JAIDS Journal of Acquired Immune Deficiency Syndromes:
α-Defensins 1, 2, and 3 Are Expressed by Granulocytes in Lymphoid Tissues of HIV-1-Seropositive and -Seronegative Individuals
Folkvord, Joy M. MS*; McCarter, Martin D. MD†; Ryder, John MD‡; Meditz, Amie L. MD*; Forster, Jeri E. MS§; Connick, Elizabeth MD*
From the *Division of Infectious Diseases, Department of Medicine, †Department of Surgery, ‡Department of Pathology, and §Department of Biostatistics, University of Colorado Health Sciences Center, Denver, CO.
Received for publication June 30, 2004; accepted April 13, 2006.
This work was supported by Public Health Services grants R29AI-42499, AI41536, AI55356, and AI054907 from the National Institutes of Health.
These data were presented in part at the 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, CA [abstract A-104].
Reprints: Elizabeth Connick, MD. University of Colorado Health Sciences Center, Division of Infectious Diseases, Box B168, 4200 E. Ninth Avenue, Denver, CO 80262 (e-mail: email@example.com).
Summary: α-Defensins 1, 2, and 3 exert antiretroviral activity in vitro, but their role in controlling HIV-1 replication in vivo and the cells that produce them are controversial. This study sought to determine whether α-defensins are present in HIV-1-infected individuals' lymphoid tissues, the major site of HIV-1 replication, and to identify the cells that express them. α-Defensin expression was evaluated by immunostaining inguinal lymph node sections from 19 untreated HIV-1-infected individuals and 8 individuals at low risk or seronegative for HIV-1 infection. Percentages of tissue sections that stained positively for α-defensins were not significantly different between HIV-seropositive (median, 7.6%) and -seronegative (median, 5.5%) individuals. Conditions that could have produced lymph node inflammation were present in most seronegative subjects, and their lymph node weights correlated with α-defensin expression (Spearman ρ = 0.833; P = 0.010). A median of 100% (range, 95%-100%) of α-defensin-expressing lymph node cells from 8 subjects coexpressed the granulocyte marker, CD15. CD15 and α-defensin staining correlated (Spearman ρ = 0.622; P < 0.001). These data suggest that α-defensins within lymphoid tissue are expressed by granulocytes and are prevalent in HIV-1-seronegative individuals with inflammatory processes as well as HIV-1-infected individuals. The role of α-defensins in controlling HIV-1 replication merits further investigation.
Human α-defensins 1, 2, and 3 are small cationic peptides that exert broad-spectrum antimicrobial activity.1 In vitro, they have been shown to inhibit replication of some bacteria,2 fungi,3 and viruses,4 including HIV-1.5 Cytotoxicity against bacteria occurs through membrane permeabilization, although other interactions between α-defensins and the cell membrane or internal structures may contribute as well.1 The mechanism of inhibition of HIV-1 replication by α-defensins is controversial. One group5 initially reported that α-defensins are the soluble CD8 cell antiviral factor that inhibits HIV-1 transcription.6 Subsequent studies by this group7,8 and others,9 however, demonstrated that α-defensins are not produced by CD8+ cells and that their mechanism of action is distinct from that of CD8 cell antiviral factor.10 A recent study demonstrated that α-defensin 1 at physiologic concentrations has both direct and indirect effects on HIV-1 infectivity.11 It inhibits HIV-1 infectivity by a direct action on the virion, which is abolished by serum. It also interferes with HIV-1 replication in CD4+ T cells at a step after reverse transcription, but before nuclear import and integration. This inhibition is thought to be mediated by impairment of protein kinase C signaling, and it is not sensitive to serum.
Substantial controversy exists concerning the role of α-defensins in HIV-1 infection and the cells that produce them. Neutrophils are the major sources of α-defensins 1, 2, and 3 in humans, although other cell types, including monocytes, B cells, γδ T cells, and natural killer cells, have been reported to produce them as well.12 α-Defensins have been reported in CD8+ cells, neutrophils, and monocytes of HIV-1-infected individuals.5,7-9,13 The aims of the present study were to determine if α-defensins are present in HIV-1-infected individuals' lymphoid tissue, the major site of HIV-1 replication,14,15 and to identify the cells that express them.
