The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pierrot, C.
Right arrow Articles by Khalife, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pierrot, C.
Right arrow Articles by Khalife, J.
The Journal of Immunology, 2007, 178: 1713-1722.
Copyright © 2007 by The American Association of Immunologists, Inc.

Contribution of T Cells and Neutrophils in Protection of Young Susceptible Rats from Fatal Experimental Malaria1

Christine Pierrot*, Estelle Adam2,*, David Hot{dagger}, Sophia Lafitte*, Monique Capron*, James D. George{ddagger} and Jamal Khalife3,*

* Institut National de la Santé et de la Recherche Médicale Unité 547, Institut Pasteur de Lille, Lille, France; {dagger} Laboratoire des Biopuces, Institut Pasteur de Lille, Lille, France; and {ddagger} GE Healthcare, Piscataway, NJ 08855


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In human malaria, children suffer very high rates of morbidity and mortality. To analyze the mechanisms involved in age-dependent protection against malaria, we developed an experimental model of infection in rats, where young rats are susceptible to Plasmodium berghei and adult rats control blood parasites and survive thereafter. In this study, we showed that protection of young rats could be achievable by adoptive transfer of spleen cells from adult protected rats, among which T cells could transfer partial protection. Transcriptome analysis of spleen cells transferring immunity revealed the overexpression of genes mainly expressed by eosinophils and neutrophils. Evaluation of the role of neutrophils showed that these cells were able to transfer partial protection to young rats. This antiparasitic effect was shown to be mediated, at least in part, through the neutrophil protein-1 defensin. Further adoptive transfer experiments indicated an efficient cooperation between neutrophils and T cells in protecting all young recipients. These observations, together with those from in vitro studies in human malaria, suggest that the failure of children to control infection could be related not only to an immaturity of their adaptive immunity but also to a lack in an adequate innate immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In human malaria caused by Plasmodium falciparum infection, there are at least 1–2 million deaths annually, mainly in children under the age of 5 years. To understand this age-related susceptibility and protection of humans to malaria infection, several epidemiological studies have addressed the issue of whether the immune response to malaria in children and adults is different. There may be several mechanisms potentially involved in immune hyporesponsiveness in young mice and humans, such as developmental immaturity of APCs thus influencing establishment of effective T cell-APC interactions (1, 2), incomplete signaling because of low expression of CD40L on T cells (3), and/or impaired responsiveness to TLR ligands leading to a lack of appropriate cytokine production (4). Whether these deficiencies are still present in 1- to 5-year-old infants has not yet been established.

Comparison of cell-mediated activity or circulating cytokine levels in children and adults in different areas of malaria transmission found Th1-like responses in childhood tending to a Th2-like response in adulthood (5, 6, 7, 8, 9, 10). Similarly, analysis of IgG subclasses indicated a predominance of IgG1, IgG3, and IgE during adulthood, features of a Th2-like response (11, 12, 13, 14, 15, 16, 17). These studies were conducted under conditions of chronic exposure to malaria, and there have been no studies characterizing immune responses in detail in children and adults after a primary exposure to malaria. However, in newly infected individuals, Baird (18) observed that protective immunity developed more rapidly in adults than in children.

In experimental malaria, the most extensive work on immune responses has been conducted using adult mouse models with no clear studies on the effects of age. From these studies, it appears that cellular and humoral responses are essential actors in the control and clearance of malaria parasites (for review, see Refs. 19, 20, 21). Analysis of cellular immunity demonstrated that CD4+ T cells (22, 23, 24) and B cells (22, 25) are associated with protection against malaria through the control of growth of blood parasite stages. Although there has been some controversy as to the function of NK T cells (26, 27, 28, 29), it has been reported that NK cells play a crucial role during the early phase of infection (30, 31, 32). In a recent work, we showed that the mouse model was not suitable to study the effect of age on the course of malaria infection because different parasitological and clinical parameters were found age-independent (33). However the course and outcome of a primary infection in the rat was clearly dependent on age (33), where an increase in the number of CD8+ T cells and NK T cells was associated with the control of infection in adult rats. In contrast, high levels of circulating IL-10 and persistence of CD4+CD25+ T cells were observed in young susceptible rats. In vivo neutralization of IL-10 or administration IFN-{gamma} was unable to protect young infected rats from death (34). Nevertheless, susceptibility of young rats can be reversed by adoptive spleen cell transfer from immune adult rats, indicating that the cellular response present in the spleen of adult host after parasite resolution is capable of controlling infection in young host.

The studies reported here were designed to define in more detail the effector cells responsible for the transfer of protective immunity to young host, and to investigate the genes expressed in spleen cells of adult protected rats capable of transferring protection. Adoptive transfer revealed that T cells obtained from adult immune rats partially protect young recipient rats, whereas microarray analysis indicated the presence of a more efficient pathway in protection that involves neutrophils and defensin neutrophil protein-1 (NP-1).4


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Parasites and animals

Plasmodium berghei ANKA strain (uncloned line from I. Landau, Museum d’Histoire Naturelle, Paris, France) used in this study was described previously (34). This parasite strain was adapted to Fischer rats by at least three successive passages (by i.p. injection). Four- and 8-wk-old F344 rats were purchased from Harlan and raised in the specific pathogen-free animal facility of the Institut Pasteur de Lille. Plasmodium infections were performed by i.p. injection of 107 parasitized RBC. Tail vein blood was used to measure parasitemia on thin film blood smears stained with eosin-thiazine (Diff Quick II kit; Dade Behring). The absence of other infections including rodent viruses, bacteria, and parasites was checked in infected animals by the International Council for Laboratory Animal Science Virus Reference Centre (The Netherlands) and the Centre de Développement des Techniques Avancées pour l’Expérimentation Animale (France). Animal work was conducted in accordance with the guidelines of laboratory animal care published by the French Ethical Committee.

Spleen cell and neutrophil preparations and transfer experiments

Spleens were removed from infected adult rats and from uninfected age-matched controls 1 wk after parasite clearance (day 25 postinfection (p.i.)). Total spleen cells were prepared in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS (AbCys). RBC were lysed by hyperosmotic treatment (0.2 M ammonium chloride, 10 mM sodium carbonate, 0.1 mM EDTA buffer). After three washes in RPMI 1640, spleen cell viability was evaluated by trypan blue dye exclusion. Neutrophils were recovered in the peritoneal cavity 4 h after i.p. injection of 5 ml of 5% thioglycollate (Difco Laboratories). Examination of cytospins and flow cytometry analysis showed that peritoneal cells contained a mean of 92% neutrophils, 4% eosinophils, and 4% macrophages. All cells were resuspended in RPMI 1640 and used in transfer experiments by i.v. route at the day of infection. Spleen cells from age-matched uninfected rats were prepared and used as control.

Spleen T cell enrichment and depletions

T cells were purified from whole spleen cells prepared from adult rats at day 25 p.i. To this end, we used Rat T cell Enrichment Columns (R&D Systems) based on high-affinity negative selection, and followed the manufacturer’s recommendations. Enriched population was checked by flow cytometry analysis and TCR{alpha}beta+ cells ranged between 85 and 90%.

