Increased deposition of C3b on red cells with low CR1 and CD55 in a malaria-endemic region of western Kenya: implications for the development of severe anemia.

BACKGROUND: Severe anemia due to Plasmodium falciparum malaria is a major cause of mortality among young children in western Kenya. The factors that lead to the age-specific incidence of this anemia are unknown. Previous studies have shown an age-related expression of red cell complement regulatory proteins, which protect erythrocytes from autologous complement attack and destruction. Our primary objective was to determine whether in a malaria-endemic area red cells with low levels of complement regulatory proteins are at increased risk for complement (C3b) deposition in vivo. Secondarily, we studied the relationship between red cell complement regulatory protein levels and hemoglobin levels.
METHODS: Three hundred and forty-two life-long residents of a malaria-holoendemic region of western Kenya were enrolled in a cross-sectional study and stratified by age. We measured red cell C3b, CR1, CD55, and immune complex binding capacity by flow cytometry. Individuals who were positive for malaria were treated and blood was collected when they were free of parasitemia. Analysis of variance was used to identify independent variables associated with the %C3b-positive red cells and the hemoglobin level.
RESULTS: Individuals between the ages of 6 and 36 months had the lowest red cell CR1, highest %C3b-positive red cells, and highest parasite density. Malaria prevalence also reached its peak within this age group. Among children </= 24 months of age the %C3b-positive red cells was usually higher in individuals who were treated for malaria than in uninfected individuals with similarly low red cell CR1 and CD55. The variables that most strongly influenced the %C3b-positive red cells were age, malaria status, and red cell CD55 level. Although it did not reach statistical significance, red cell CR1 was more important than red cell CD55 among individuals treated for malaria. The variables that most strongly influenced the hemoglobin level were age, the %C3b-positive red cells, red cell CR1, and red cell CD55.
CONCLUSION: Increasing malaria prevalence among children >6 to <or= 36 months of age in western Kenya, together with low red cell CR1 and CD55 levels, results in increased C3b deposition on red cells and low hemoglobin. The strong contribution of age to C3b deposition suggests that there are still additional unidentified age-related factors that increase the susceptibility of red cells to C3b deposition and destruction.

Background

Plasmodium falciparum malaria is responsible for 1 to 2 million deaths per year, with most in sub-Saharan Africa [1]. One unexplained but consistent feature of the epidemiology of clinical malaria is the age distribution of syndromes of severe disease. Severe anemia is most common in areas of intense transmission of P. falciparum and tends to occur in children <3 years of age, whereas cerebral malaria occurs in areas where the annual inoculation rate is low, and it occurs in older children and adults [2-5].

The pathogenesis of severe anemia in malaria is complex. However, a number of observations suggest that the destruction of uninfected red cells is a significant contributor to, if not the major cause of, this anemia: uninfected red cells have a decreased life span in patients with P. falciparum [6] and the hematocrit can continue to decrease for days following treatment [7]. Further, the fact that the life span of erythrocytes is 120 days suggests that bone marrow dysfunction would have to be very prolonged to make a significant contribution to this anemia. A mathematical model of severe malarial anemia has revealed that with each lysed infected erythrocyte a further 8.5 uninfected erythrocytes are destroyed [8]. These observations suggest that destruction of uninfected red cells takes place during malaria infection. Complement activation and its deposition on red cells are prime suspects in this process. Complement is activated during malaria infection [9,10] and C3d and IgG, molecules that are implicated in the removal of senescent red cells via erythrophagocytosis [11], have been detected on red cells of children with severe malaria [12,13].

In order to understand the nature of the susceptibility of red cells to complement activation during malaria infection we have investigated the expression of red cell complement regulatory proteins in persons living in a malaria-endemic area. Complement receptor 1 (CR1, CD35) is a 200 kDa protein that is found on red cells and most leukocytes [14]. It also accelerates the decay of C3 and C5 convertases, protein complexes that catalyze the cleavage of C3 and C5 into C3b and C5b respectively [15]. Red cells are able to bind C3b-bearing immune complexes (ICs) via CR1 and carry them to the liver and spleen where they are removed from circulation [16,17]. Therefore, CR1 prevents the deposition of C3b on cell surfaces and has a critical role in the removal of ICs from circulation.

The other red cell complement regulatory protein that we have studied is decay accelerating factor (DAF, CD55) [18]. It is a 70 kDa protein linked to the cell membrane via glycosyl-phosphatidyl-inositol (GPI). CD55 catalyzes the degradation of C3 convertases and also promotes the inactivation of C3b [19,20]. Deficiency in membrane DAF and CD59, a third red cell complement regulator, leads to paroxysmal nocturnal hemoglobinuria, a condition characterized by massive hemolysis due to complement activation on red cells. Consequently, red cell complement regulatory proteins may play an important role in protecting red cells from complement-mediated destruction as a result of IC formation and complement activation that occur during malaria infection [9,21]. Consistent with this hypothesis, we have found that red cells of children with P. falciparum infection and severe anemia have acquired deficiencies in CR1 and CD55, and increased C3b deposition [13,22]. Excessive C3b deposition on red cells during malaria infection as a result of deficiency in red cell complement regulatory proteins may result in increased destruction of red cells by phagocytosis and severe anemia [15].