HIV-1-infected individuals who were not receiving antiretroviral therapy were recruited to donate inguinal lymph nodes. None of these subjects had an active opportunistic infection or malignancy or a sexually transmitted disease other than HIV-1. Inguinal lymph node excisional biopsies were performed under local anesthesia, and portions of the lymph nodes were snap frozen in OCT compound (VWR, Denver, CO) and stored at −70°C, as previously described.16 The remaining lymph node tissue was minced in phosphate-buffered saline (Life Technologies, Grand Island, NY), and the cell suspension was cryopreserved immediately and stored in liquid nitrogen. Informed consent was obtained from all participants in accordance with the Colorado Multiple Institutional Review Board. Lymph nodes from control subjects, who were individuals at low risk or seronegative for HIV-1 infection, were obtained by informed consent preceding a nonemergent surgical procedure in the groin area or as discarded surgical specimens, with permission of the Colorado Multiple Institutional Review Board. These lymph nodes were processed identically to lymph nodes obtained from HIV-1-seropositive subjects.
Plasma Virus Measurements
HIV-1 RNA was measured in plasma using the HIV-1 Monitor Assay (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions.
Immunohistochemical Staining for α-Defensins 1, 2, and 3 and CD15
Six-micrometer sections of cryopreserved lymph node tissue were thaw mounted on slides and fixed in 2% formaldehyde for 20 minutes. Sections were incubated at room temperature for 1 hour with mouse antihuman α-defensins 1, 2, and 3 antibody (Novacastra, Newcastle upon Tyne, UK), CD15 (Becton Dickinson, San Jose, CA), or normal mouse ascites (Sigma, St Louis, MO), as a control. Sections were then treated with biotinylated antimouse IgG (Vector Laboratories, Burlingame, CA) followed by alkaline phosphatase-labeled streptavidin (Vector Laboratories) and developed with Alkaline Phosphatase Substrate Kit I (Vector Laboratories), according to the manufacturer's instructions. Quantification of α-defensins 1, 2, and 3 and CD15 was performed by imaging 5 to 10 randomly selected, 1.25-mm2 fields and calculating the proportion of the field that stained positively for α-defensins or CD15 using a computerized image analysis system (Leica Q5001W Image Analysis; Leica, Cambridge, UK), as previously described.16 To demonstrate the specificity of α-defensins 1, 2, and 3 staining, antibody was preincubated overnight with 200 μg/mL α-defensins 1, 2, and 3 (Peprotech, Rocky Hill, NJ) before its use in staining in a subset of specimens. To define the distribution of α-defensin staining, α-defensin-stained specimens were stained with mouse antihuman CD20 (DAKO, Carpinteria, CA), a B-cell antibody that is useful in defining follicular regions of lymph node, as we have previously described.17
Immunofluorescent Double Staining to Identify the Phenotype and Location of Cells that Express α-Defensins 1, 2, and 3
Cryopreserved lymph nodes were partitioned into 6-μm sections, fixed in 2% formaldehyde, and incubated at room temperature with antibody to α-defensins 1, 2, and 3. Sections were treated with biotinylated antimouse IgG (Vector Laboratories) and then Alexa Fluor 594-labeled streptavidin (Molecular Probes, Eugene, OR), followed by direct staining with fluorescein isothiocyanate-conjugated antibodies to the granulocyte marker, CD15 (Becton Dickinson); CD68 (DAKO), which is found on macrophages as well as many other types of cells; or CD8 (Becton Dickinson). Tissue sections were viewed under epifluorescence, and 50 to 100 α-defensin-staining cells were identified and assessed for colocalization with CD15, CD68, or CD8. To further delineate the cell types associated with α-defensins, disaggregated lymph node cells that had previously been cryopreserved were thawed, spread on slides, air dried, fixed, and permeabilized in 2% formaldehyde containing 1% Igepal CA-630 (Sigma). Cells were double stained and evaluated for colocalization in the same way as described above for tissue sections. To determine whether CD15-positive cells were within blood vessels or the lymph node parenchyma, tissue sections were stained with unconjugated CD15 antibody and detected with Alexa Fluor 594-labeled streptavidin as described above, followed by direct staining with fluorescein isothiocyanate-conjugated factor VIII (DAKO), which identifies vascular endothelial cells.