Depletions of T and B cells from spleen cells of Fischer F344 rats were performed by positive selection. The spleen cell suspension was incubated with mouse IgG1 mAbs R73 (anti-{alpha}beta TCR) or OX33 (anti-CD45RA, expressed mainly on B cells) (I. Anegon, Institut National de la Santé et de la Recherche Médicale Unité 437, Nantes, France). Unlabeled cells were purified using magnetic beads coated with rat anti-mouse IgG1 (Dynal Biotech). Quality of the depletion was checked by flow cytometry analysis.

Flow cytometry

Spleen cells were analyzed by one- or two-color fluorescence-activated cell sorting immunophenotyping, performed using FITC-, PE-, or biotin-conjugated mAbs: mouse anti-TCR (R-73), anti-CD45RA (OX33), anti-CD25 (OX39), anti-granulocytes (His48), anti-macrophages (ED2-like, His36) (BD Pharmingen), anti-CD4 (W3/25), anti-CD8 (OX8), anti-CD172a (OX41), anti-CD134 (OX40), and anti-NKR-P1A (clone 3.2.3) (I. Anegon, Institut National de la Santé et de la Recherche Médicale Unité 437, Nantes, France). Spleen cells (5 x 105 cells) were stained for 30 min on ice with these different Abs. After two washes in staining buffer (PBS with 2% FCS), streptavidin-allophycocyanin was added for 10 min on ice to detect biotinylated mAb. Fluorescence analysis was performed with CellQuest software (FACScan; BD Biosciences) on a total of 10,000 acquired events. Results are expressed as absolute number of cells within 150 x 106 transferred spleen cells.

Microarray analysis

Total RNA was extracted using RNAplus (Qbiogene) from spleen cells of protected adult rats that have been shown to transfer protection to young rats and from spleens of uninfected age-matched rats. To ensure RNA quality of each sample, integrity and purity were assessed by use of the Agilent Bioanalyser (Agilent Technologies). Ten micrograms of purified total RNA was then used for linear amplification following the CodeLink Gene Expression System Protocol (GE Healthcare). Ten micrograms of the resulting cRNA was hybridized to each CodeLink Rat UniSet I Bioarray following the CodeLink Gene Expression System Protocol without any modification. Each bioarray contains 9911 rat sequences and 300 negative bacterial control probes. The bioarrays were scanned using an Axon GenePix 4000B scanner at 635 nm, 10 µm resolution and a PMT value of 600. The resulting image was analyzed using the CodeLink Expression Analysis Software version 2.3, and data from nonexploitable and control spots were filtered out.

The mean intensity of data from the three noninfected animals was used as reference, and intensities of each animal (infected and noninfected) were compared with this reference to calculate M and A values. The SNOMAD (Standardization and NOrmalization of MicroArray Data) package (35) was then used in the statistical language R (36) to normalize data. MA-plot was drawn and the "global loess" function was applied to the data to correct for bias (37). Genes with absolute mean M value for noninfected rat above 0.5 were removed from analysis. M values of the remaining genes of the four infected rats were analyzed using the Linear Model for Microarray Data library in R (38). A classification of statistically significant modulations was obtained using moderated t statistic with empirical Bayes shrinkage of the SE (39). Because of multiple testing, obtained p values were corrected using the Benjamini and Hochberg method (40) to control the false discovery rate.

Confirmation of increased gene expression by quantitative PCR (Q-PCR)

For Q-PCR analyses, cDNA were prepared from total RNA using a Superscript II kit (Invitrogen Life Technologies), according to the manufacturer’s recommendations. Q-PCR was conducted on an Applied Biosystems 7000 PCR apparatus using SYBR Green dye (Applied Biosystems) to quantify the products over the course of the amplification reaction. Primers were designed using Primer Express software (ABI Prism) and were selected on the basis of their melting point and whether they produce an amplicon in the range of 120–150 bp. Primers pairs used for Q-PCR are shown in Table I. The cDNA samples were diluted with sterile Dnase-free water and used at the equivalent of 40 ng of total RNA used in transcription for quantification of target transcript levels. For each primer/cDNA combination, reactions were conducted in triplicate with controls including primers alone or primers/RNA. Q-PCR mixtures (25 µl) contained 2.5 µl of cDNA, 7.5 pmol of both primers, and 12.5 µl of 2 x SYBR Green PCR Master Mix containing the ROX-1 dye labeled. Reactions were done as follows: 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Dissociation curves were conducted at the end of each run to verify the absence of DNA contamination. All data were analyzed with ABI Prism 7000 SDS software (Applied Biosystems). The amplification threshold was set at 0.2 and the background fluorescence was determined during cycle 5–10. The mean of raw cycle threshold (Ct) values obtained for the control beta-actin transcript was used to adjust the Ct values of the gene of interest for each sample/primer combination ({Delta}CT). We calculated the ratio of infected rat:control as a 2{Delta}CT value (where {Delta}CT = CT of infected rat – CT of control rat) for each gene to evaluate the fold increase.


View this table:
[in this window]
[in a new window]

  		Table I. List of primers used for Q-PCR

 
Synthesis of rat defensin NP-1 and administration to young rats

The active rat defensin NP-1 is composed of a mature peptide of 32 aa (VTCYCRRTRCGFRERLSGACGYRGRIYRLCCR) (41, 42). Given that the Cys residues present in the mature peptide are fully dispensable for the function of defensin (43) and that they are known to be involved in the oxidation, dimerization, and solubility of synthetic peptides, we replaced them by Ser residues. The synthetic mature peptide with the following sequence (VTSYSRRTRSGFRERLSGASGYRGRIYRLSSR) was then produced using Fmoc solid-phase synthesis (Genepep). The peptide was purified to homogeneity (purity >98%) and the molecular mass was confirmed by mass spectrometry. The peptide dissolved in PBS LPS free (1 mg/ml/rat) was injected (i.p.) on the day of infection and at day 6 postinfection with the same dose. As control, young rats were injected either with PBS alone or a control peptide (AKFEVNNPQVQRAFNELIRVVHQL LPESSLRKRKRSR) under the conditions described above. In parallel, a set of experiments was performed to evaluate the direct effect of defensin on blood parasites. To this end, 107 infected RBC were preincubated with 300 µg of defensin or vehicle for 1 h at 37°C before infection. The course of infection was compared with that observed in rats administered with 300 µg of defensin i.p. at the day of infection.

Statistical analysis

The Mann-Whitney U test for nonparametric data was used for statistical comparisons of cellular numbers in spleen cells from protected rats and from age-matched uninfected controls.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Protection of young susceptible rats by transfer of spleen cells of adult protected rats

The course of parasitemia and survival in young infected rats (4 wk old at the beginning of experiment) that received spleen cells from adult rats prepared 1 wk after resolution of infection (day 25 p.i.) were monitored. All infected young rats receiving 150 x 106 immune spleen cells controlled blood parasite growth and survived with no parasite recrudescence (Fig. 1A), and protection appeared to be dose dependent as 10 x 106 transferred cells did not protect, 50 x 106 protected 50% of animals, and >80 x 106 conferred protection to all rats (Fig. 1B). In contrast, infected young rats that received 150 x 106 spleen cells from uninfected healthy adult rats were still susceptible to infection.