To understand the age predilection of severe anemia during P. falciparum infection, we have studied the relationship between red cell CR1 and CD55 and age. Our studies suggest that the expression of red cell CR1 and CD55 are age-dependent, decreasing from birth to a nadir somewhere between 1 and 2 years of age and rising thereafter [23]. This pattern has been observed in populations of diverse ethnic origin and is independent of malaria infection. We have proposed that the changes in the expression of red cell complement regulatory proteins with age may influence the susceptibility of red cells to complement-mediated damage and, by extension, the age predilection for severe anemia during malaria infection [24]. To test whether the susceptibility of red cells to C3b deposition is influenced by the age-related changes in complement regulatory proteins we carried out a cross-sectional study of individuals in a malaria-holoendemic region of western Kenya, measured their red cell CR1, CD55, and IC binding capacity, and determined the %C3b-positive red cells.

Methods

Study design and population

The study was cross-sectional in design and was approved by the Walter Reed Army Institute of Research Human Use Research Committee and by the National Ethical Review Committee, Nairobi, Kenya. It was carried out from October through December 2004 in Kombewa Division, an area off the shores of Lake Victoria within Kisumu District. Malaria is holoendemic in this region, occurring throughout the year with peak seasons during the long rains (March through August) and during the short rains (October through December) [25]. The area is divided into four sub-Divisions and 12 political locations with approximately 62,000 inhabitants. The majority of the inhabitants are of the Luo ethnic group and dedicate themselves to subsistence farming and fishing.

Enrollment was stratified into nine age groups selected based on the age distribution of children with severe anemia (Hgb ≤ 5.0 g/dl) and P. falciparum malaria from Kisumu District, western Kenya, who participated in our studies from 1998 to 2001 [13,26,27]: 0 to ≤ 6 months, >6 to ≤ 12 months, >12 to ≤ 18 months, >18 to ≤ 24 months, >24 to ≤ 36 months, >36 to ≤ 72 months, >72 to ≤ 144 months, >144 to ≤ 288 months, and >288 to ≤ 540 months. Since we had no previous data on the levels of C3b deposition on red cells, enrollment targets for each group were set to maximize the probability of detecting significant differences between the >6 to ≤ 12 month group, where we had seen the lowest level of CR1 in a previous study [28], and other groups. The inclusion criterion was any person male or female of age 45 years or younger who was a life-long resident of the study area. As many conditions including malaria [13], HIV infection [29], and others [30,31] can alter the level of red cell complement regulatory proteins, the following exclusion criteria were established: 1) evidence on clinical grounds of malnutrition manifested by marasmus or kwashiorkor; 2) immunocompromised status manifested by weight loss, thrush, or diffuse adenopathy; 3) severe anemia (Hgb ≤ 5.0 g/dl); 4) bacterial infection (for example, pneumonia); 5) malignancy; and 6) blood transfusion within 3 months preceding the study.

A preliminary census was carried out within selected villages to identify potential participants. All individuals who met the entry criteria were invited to participate. On the days of recruitment individuals or parents with children self-reported to the clinic for enrollment in the study. Informed consent was obtained prior to any procedures. Individuals underwent a standard medical evaluation. Date of birth was confirmed by national registration card, baptismal certificate, or birth certificate. During the initial screening, blood obtained by finger prick was used to prepare thick and thin Giemsa-stained blood smears. If an individual was positive for malaria, he or she was treated with artemether/lumefantrine [32] and asked to return 2 weeks later for re-evaluation. Individuals who were febrile (oral or axillary temperature >37.5°C) without malaria were evaluated and treated for any minor illness identified and asked to return 2 weeks later. Individuals who returned for re-evaluation were screened for malaria again. If found to be malaria positive or febrile at this second screen, they were again evaluated, treated, and asked to come back 2 weeks later.

Blood samples and smears

Malaria smears were read by trained and certified microscopists. All smears were read in duplicate by independent microscopists who were unaware of the status of the participants. Discrepancies were settled by a third independent microscopist. A smear required a minimum of 200 high power fields scanned prior to being considered negative. Once an individual was deemed well and asymptomatic, a 2.5 ml ethylenediaminetetraacetic acid (EDTA)-anticoagulated sample of venous blood was obtained. An aliquot was used for complete blood count determination using an automated hematology analyzer (Coulter, Hialeah, FL). The remaining sample was centrifuged and the plasma removed. The red blood cell pellet was then cryopreserved until used [33].