Lymph nodes from 19 HIV-1-infected individuals who were not receiving antiretroviral therapy were evaluated for this study. Clinical characteristics of these individuals are shown in Table 1 and have been previously reported for some of these subjects.16-18 The majority (84%) of the subjects were men. The median CD4+ T-cell count of this group was 464 cells/μL (range, 107-913 cells/μL), and the median plasma HIV-1 RNA concentration was 22,389 copies/mL (range, 538-181,000 copies/mL). Four subjects (21%) had been infected for less than 1 year (subjects 19, 24, 27, and 77), and the remainder (79%) had been infected longer. The majority (63%) of HIV-1 seropositive study subjects were men who reported a history of sex with men, and nearly half (47%) reported a history of intravenous drug use. Two of 3 female subjects reported heterosexual exposure.
Inguinal lymph nodes from 8 individuals who were at low risk or seronegative for HIV-1 infection were evaluated for this study, and the clinical characteristics of these individuals are shown in Table 1. These individuals were 4 male and 4 female adults who were undergoing surgery in the inguinal region for multiple reasons, as outlined in Table 1.
Expression of α-Defensins 1, 2, and 3 in Lymph Nodes
Staining for α-defensins was abundant and found primarily in the T-cell regions of the lymph node in both HIV-1-seropositive subjects and individuals who were seronegative or at low risk for HIV-1 infection (Fig. 1 and Table 1). Among HIV-1-seropositive subjects, a median of 7.6% (range, 1.6%-23%) of tissue area stained positively for α-defensins. Control sections stained with normal mouse ascites were negative in all instances. Preincubation of α-defensin antibody with α-defensins 1, 2, and 3 before the staining procedure abrogated staining in all 4 lymph node sections in which this was performed, demonstrating the specificity of the antibody. There was no statistically significant correlation between either plasma HIV-1 RNA concentration (Spearman ρ = 0.046; P = 0.853) or CD4+ T-cell count (Spearman ρ = 0.380; P = 0.108) in HIV-1-seropositive subjects and the proportion of tissue area that stained positively for α-defensins. Furthermore, there was no significant difference in expression of α-defensins in subjects infected for less than 1 year compared with subjects infected for more than 1 year.
In lymph nodes from individuals who were low risk or HIV-1 seronegative, a median of 5.5% (range, 1.0%-12.9%) of tissue area stained positively for α-defensins. Differences in α-defensin staining between seropositive and seronegative subjects in the present study were not statistically significant (Wilcoxon test P = 0.382). These findings are in contrast to a previous study in which we reported that expression of α-defensins 1, 2, and 3 is up-regulated in lymph nodes of HIV-1-seropositive individuals compared with individuals who are at low risk or HIV-1 seronegative.17 Staining for α-defensins was similar in HIV-1-seropositive subjects in these 2 studies but was more than 10-fold lower among low-risk or HIV-1-seronegative subjects in the earlier study compared with the present one. Comparison of the low-risk and seronegative subjects in the 2 studies revealed important clinical differences that likely account for these divergent findings. The 5 low-risk or HIV-1-seronegative subjects in the previous study were individuals who had lymph nodes removed during coronary artery bypass surgery (4) and inguinal hernia repair (1), conditions that would not be anticipated to lead to lymph node inflammation. Lymph nodes from 2 of these subjects (subjects 34 and 49) were included in the present study; staining results for these 2 subjects were similar in the 2 studies and some of the lowest in the present study. Most of the seronegative subjects included in the present study, however, were recruited because they were undergoing stripping of varicose veins in their lower extremity, and one was undergoing bypass of the femoral artery (Table 1). The majority of these subjects had clinical evidence of a process in the inguinal region or leg that could have led to