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 1. Protection of young rats infected with P. berghei by i.v. transfer of spleen cells from adult protected rats (obtained at day 25 p.i.). A, Course of parasitemia observed in young infected rats after transfer of 150 x 106 spleen cells purified either from adult protected rats or from control uninfected rats. Data are represented as mean parasitemia ± SEM (n = 12 rats). B, Course of parasitemia and cumulative survival at day 25 p.i. (insert) of young infected rats that received 10 x 106, 50 x 106, 80 x 106, or 150 x 106 spleen cells, respectively. For clarity of the figure, the SEs are not shown. Data are representative of two experiments (n = 6 in each group). {dagger}, Represents death of all rats.

 
Analysis of cell distribution in spleen cells transferring protection

The distribution of spleen cells transferring protection was investigated and compared with cells from uninfected age-matched rats. A comparison of either cell percentages or absolute numbers of CD45RA+, CD4+, CD8+, neutrophils, and NK T with those of age-matched controls did not show any significant differences (Fig. 2A). However, the number of macrophages His36+ present in the spleen decreased in adult rats after parasite clearance. By contrast, a significantly lower number of cells expressing CD25 was observed among the CD4+ T cells of protected adult rats, when compared with CD4+CD25+ T cells in uninfected age-matched controls (Fig. 2B). With regard to the expression of OX40, a marker of activated T cells, we observed a significant higher number within the spleen cells transferring protection when compared with uninfected age-matched rats (Fig. 2B).


Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 2. A, Cellular distribution of spleen cells from adult protected rats upon resolution of blood parasites. B, Distribution of CD4+CD25+ and T OX40+ cells. Results are presented as absolute number of each cell population present within 150 x 106 spleen cells used for adoptive transfer experiments. {blacksquare}, Represents spleen cells from adult protected rats; Figure 2, represents spleen cells from uninfected age-matched control rats. Data (mean + SEM) are of three independent experiments (n = 6 in each experiment). *, p < 0.001.

 
Contribution of B and T cells in protection of young susceptible rats

Next, we evaluated the contribution of B and T cells because they represent the dominant population in the spleen of adult immune rats transferring protection. When using whole spleen cells depleted in B cells before transfer to young rats, parasitemia levels were still low and comparable to those observed for the parental whole spleen cells (Fig. 3). In both cases, as expected, all rats recovered from infection. With respect to the role of T cells, young infected rats receiving 80 x 106 cells depleted in T cells developed high parasitemia and 37% of rats survived to infection, suggesting a partial contribution of T cells in protection. To confirm this role, adoptive transfer experiments with enriched T cells from adult immune rats were performed. Transfer of whole spleen cells, as expected, dramatically and rapidly decreased the level of infected RBC and all rats survived (Fig. 4A). However, young rats receiving enriched T cells (>85% purity) exhibited high parasitemia similar to that observed in the infected control group. After 15 days p.i., the parasitemia decreased in the young rats receiving T cells, and 58% eliminated their parasites and survived while all infected control rats died (Fig. 4B). The fact that parasitemia was high and required more time to be cleared in the young rats receiving purified T cells likely suggest an activation by blood parasites and/or a crosstalk with cells of recipients. All together, although these data suggest a partial role of T cell in the transfer of protection, they support the role of other spleen cells in the early control of parasite growth.


Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 3. T depletion, but not B depletion of spleen cells before transfer, affects course of parasitemia and survival rate of young recipient rats infected with P. berghei. Course of parasitemia and cumulative survival rate at day 25 p.i. (inset) observed in young rats receiving whole spleen cells, T-depleted or B-depleted spleen cells obtained from adult protected rats. Data are mean + SEM (n = 8 in each group). {dagger}, Represents death of rats.

 

Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 4. Protection of P. berghei-infected young rats after transfer of immune T-enriched cells. Course of parasitemia (A) and survival rate (B) observed in young infected rats after transfer of 40 x 106 immune spleen T cells (proportion of T cells in 80 x 106 of whole spleen cells) or 80 x 106 whole spleen cells obtained from adult protected rats upon resolution of blood parasites. x, Represents young infected control rats. Data are mean + SEM (n = 12). {dagger}, Represents death of rats.

 
Gene expression profiling of spleen cells transferring protection to young rats

Having observed a dramatic difference in the control of parasitemia levels between the transfer of purified T cells and that of whole spleen cells, it is conceivable that a rapid T-independent mechanism may be involved in the protection of young rats. To investigate the molecular underlying mechanisms and thereby the cells involved, we conducted a microarray analysis of the mRNA of spleen cells from immune adult rats obtained 1 wk after resolution of infection (day 25 p.i.) and compared them with uninfected rat spleen cells. For this purpose, we used the GE Healthcare Codelink Uniset Rat array. Gene expression data generated from spleen cells of adult protected rats (day 25 p.i.) allowed the identification of a number of genes that are up- and down-regulated upon resolution of infection when compared with the transcriptome obtained from age-matched uninfected rats. A complete list of genes has been deposited in National Center for Biotechnology Information’s Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession no. GSE4552. Genes with significant modulation of expression (p value <0.02) and with a log2-ratio (M value) above 2.0 or below –2.0 (fold equal to or less than –4 or ≥4) are listed in Table II. We have grouped the genes as up-regulated (10 genes in the left column, Table II) or down-regulated (31 genes in the right column, Table II) and have delineated known and unknown genes. Six of the known increased genes are mainly expressed by neutrophils (lipocalin 2, NM_130741; peptidoglycan recognition protein (PGRP), NM_053373; {alpha} defensin ratNP-1, U16686, matrix metalloproteinase 9 (MMP9), NM_031055), eosinophils (proteoglycan 2 (PRG2), NM_031619, referred to as major basic protein (MBP)), and macrophages (natural resistance-associated macrophage protein 1 (NRAMP1), NM_031537). We also observed an increase of haptoglobin (Hp) (NM_012582.1), an acute phase protein described as a marker of inflammation, infection, and trauma. It is important to point out that beta defensins 1 and 2 that are mapped closely to the {alpha} defensin gene (44) were not overexpressed. This suggests that the increase of NP-1 is specific and is not due to a general boost of defensins. Interestingly, we observed an increased expression of lipocalin (>17-fold), secreted mainly by neutrophils, described to bind MMP9 to protect its activity. Intriguingly, many genes that are associated with T lymphocyte activation or cytokines were not found to be enhanced in spleen cells from adult protected rats when compared with healthy uninfected rats, even if we consider the threshold ≥2-fold. This result could likely be explained by the fact that the transcriptional profiling was performed after parasite clearance, during the recovery period p.i.