Flow cytometric measurement of CR1 and CD55

Erythrocyte CR1 and CD55 levels were determined using frozen samples. In preliminary experiments, we observed no difference in the level of red cell complement regulatory proteins between fresh and frozen samples. All primary antibodies were titered to saturation. 10 μl of thawed erythrocyte pellet was washed twice in 1 ml of Alsever's buffer and resuspended in the same volume of buffer. Unless otherwise stated, all procedures were as previously described [34]. The following primary antibodies were used in dilutions of 1:20: anti-CR1 clone E11, anti-CD55 clone IA10, and isotype controls for each (Becton-Dickinson, Belgium). A secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Becton-Dickinson, San Diego, CA) was used at a dilution of 1:50.

Flow cytometry was carried out using a FACScan flow cytometer (Becton-Dickinson). Analysis was done using FCS Express v2.5 (De Novo Software, Los Angeles, CA). Red cells were gated on the basis of their forward and side scatter characteristics using logarithmic amplification. The median fluorescence intensity (MFI) of each sample was measured using logarithmic amplification. The MFI values for CR1 and CD55 were normalized to the mean of the MFI of the red cell standard using the formula

where 'CorrMFIs' and 'MFIs' are the corrected and uncorrected sample MFI respectively, 'MFIcmean' is the mean of all the MFI values of the standard control, and 'MFIc' is the mean of the control obtained in parallel with the sample. The number of molecules of CR1 per red cell was derived from a fluorescence standard curve created using cells with known CR1 numbers. Red cell anti-CD55 antibody binding capacities were derived from a standard curve created using beads of known antibody binding capacity (Bangs Lab, Fishers, IN) [35].

Preparation of ICs

50 μl of 49 mg/ml rabbit anti-BSA (Sigma-Adrich, St. Louis, MO) and 3 μl of 5 mg/ml BSA-FITC (Accurate Chemical and Scientific Corp., Westbury, NY) were added to 950 μl of RPMI1640 (Sigma-Aldrich). This combination was noted to be the point of equivalence in preliminary experiments. The mixture was incubated at 37°C for 1 hr and overnight at 4°C. The next day, the IC preparation of soluble and insoluble IC was aliquoted and stored at -20°C.

Measurement of C3b on red cells

All centrifugation steps were at × 500 g for 5 min. Rabbit polyclonal anti-C3a (negative control antibody) and anti-C3b (Accurate) were pre-adsorbed ×3 by adding a 1:50 dilution of antibody in phosphate buffered saline (PBS) pH 7.4 to an equal volume of packed pre-washed erythrocytes from the normal standard control. The cells were incubated for 1 hr at 37°C with constant rocking followed by centrifugation. The pre-adsorbed antibody was frozen at -20°C in single-use aliquots. 100 μl of pre-washed freshly thawed erythrocytes at 1% hematocrit in Alsever's buffer was added to wells of a 96-well plate and resuspended in 50 μl of pre-adsorbed rabbit anti-C3b, anti-C3a, or in PBS (unstained control), and incubated for 10 min at 37°C. After two washes in PBS, the cells were resuspended in 1:50 anti-rabbit PE (Sigma-Aldrich) for 30 min at room temperature, washed twice, and resuspended again in PBS. Acquisition was carried out as above. The %C3b-positive cells were calculated by Overton subtraction [36] of the baseline C3a histogram from the baseline C3b histogram.

Measurement of IC binding capacity

For IC opsonization, 5 μl of stock IC or RPMI 1640 (unstained control) was incubated in a total volume of 100 μl containing 30% AB+ serum in wells of a 96-well plate. For a negative control, a separate set of wells contained IC plus 10 mM EDTA. Following incubation at 37°C for 30 min with constant rocking motion, 100 μl of 1% hematocrit suspension of freshly thawed red cells from each study participant or from a standard aparasitemic control in RPMI 1640 was added to each of the wells of the above 96-well plate. This was followed by further incubation for 30 min at 37°C. The erythrocytes were then washed twice in 200 μl of ice-cold RPMI 1640, resuspended in PBS containing 1% paraformaldehyde, and stored at 4°C until acquisition. After gating, the erythrocyte FITC fluorescence was measured using logarithmic amplification and the positive cutoff was set using the unstained cells. The percent of positive red cells (IC binding capacity) was calculated based on this cutoff. To control for day-to-day variation, the IC binding capacity was normalized to the mean IC binding capacity of the red cell standard used throughout using a formula similar to the one used for correction of CR1 and CD55 (above).

