading favors experimental candidal and mycobacterial infections [40-42]; increased plasma iron may be a risk factor for candidiasis [43]; and high iron stores may be a risk factor for mycobacterial infections in patients with and without HIV infection [5,6,44]. Withholding iron is also an effective therapy for experimental candidal and Pneumocystis infections [40,45]. In mice, resistance or susceptibility to infections caused by mycobacteria may be linked to the BcgR or BcgS status, respectively, whereby macrophages from BcgR mice are endowed with an active Nramp 1 protein. Recent results suggest that this protein plays a role in removing iron from the mycobacterial-containing phagosome [46].

In unadjusted and adjusted analyses, a statistical association was consistently found between macrophage iron grade and mortality rate. This association existed whether survival time was analysed from the time of bone marrow aspiration or from the time of the determination of HIV seropositivity. These observations are in accordance with the recently reported effect of haptoglobin polymorphisms on the outcome of HIV infection [47]. In that study, HIV-infected patients with haptoglobin 2-2 were shown to accumulate more iron, to oxidize more vitamin C, to have higher HIV-1 RNA levels and to have shortened survival times compared with individuals carrying haptoglobin 1-1 and 2-1.

Despite the limitations of this retrospective study, the fact that this cohort of HIV-seropositive patients was selected because they required a bone marrow study might decrease the possibility of bias as a result of the varying severity of the HIV infection. The need for a bone marrow study would tend to preclude the inclusion in the cohort of patients with early-stage HIV infection, but would rather tend to ensure that all patients had progressed to the point that an invasive investigation of an unexplained fever, hematological disorder, or malignancy became necessary.

Conclusion

This study raises the possibility that high macrophage iron stores help to accelerate the clinical course of HIV infection. If an adverse effect of high iron stores is confirmed by prospective studies, it may be possible to prevent or reduce iron loading in HIV-seropositive patients as a means of slowing the progression of the infection.

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Keywords:

; bone marrow; Candida spp.; HIV; iron stores; Mycobacterium spp.; opportunistic infections; Pneumocystis carinii; survival

© 1999 Lippincott Williams & Wilkins, Inc.