View this table:
[in this window]
[in a new window]

  		Table II. Genes for which transcript levels change in spleen cells from adult immune rats following a primary infection with P. bergheia

 
Expression of mRNA for many enzymes was decreased 4- to 9-fold when compared with controls. As shown in Table II, we observed an 8-fold decrease of PGD2 synthase (NM_031644) that can be linked, at least in part, to the decrease of CASP1 (NM_012762) because the product of the latter (IL-1beta) was shown to up-regulate the expression of PGD2 synthase (45). Other down-regulated genes include one transcript associated with protection against oxidative stress (oxidation resistance 1, NM_057153). This gene is targeted to the mitochondria, suggesting that the function of these organelles may be impaired following Plasmodium infection.

Confirmation of increased genes by Q-PCR

The transcriptional changes of several genes reflecting the activation of myeloid cells, and particularly neutrophils, were evaluated using real-time PCR on independently generated RNA samples (beta-actin was used as an internal control for each sample). Results presented in Table III show that lipocalin, PGRP, defensin rat NP-1, MMP9, eosinophils PRG2, NRAMP1, and Hp transcript levels were significantly higher in the spleen cells of protected adult rats when compared with uninfected age-matched controls. The increase in gene expression intensity did not correspond to a significant increase in neutrophil or eosinophil numbers. Indeed, differential spleen cell count using stained-cytospin preparations (data not shown) or flow cytometry analysis of neutrophil His48+ cells did not show significant differences (see Fig. 2A), suggesting an increase of cell activity rather than cell number.


View this table:
[in this window]
[in a new window]

  		Table III. Q-PCR analysis of increased gene expression in spleen cells of adult protected rats

 
Role of neutrophils in protection of young rats

Transcriptome analysis indicated that neutrophils of adult immune rats may be one of the cell populations that may participate in the early defense of young susceptible rats. For this reason, we evaluated the role of thioglycolate-elicited peritoneal neutrophils obtained from adult protected rats in protection. As shown in Fig. 5A, neutrophils transferred to young rats exerted a significant dose-dependent antiparasite effect because the parasitemia level was significantly decreased when compared with either rats that received peritoneal neutrophils from uninfected rats or vehicle. The follow-up of survival revealed that 33 and 75% of young rats receiving 20 or 35 x 106 neutrophils, respectively, resolved the infection. The mortality was 75 and 100% in young rats that received 35 x 106 neutrophils from uninfected rats or vehicle, respectively (Fig. 5A, inset). The role of other contaminating cells was excluded because the transfer of peritoneal cells from immune rats without thioglycolate induction did not control the parasitemia levels (data not shown).


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 5. Role of neutrophils in the transfer of protection to P. berghei-infected young rats. A, Course of parasitemia and cumulative survival rate at day 25 p.i. (inset) observed in young infected rats after transfer of 35 x 106 neutrophils (polymorphonuclear cell; PMN) obtained from adult protected rats or from uninfected age-matched controls. B, Course of parasitemia and cumulative survival rate at day 25 p.i. (inset) observed in young infected rats after transfer of 20 x 106 neutrophils (PMN) and 40 x 106 T cells alone or combined, obtained from adult protected rats. x, Represents young infected control rats. Data are mean + SEM (n = 6 in each group). {dagger}, Represents death of rats.

 
Next, we conducted adoptive cotransfer of neutrophils and T cells to see whether the protection levels observed with each cell population may be enhanced. As can be seen from Fig. 5B, parasitemia of cotransferred recipients was undetectable until day 13 p.i., followed by a low level of blood parasites between day 13 and 21 p.i. and a total clearance of parasites thereafter. Under the same conditions, young rats transferred with either neutrophils or T cells were only partially protected (Fig. 5B, inset). These data demonstrate that neutrophils together with T cells can actively control the early parasite growth and prevent rats from death.

Role of synthetic defensin NP-1 in protection of young rats

To further investigate the mechanism of protection at the molecular level, we decided to examine the effects of defensin NP-1 on a lethal P. berghei challenge in young rats. This was motivated by the fact that the expression of rat defensin NP-1 mRNA was up-regulated, and because defensins are known to play a key role not only in the defense against microbial infections but also against malaria parasite stages in the mosquito (46, 47). Administration of 2 x 1 mg/rat NP-1 (days 0 and 6 p.i.) allowed young rats to control parasitemia levels rapidly and protected all rats from death (Fig. 6). A single injection of 300 µg/rat of synthetic NP-1 did not affect growth of blood parasites nor confer protection to rats from death. However, a preincubation of infected RBC with 300 µg of NP-1 before infection led to a significant increase of protection levels (Fig. 6B).


Figure 6
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 6. Synthetic rat defensin NP-1 protects young rats from a lethal infection with P. berghei. Course of parasitemia (A) and survival rate (B) observed in young infected rats after administration of synthetic defensin. Each rat received at day 0 and day 6 p.i. 1 mg of defensin or control peptide (indicated by arrows). Survival rate of rats infected with P. berghei parasites preincubated with 300 µg of defensin 1 h at 37°C was compared with that observed in rats administered with 300 µg of defensin i.p. at the day of infection (B). Data are mean + SEM (n = 6 in each group). {dagger}, Represents death of rats.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
There is a critical need to establish approaches for effective and rapid protection from infection of young hosts, because their immune responses are immature. In this study, the combination of cellular approaches and gene profiling of the host response to experimental malaria showed that the protection of young rats from lethal infection can be accomplished in two ways: 1) the transfer of T cells obtained from spleen cells of adult protected rats after parasite resolution, and 2) the transfer of peritoneal neutrophils elicited by thioglycollate from adult protected rats or the administration of NP-1, known to be an antimicrobial peptide. The latter finding has implications for young hosts with deficient immune system who may generate poor T cell immunity against intracellular infections.

Experimental malaria studies performed exclusively in adult hosts indicated that both CD4+ and CD8+ T cells play an important role in the defense against malaria. To address the role of these cells in an age-dependent model, where young susceptible rats can be protected by the transfer of whole spleen cells from adult protected rats, TCR+ cells were transferred to young infected rats. These experiments indicated that 58% of young rats recovered from infection after T cell transfer. In this context, our results demonstrated that expression of OX40, a marker restricted to rat-activated CD4+ T cells (48), was significantly increased in the spleen of protected rats. The OX40 molecule is known to bind to rat splenic B cells and dendritic cells via OX40L and to induce proliferation and IL-2, IFN-{gamma}, IL-10, and IL-13 production (49), suggesting a possible involvement in cellular and humoral immune responses in the rat system. In parallel to this increase, analysis of CD25 cells, a marker for the regulating phenotype, showed that the CD4+CD25+ cells were decreased in the spleen of adult protected rats when compared with uninfected age-matched rats. Together, these data suggest that a down-regulation of CD4+CD25+ along with an increase of OX40+ T cells in adult protected rats may be involved, at least in part, in the transfer of protection to young rats.