Statistical analysis

Statistical analysis was carried out using SPSS v11.5 (SPSS Inc., Chicago, IL). The flow cytometry and parasite density data are presented graphically for each age group as box plots, where the box represents the boundaries between the 25th and 75th percentile, the line through the box represents the median, the whiskers the 10th and 90th percentile limits, and the dots are the outliers. Analysis of variance (ANOVA) was used to detect differences across age groups adjusting for factors and covariates as indicated in the figure legends. P values were adjusted for multiple comparisons using the Sidak correction. The independent samples t-test was used for comparisons of normal continuous data between two groups. The chi-square test was used to compare proportions across groups. The general linear model procedure was used to carry out univariate ANOVA and analysis of covariance (ANCOVA) to identify independent variables associated with the %C3b-positive red cells and the hemoglobin level. Age groups, red cell CR1, red cell CD55, parasite density, malaria status, and IC binding capacity were the independent predictor variables included in the models. %C3b-positive red cells were also used as an independent variable for the hemoglobin level. Categorical variables were transformed into dummy variables and the B coefficient for the last group set to '0' for reference. All tests were two-sided with α ≤ 0.05.

Results

Demographic and clinical characteristics of the study population

Four hundred and two potential participants or parents of children who met the inclusion criterion signed the consent form and were screened. Of these, 344 met none of the exclusion criteria, attended the screening sessions, and agreed to provide blood samples (Table 1). Blood samples could not be drawn in sufficient quantities from two individuals, making the final number of samples available for the study 342. All individuals who were parasitemic were treated for malaria and in most cases the blood sample was collected 2 weeks later. Table 2 summarizes the demographic characteristics of the enrolled participants that contributed samples. We observed no difference in the proportion of locations or villages represented across the groups (data not shown).

Table 1

Disposition of participants

	Enrolled (N)	Deferred to Next Screen		Excluded
		N	Reason for Deferral	N	Reason for Exclusion
Screen 1	190	194	190- positive blood smear,	18	3- refused blood draw
			4- non-malaria fever		12- malnourished or immunocompromised
					2- pneumonia
					1- severe anemia
Screen 2	151	10	All positive smear	33	1- age cohort full
					32- no shows
Screen 3	3	0		7	4- no shows,
					3- age cohort full
Total	344*			58

*Two individuals excluded due to insufficient sample.

Table 2

Population demographics

Age Groups (Months)	Total N	No. Female (%)	Median Age in Months (Range)
0 to ≤ 6	30	17 (56.7)	2.6 (0.4 to 0.6)
>6 to ≤ 12	60	29 (48.3)	10.9 (6.1 to 12.0)
>12 to ≤ 18	34	13 (38.2)	14.2 (12.1 to 17.5)
>18 to ≤ 24	27	14 (51.9)	21.3 (18.3 to 23.7)
>24 to ≤ 36	31	14 (45.2)	28.6 (24.2 to 36.0)
>36 to ≤ 72	42	22 (52.4)	42.7 (36.8 to 70.5)
>72 to ≤ 144	39	17 (43.6)	99.2 (73.1 to 143.9)
>144 to ≤ 288	24	12 (50.0)	248.9 (145.1 to 288.0)
>288 to ≤ 540	55	31 (56.4)	393.4 (290.7 to 540.0)
Total	342	169 (49.4)	29.7 (0.4 to 540.0)

Red cell CR1, CD55, and IC binding capacity

The nadir of hemoglobin level occurred in the >12 to ≤ 18 month group (Figure 1a). For multiple comparison testing we collapsed the age groups for age >6 to ≤ 36 months, which seemed to form a homogeneous subset in most analyses (data not shown), to reduce the number of comparisons and compared all other groups to this new group (Figure 1). The changes in hemoglobin level across groups were highly statistically significant. Although overall unadjusted changes in CR1, CD55, and IC binding capacity across age groups were statistically significant, only a limited number of comparisons were statistically significant after adjustment for malaria status and parasite density (Figure 1). Nonetheless, these findings confirm our previous results [37]. In the present study, children of between 6 and 36 months of age had the lowest red cell CR1 (Figure 1b) whereas children between 24 and 36 months of age had the lowest red cell CD55 (Figure 1c). The red cell IC binding capacity showed a pattern similar to that of CR1 but with a less marked rise in adulthood (Figure 1d).

Figure 1

Age-related changes in hemoglobin concentration, red cell CR1, CD55, and IC binding capacity. For multiple comparison testing all groups were compared with the group formed by collapsing the groups ranging in age from >6 to ≤ 36 months (bracket). P values were adjusted using the Sidak correction for multiple comparisons. * = 0.01

Age-related changes in hemoglobin concentration, red cell CR1, CD55, and IC binding capacity. For multiple comparison testing all groups were compared with the group formed by collapsing the groups ranging in age from >6 to ≤ 36 months (bracket). P values were adjusted using the Sidak correction for multiple comparisons. * = 0.01