Although the transfer of enriched T cells can protect young rats, surprisingly the parasitemia levels and the time required for parasite clearance by these cells greatly differed from that obtained after total spleen cell transfer. The slow and partial effect of the transfer of purified T cells may be attributed to a delay of activation of the immune system, suggesting that they play the role of delivery vector to induce cell-mediated immunity of young recipient rats. Another explanation for this difference could be the possible implication of other cells that act more rapidly and provide a quick control of blood parasite growth. The participation of B cells or NK/NK T cells (data not shown) can be excluded because their depletion before transfer did not affect the protection of young infected rats. These results contrast with cells required to confer resistance in infected adult mice (20, 21) and emphasize the need for caution when extrapolating immune mechanisms from adult to young hosts.

The comparison of the transfer of T cells with that of whole spleen cells from adult protected rats suggests that other cells than T cells are involved in the rapid protection of parasite growth and protection of young susceptible rats. At this stage, it is reasonable to propose that the nonlymphoid compartment of the spleen containing myeloid cells could be able to confer protection in adoptive transfer to young rats. Based on the genome-wide analysis of expression in spleens of adult protected rats, it appears that neutrophils may be involved in the transfer of an early control of blood parasites in young rats because several related genes were up-regulated. The activation of neutrophils in infected rats is well supported by a recent study in which transcriptome analysis of whole blood from humans infected with P. falciparum identified a gene expression profile related to neutrophil activity (50). Although neutrophilia has been linked with acute malaria (51), their role in host defense to malaria has been relatively underinvestigated (52, 53, 54). To clarify the role of neutrophils in adoptive immunity of infected rats, thioglycollate-elicited neutrophils from adult protected rats were used in transfer experiments. First, the transfer of these cells to adult-infected rats contributed to the control of parasitemia growth, suggesting an antiparasitic effect of these neutrophils (data not shown). With respect to young rats, adoptive transfer of these cells kept the parasitemia levels very low and allowed to resolve the infection in 33–75% of rats according to the number of neutrophils administered. It is noteworthy that the i.v. coadministration of neutrophils together with T cells significantly increased the survival rate of young rats from fatal infection when compared with single administration of each cell population. This result suggests that donor T cells and donor neutrophils act, at least additively, to increase the protection. In this context, neutrophils have been shown to prime in vivo effector T cells in response to bacterial Ags that are not directly accessible to the classical presentation pathway (55).

The gene expression intensity showed a significant increase of NP-1, referred to as defensin. Many studies have shown that parasites are susceptible to mosquito defensins, dermaseptin S3, and some synthetic peptides designed on the known structure of natural antimicrobial peptides (56, 57). In this study, we investigated the capacity of synthetic defensin NP-1 to protect young rats from a lethal infection. Its injection revealed that all treated young rats significantly reduced the peak of parasitemia, cleared total blood parasites within 3 wk, and survived the infection. Defensins are cytotoxic peptides and are believed to permeabilize the membranes of a wide range of organisms (58) including virus and parasites (59, 60). It has been shown that some synthetic peptides were able to kill intracellular blood-stage forms of the malaria parasite (56). It is conceivable that defensin may have a direct antimalarial effect after schizogony, when merozoites are released to infect further erythrocytes. Defensin may also have an indirect effect either by inducing NO synthase (61), which in turn increases NO production, and/or by enhancing Th1- and Th2-type cytokines (62). All together, these observations indicated an effective role of neutrophil in early protection of young rats. An additional argument in favor of a role for activated neutrophils in the adoptively transferred protection is the increased transcription of lipocalin. This molecule has been shown to play a role in both iron transport (63) and binding of iron siderophores as part of the innate antimicrobial defense mechanism (64). However, high levels of lipocalin were detected in sera from infected patients with P. falciparum with severe malaria and may have pathologic consequences resulting from alteration of iron homeostasis (65). This suggests that host protection vs disease promotion played by lipocalin is dose-dependent. A role for other molecular mediators is suggested because their expression was found to be up-regulated in spleen cells transferring protection. Indeed, microarray analysis revealed the overexpression of Hp and MBP (or PRG2) expressed by eosinophils. Interestingly, it has been shown that eosinophil granule proteins including eosinophil cationic protein and MBP are toxic for P. falciparum parasites (66). Subsequent studies revealed that MBP together with hemozoin, produced in malarial infection, can directly activate neutrophils (67, 68). With respect to Hp that is produced by different cell types including macrophages and eosinophils (69), there are several arguments that well support its role in the transfer of protection. First, it has been shown in vitro to have a deleterious effect on the growth of P. falciparum (70). Second, its role in vivo for antiparasitic effect comes from experiments showing that blood parasites burden were significantly greater in Hp knock-out mice compared with wild-type mice (71). Finally, studies investigating its relationship with immune response revealed that Hp plays a modulating role on the Th1/Th2 balance by promoting a dominant Th1 cellular response (72), which is described to participate in defense against intracellular parasites. Combining the in vivo data presented in this study, together with those obtained from human in vitro studies, it can be strongly suggested that both neutrophils and eosinophils participate in natural immunity to human malaria until the adaptive immune system becomes effective.

In summary, this study has demonstrated the efficient way to protect young rats infected with a lethal Plasmodium strain. It has provided us with specific insight on the role of neutrophils and their effector proteins in the control of early phase blood parasite growth, very likely associated with T cells to clear the infection. Further investigations during infection are now necessary to elucidate at the molecular levels why young rats exhibit heavy parasite burden leading to a fatal outcome, whereas adult rats control parasitemia levels and resolve completely the infection.


   Acknowledgments
 
We thank Dr. Jean Langhorne, Prof. David Dunne, and Dr. Laurent Renia for their comments and critical reading of the manuscript and C. Godin for his technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Institut National de la Santé et de la Recherche Médicale Unité 547 and Institut Pasteur de Lille. C.P. is a member of Institut Pasteur de Lille and J.K. is a member of Centre National de la Recherche Scientifique. Back

2 Current address: Endotis Pharma, Bioincubateur, Parc Eurasanté, 70 rue du Dr Yersin, 59120 Loos, France. Back

3 Address correspondence and reprint requests Dr. Jamal Khalife, Institut National de la Santé et de la Recherche Médicale Unité 547, Institut Pasteur de Lille, 1 rue du Prof. Calmette, 59019 Lille, France. E-mail address: jamal.khalife{at}pasteur-lille.fr Back

4 Abbreviations used in this paper: NP-1, neutrophil protein-1; p.i., postinfection; Q-PCR, quantitative PCR; Ct, cycle threshold; PGRP, peptidoglycan recognition protein; MMP9, matrix metalloproteinase 9; PRG2, proteoglycan 2; MBP, major basic protein; NRAMP1, natural resistance-associated macrophage protein 1; Hp, haptoglobin. Back

Received for publication March 29, 2006. Accepted for publication November 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Hunt, D. W., H. I. Huppertz, H. J. Jiang, R. E. Petty. 1994. Studies of human cord blood dendritic cells: evidence for functional immaturity. Blood 84: 4333-4343. [Abstract/Free Full Text]
  2. Marshall-Clarke, S., L. Tasker, R. M. Parkhouse. 2000. Immature B lymphocytes from adult bone marrow exhibit a selective defect in induced hyperexpression of major histocompatibility complex class II and fail to show B7.2 induction. Immunology 100: 141-151. [Medline]
  3. Brugnoni, D., P. Airo, D. Graf, M. Marconi, M. Lebowitz, A. Plebani, S. Giliani, F. Malacarne, R. Cattaneo, A. G. Ugazio, et al 1994. Ineffective expression of CD40 ligand on cord blood T cells may contribute to poor immunoglobulin production in the newborn. Eur. J. Immunol. 24: 1919-1924. [Medline]
  4. Levy, O., K. A. Zarember, R. M. Roy, C. Cywes, P. J. Godowski, M. R. Wessels. 2004. Selective impairment of TLR-mediated innate immunity in human newborns: neonatal blood plasma reduces monocyte TNF-{alpha} induction by bacterial lipopeptides, lipopolysaccharide, and imiquimod, but preserves the response to R-848. J. Immunol. 173: 4627-4634. [Abstract/Free Full Text]
  5. Riley, E. M., P. H. Jakobsen, S. J. Allen, J. G. Wheeler, S. Bennett, S. Jepsen, B. M. Greenwood. 1991. Immune response to soluble exoantigens of Plasmodium falciparum may contribute to both pathogenesis and protection in clinical malaria: evidence from a longitudinal, prospective study of semi-immune African children. Eur. J. Immunol. 21: 1019-1025. [Medline]
  6. Riley, E. M., S. J. Allen, J. G. Wheeler, M. J. Blackman, S. Bennett, B. Takacs, H. J. Schonfeld, A. A. Holder, B. M. Greenwood. 1992. Naturally acquired cellular and humoral immune responses to the major merozoite surface antigen (PfMSP1) of Plasmodium falciparum are associated with reduced malaria morbidity. Parasite Immunol. 14: 321-337. [Medline]
  7. Elghazali, G., F. Esposito, M. Troye-Blomberg. 1995. Comparison of the number of IL-4 and IFN-{gamma} secreting cells in response to the malaria vaccine candidate antigen Pf155/RESA in two groups of naturally primed individuals living in a malaria endemic area in Burkina Faso. Scand. J. Immunol. 42: 39-45. [Medline]
  8. Troye-Blomberg, M., E. M. Riley, L. Kabilan, M. Holmberg, H. Perlmann, U. Andersson, C. H. Heusser, P. Perlmann. 1990. Production by activated human T cells of interleukin 4 but not interferon-{gamma} is associated with elevated levels of serum antibodies to activating malaria antigens. Proc. Natl. Acad. Sci. USA 87: 5484-5488. [Abstract/Free Full Text]
  9. Mshana, R. N., J. Boulandi, N. M. Mshana, J. Mayombo, G. Mendome. 1991. Cytokines in the pathogenesis of malaria: levels of IL-Ibeta, IL-4, IL-6, TNF-{alpha} and IFN-{gamma} in plasma of healthy individuals and malaria patients in a holoendemic area. J. Clin. Lab. Immunol. 34: 131-139. [Medline]
  10. Luty, A. J., B. Lell, R. Schmidt-Ott, L. G. Lehman, D. Luckner, B. Greve, P. Matousek, K. Herbich, D. Schmid, F. Migot-Nabias, et al 1999. Interferon-{gamma} responses are associated with resistance to reinfection with Plasmodium falciparum in young African children. J. Infect. Dis. 179: 980-988. [Medline]
  11. Rzepczyk, C. M., K. Hale, N. Woodroffe, A. Bobogare, P. Csurhes, A. Ishii, A. Ferrante. 1997. Humoral immune responses of Solomon Islanders to the merozoite surface antigen 2 of Plasmodium falciparum show pronounced skewing towards antibodies of the immunoglobulin G3 subclass. Infect. Immun. 65: 1098-1100. [Abstract]
  12. Shi, Y. P., U. Sayed, S. H. Qari, J. M. Roberts, V. Udhayakumar, A. J. Oloo, W. A. Hawley, D. C. Kaslow, B. L. Nahlen, A. A. Lal. 1996. Natural immune response to the C-terminal 19-kilodalton domain of Plasmodium falciparum merozoite surface protein 1. Infect. Immun. 64: 2716-2723. [Abstract]
  13. Taylor, R. R., D. B. Smith, V. J. Robinson, J. S. McBride, E. M. Riley. 1995. Human antibody response to Plasmodium falciparum merozoite surface protein 2 is serogroup specific and predominantly of the immunoglobulin G3 subclass. Infect. Immun. 63: 4382-4388. [Abstract]
  14. Desowitz, R. S.. 1989. Plasmodium-specific immunoglobulin E in sera from an area of holoendemic malaria. Trans. R. Soc. Trop. Med. Hyg. 83: 478-479. [Medline]
  15. Desowitz, R. S., J. Elm, M. P. Alpers. 1993. Plasmodium falciparum-specific immunoglobulin G (IgG), IgM, and IgE antibodies in paired maternal-cord sera from east Sepik Province, Papua New Guinea. Infect. Immun. 61: 988-993. [Abstract/Free Full Text]
  16. Perlmann, H., H. Helmby, M. Hagstedt, J. Carlson, P. H. Larsson, M. Troye-Blomberg, P. Perlmann. 1994. IgE elevation and IgE anti-malarial antibodies in Plasmodium falciparum malaria: association of high IgE levels with cerebral malaria. Clin. Exp. Immunol. 97: 284-292. [Medline]
  17. Bouharoun-Tayoun, H., P. Druilhe. 1992. Plasmodium falciparum malaria: evidence for an isotype imbalance which may be responsible for delayed acquisition of protective immunity. Infect. Immun. 60: 1473-1481. [Abstract/Free Full Text]
  18. Baird, J. K.. 1995. Host age as a determinant of naturally acquired immunity to Plasmodium falciparum. Parasitol. Today 11: 105-111. [Medline]
  19. Taylor-Robinson, A. W.. 1995. Regulation of immunity to malaria: valuable lessons from murine models. Parasitol. Today 11: 334-342. [Medline]
  20. Li, C., E. Seixas, J. Langhorne. 2001. Rodent malarias: the mouse as a model for understanding immune responses and pathology induced by the erythrocytic stages of the parasite. Med. Microbiol. Immunol. 189: 115-126. [Medline]
  21. Langhorne, J., S. J. Quin, L. A. Sanni. 2002. Mouse models of blood-stage malaria infections: immune responses and cytokines involved in protection and pathology. Chem. Immunol. 80: 204-228. [Medline]
  22. Meding, S. J., J. Langhorne. 1991. CD4+ T cells and B cells are necessary for the transfer of protective immunity to Plasmodium chabaudi chabaudi. Eur. J. Immunol. 21: 1433-1438. [Medline]
  23. Weiss, W. R., M. Sedegah, J. A. Berzofsky, S. L. Hoffman. 1993. The role of CD4+ T cells in immunity to malaria sporozoites. J. Immunol. 151: 2690-2698. [Abstract]
  24. Phillips, R. S., K. E. Mathers, A. W. Taylor-Robinson. 1994. T cells in immunity to Plasmodium chabaudi chabaudi: operation and regulation of different pathways of protection. Res. Immunol. 145: 406-412. [Medline]
  25. von der Weid, T., N. Honarvar, J. Langhorne. 1996. Gene-targeted mice lacking B cells are unable to eliminate a blood stage malaria infection. J. Immunol. 156: 2510-2516. [Abstract]
  26. Gonzalez-Aseguinolaza, G., C. de Oliveira, M. Tomaska, S. Hong, O. Bruna-Romero, T. Nakayama, M. Taniguchi, A. Bendelac, L. Van Kaer, Y. Koezuka, M. Tsuji. 2000. {alpha}-Galactosylceramide-activated V{alpha}14 natural killer T cells mediate protection against murine malaria. Proc. Natl. Acad. Sci. USA 97: 8461-8466. [Abstract/Free Full Text]
  27. Molano, A., S. H. Park, Y. H. Chiu, S. Nosseir, A. Bendelac, M. Tsuji. 2000. Cutting edge: the IgG response to the circumsporozoite protein is MHC class II-dependent and CD1d-independent: exploring the role of GPIs in NK T cell activation and antimalarial responses. J. Immunol. 164: 5005-5009. [Abstract/Free Full Text]
  28. Pied, S., J. Roland, A. Louise, D. Voegtle, V. Soulard, D. Mazier, P. A. Cazenave. 2000. Liver CD4–CD8– NK1.1+ TCR{alpha}beta intermediate cells increase during experimental malaria infection and are able to exhibit inhibitory activity against the parasite liver stage in vitro. J. Immunol. 164: 1463-1469. [Abstract/Free Full Text]
  29. Weerasinghe, A., H. Sekikawa, H. Watanabe, K. Mannoor, S. R. Morshed, R. C. Halder, T. Kawamura, T. Kosaka, C. Miyaji, H. Kawamura, et al 2001. Association of intermediate T cell receptor cells, mainly their NK1.1– subset, with protection from malaria. Cell Immunol. 207: 28-35. [Medline]
  30. Kitaguchi, T., M. Nagoya, T. Amano, M. Suzuki, M. Minami. 1996. Analysis of roles of natural killer cells in defense against Plasmodium chabaudi in mice. Parasitol. Res. 82: 352-357. [Medline]
  31. Mannoor, M. K., A. Weerasinghe, R. C. Halder, S. Reza, M. Morshed, A. Ariyasinghe, H. Watanabe, H. Sekikawa, T. Abo. 2001. Resistance to malarial infection is achieved by the cooperation of NK1.1+ and NK1.1– subsets of intermediate TCR cells which are constituents of innate immunity. Cell. Immunol. 211: 96-104. [Medline]
  32. Mannoor, M. K., R. C. Halder, S. R. Morshed, A. Ariyasinghe, H. Y. Bakir, H. Kawamura, H. Watanabe, H. Sekikawa, T. Abo. 2002. Essential role of extrathymic T cells in protection against malaria. J. Immunol. 169: 301-306. [Abstract/Free Full Text]
  33. Pierrot, C., E. Adam, S. Lafitte, C. Godin, D. Dive, M. Capron, J. Khalife. 2003. Age-related susceptibility and resistance to Plasmodium berghei in mice and rats. Exp. Parasitol. 104: 81-85. [Medline]
  34. Adam, E., C. Pierrot, S. Lafitte, C. Godin, A. Saoudi, M. Capron, J. Khalife. 2003. The age-related resistance of rats to Plasmodium berghei infection is associated with differential cellular and humoral immune responses. Int. J. Parasitol. 33: 1067-1078. [Medline]
  35. Colantuoni, C., G. Henry, S. Zeger, J. Pevsner. 2002. SNOMAD (Standardization and NOrmalization of MicroArray Data): web-accessible gene expression data analysis. Bioinformatics 18: 1540-1541. [Abstract/Free Full Text]
  36. Ihaka, R., R. Gentleman. 1996. R: a language for data analysis and graphics. J. Computational Graphical Stat. 5: 299-314.
  37. Yang, Y. H., S. Dudoit, P. Luu, D. M. Lin, V. Peng, J. Ngai, T. P. Speed. 2002. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30: e15[Abstract/Free Full Text]
  38. Smyth, G. K., Y. H. Yang, T. Speed. 2003. Statistical issues in cDNA microarray data analysis. Methods Mol. Biol. 224: 111-136. [Medline]
  39. Lönnstedt, I., T. P. Speed. 2002. Replicated microarray data. Statistical Sinica 12: 31-46.
  40. Benjamini, Y., Y. Hochberg. 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. Roy. Stat. Soc. B. 57: 289-300.
  41. Yount, N. Y., M. S. Wang, J. Yuan, N. Banaiee, A. J. Ouellette, M. E. Selsted. 1995. Rat neutrophil defensins: precursor structures and expression during neutrophilic myelopoiesis. J. Immunol. 155: 4476-4484. [Abstract]
  42. Eisenhauer, P. B., S. S. Harwig, D. Szklarek, T. Ganz, M. E. Selsted, R. I. Lehrer. 1989. Purification and antimicrobial properties of three defensins from rat neutrophils. Infect. Immun. 57: 2021-2027. [Abstract/Free Full Text]
  43. Wu, Z., D. M. Hoover, D. Yang, C. Boulegue, F. Santamaria, J. J. Oppenheim, J. Lubkowski, W. Lu. 2003. Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3. Proc. Natl. Acad. Sci. USA 100: 8880-8885. [Abstract/Free Full Text]
  44. Jia, H. P., J. N. Mills, F. Barahmand-Pour, D. Nishimura, R. K. Mallampali, G. Wang, K. Wiles, B. F. Tack, C. L. Bevins, P. B. McCray, Jr. 1999. Molecular cloning and characterization of rat genes encoding homologues of human beta-defensins. Infect. Immun. 67: 4827-4833. [Abstract/Free Full Text]
  45. Muraki, T., K. Fujimori, M. Ishizaka, Y. Ohe, Y. Urade, F. Okajima, K. Ishikawa. 2004. Effects of interleukin-1beta and prostaglandin E2 on prostaglandin D synthase production in cultivated rat leptomeningeal cells. J. Cereb. Blood Flow Metab. 24: 409-418. [Medline]
  46. Lehrer, R. I.. 2004. Primate defensins. Nat. Rev. Microbiol. 2: 727-738. [Medline]
  47. Shahabuddin, M., I. Fields, P. Bulet, J. A. Hoffmann, L. H. Miller. 1998. Plasmodium gallinaceum: differential killing of some mosquito stages of the parasite by insect defensin. Exp. Parasitol. 89: 103-112. [Medline]
  48. Paterson, D. J., W. A. Jefferies, J. R. Green, M. R. Brandon, P. Corthesy, M. Puklavec, A. F. Williams. 1987. Antigens of activated rat T lymphocytes including a molecule of 50,000 Mr detected only on CD4 positive T blasts. Mol. Immunol. 24: 1281-1290. [Medline]
  49. Satake, Y., H. Akiba, K. Takeda, M. Atsuta, H. Yagita, K. Okumura. 2000. Characterization of rat OX40 ligand by monoclonal antibody. Biochem. Biophys. Res. Commun. 270: 1041-1048. [Medline]
  50. Griffiths, M. J., M. J. Shafi, S. J. Popper, C. A. Hemingway, M. M. Kortok, A. Wathen, K. A. Rockett, R. Mott, M. Levin, C. R. Newton, et al 2005. Genomewide analysis of the host response to malaria in Kenyan children. J. Infect. Dis. 191: 1599-1611. [Medline]
  51. Ladhani, S., B. Lowe, A. O. Cole, K. Kowuondo, C. R. Newton. 2002. Changes in white blood cells and platelets in children with falciparum malaria: relationship to disease outcome. Br. J. Haematol. 119: 839-847. [Medline]
  52. Brown, J., M. E. Smalley. 1981. Inhibition of the in vitro growth of Plasmodium falciparum by human polymorphonuclear neutrophil leucocytes. Clin. Exp. Immunol. 46: 106-109. [Medline]
  53. Kharazmi, A., S. Jepsen. 1984. Enhanced inhibition of in vitro multiplication of Plasmodium falciparum by stimulated human polymorphonuclear leucocytes. Clin. Exp. Immunol. 57: 287-292. [Medline]
  54. Taverne, J., H. M. Dockrell, J. H. Playfair. 1982. Killing of the malarial parasite Plasmodium yoelii in vitro by cells of myeloid origin. Parasite Immunol. 4: 77-91. [Medline]
  55. Tvinnereim, A. R., S. E. Hamilton, J. T. Harty. 2004. Neutrophil involvement in cross-priming CD8+ T cell responses to bacterial antigens. J. Immunol. 173: 1994-2002. [Abstract/Free Full Text]
  56. Ghosh, J. K., D. Shaool, P. Guillaud, L. Ciceron, D. Mazier, I. Kustanovich, Y. Shai, A. Mor. 1997. Selective cytotoxicity of dermaseptin S3 toward intraerythrocytic Plasmodium falciparum and the underlying molecular basis. J. Biol. Chem. 272: 31609-31616. [Abstract/Free Full Text]
  57. Arrighi, R. B., C. Nakamura, J. Miyake, H. Hurd, J. G. Burgess. 2002. Design and activity of antimicrobial peptides against sporogonic-stage parasites causing murine malarias. Antimicrob. Agents Chemother. 46: 2104-2110. [Abstract/Free Full Text]
  58. Bulet, P., C. Hetru, J. L. Dimarcq, D. Hoffmann. 1999. Antimicrobial peptides in insects; structure and function. Dev. Comp. Immunol. 23: 329-344. [Medline]
  59. Aley, S. B., M. Zimmerman, M. Hetsko, M. E. Selsted, F. D. Gillin. 1994. Killing of Giardia lamblia by cryptdins and cationic neutrophil peptides. Infect. Immun. 62: 5397-5403. [Abstract/Free Full Text]
  60. Lehrer, R. I., K. Daher, T. Ganz, M. E. Selsted. 1985. Direct inactivation of viruses by MCP-1 and MCP-2, natural peptide antibiotics from rabbit leukocytes. J. Virol. 54: 467-472. [Abstract/Free Full Text]
  61. Velasco, M., M. J. Diaz-Guerra, P. Diaz-Achirica, D. Andreu, L. Rivas, L. Bosca. 1997. Macrophage triggering with cecropin A and melittin-derived peptides induces type II nitric oxide synthase expression. J. Immunol. 158: 4437-4443. [Abstract]
  62. Lillard, J. W., Jr, P. N. Boyaka, O. Chertov, J. J. Oppenheim, J. R. McGhee. 1999. Mechanisms for induction of acquired host immunity by neutrophil peptide defensins. Proc. Natl. Acad. Sci. USA 96: 651-656. [Abstract/Free Full Text]
  63. Yang, J., D. Goetz, J. Y. Li, W. Wang, K. Mori, D. Setlik, T. Du, H. Erdjument-Bromage, P. Tempst, R. Strong, J. Barasch. 2002. An iron delivery pathway mediated by a lipocalin. Mol. Cell. 10: 1045-1056. [Medline]
  64. Goetz, D. H., M. A. Holmes, N. Borregaard, M. E. Bluhm, K. N. Raymond, R. K. Strong. 2002. The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol. Cell. 10: 1033-1043. [Medline]
  65. Mohammed, A. O., G. Elghazali, H. B. Mohammed, M. I. Elbashir, S. Xu, K. Berzins, P. Venge. 2003. Human neutrophil lipocalin: a specific marker for neutrophil activation in severe Plasmodium falciparum malaria. Acta Trop. 87: 279-285. [Medline]
  66. Waters, L. S., J. Taverne, P. C. Tai, C. J. Spry, G. A. Targett, J. H. Playfair. 1987. Killing of Plasmodium falciparum by eosinophil secretory products. Infect. Immun. 55: 877-881. [Abstract/Free Full Text]
  67. Shenoy, N. G., G. J. Gleich, L. L. Thomas. 2003. Eosinophil major basic protein stimulates neutrophil superoxide production by a class IA phosphoinositide 3-kinase and protein kinase C-{zeta}-dependent pathway. J. Immunol. 171: 3734-3741. [Abstract/Free Full Text]
  68. Graca-Souza, A. V., M. A. Arruda, M. S. de Freitas, C. Barja-Fidalgo, P. L. Oliveira. 2002. Neutrophil activation by heme: implications for inflammatory processes. Blood 99: 4160-4165. [Abstract/Free Full Text]
  69. Yang, F., A. J. Ghio, D. C. Herbert, F. J. Weaker, C. A. Walter, J. J. Coalson. 2000. Pulmonary expression of the human haptoglobin gene. Am. J. Respir. Cell. Mol. Biol. 23: 277-282. [Medline]
  70. Imrie, H., D. J. Ferguson, K. P. Day. 2004. Human serum haptoglobin is toxic to Plasmodium falciparum in vitro. Mol. Biochem. Parasitol. 133: 93-98. [Medline]
  71. Hunt, N. H., C. Driussi, L. Sai-Kiang. 2001. Haptoglobin and malaria. Redox Rep. 6: 389-392. [Medline]
  72. Arredouani, M., P. Matthijs, E. Van Hoeyveld, A. Kasran, H. Baumann, J. L. Ceuppens, E. Stevens. 2003. Haptoglobin directly affects T cells and suppresses T helper cell type 2 cytokine release. Immunology 108: 144-151. [Medline]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
N. Kesteman, G. Vansanten, B. Pajak, S. M. Goyert, and M. Moser
Injection of lipopolysaccharide induces the migration of splenic neutrophils to the T cell area of the white pulp: role of CD14 and CXC chemokines
J. Leukoc. Biol., March 1, 2008; 83(3): 640 - 647.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map