inflammation in their inguinal lymph nodes. Subject 58 had recently undergone a failed femoral artery bypass in the inguinal region, subject 59 had a recent history of superficial thrombophlebitis, subjects 68 and 72 both had a history of chronic venous stasis ulcers, and subject 69 had a history of deep venous thrombosis in the calf. Three of the lymph nodes from seronegative subjects (subjects 58, 59, and 69) included in the present study, but none in the previous one, were enlarged (diameter, >1.5 cm). The median weight (494 mg) of lymph nodes from individuals who were at low risk or HIV-1 seronegative in the present study was more than 50% higher than the median weight (297 mg) of lymph nodes from low-risk or seronegative subjects in the previous study (Table 1). Lymph node weight correlated significantly with expression of α-defensins in individuals who were at low risk or seronegative for HIV-1 infection (Fig. 2) in the present study, but not in seropositive subjects (Spearman ρ = 0.127; P = 0.709).
To identify the cell type that produced α-defensins 1, 2, and 3, immunofluorescent double staining was performed in tissue sections from 3 subjects. All CD15-expressing cells expressed α-defensins. The majority of α-defensin-expressing cells (range, 93%-95%) coexpressed CD15. However, due to the diffuse nature of the α-defensin stain, which not only colocalized with cells, but also appeared to spill out into adjacent tissues (Fig. 3A), it was not possible to definitively identify the cells that expressed α-defensins in all instances. To further address this question, disaggregated lymph node cells from 6 seropositive and 2 seronegative subjects were evaluated for colocalization of α-defensins with CD15, CD68, and CD8. A median of 100% (range, 95%-100%) of disaggregated cells that expressed α-defensins also expressed the granulocyte marker, CD15 (Fig. 3B). Colocalization of α-defensins with CD68 was rare (median, 0%; range, 0%-1.3%; Fig. 3C) and was never observed with CD8.
To further evaluate the relationship between CD15 expression and α-defensins, adjacent sections of lymph node were stained for these markers. The percentages of tissue area that stained positively for CD15 and α-defensins 1, 2, and 3 (Table 1) were strongly correlated (Fig. 4) in both HIV-1-seropositive subjects and subjects at low risk or seronegative for HIV-1. Furthermore, in all subjects, the pattern of α-defensin expression closely paralleled the pattern of CD15 staining within the same lymph node, as shown in representative tissue sections in Figure 5. To assess whether the granulocytes detected by anti-CD15 were within the lymph node parenchyma or within blood vessels that supplied the tissue, lymph node sections from 4 subjects were double stained with anti-CD15 antibody and factor VIII antibody. The vast majority (94%; range, 91%-98%) of CD15-staining cells were not adjacent to cells that stained positively for factor VIII (Fig. 3D).
Up-regulation of α-defensins in peripheral blood, cervix, plasma, breast milk, and other body fluids of humans with a range of infections,19 including HIV-1,13,20 has been observed. The present study is only the second report of expression of α-defensins in lymphoid tissues from either HIV-1-seropositive or -seronegative persons and the most extensive evaluation to date. We previously reported that expression of α-defensins 1, 2, and 3 is significantly higher in lymph nodes of HIV-1-infected individuals compared with lymph nodes of HIV-1-seronegative individuals17 but did not observe a similar disparity in expression of α-defensins in the present study. This difference between the present study and our previous one is likely related to important clinical differences between HIV-1-seronegative subjects in the 2 studies rather than a true discrepancy in results. In the present study, most individuals who were at low risk or HIV-1 seronegative had clinical evidence of inflammatory processes involving the lower extremity or inguinal area that were directly related to their recruitment and inclusion in the study. Indeed, 3 had pathologically enlarged lymph nodes, which is strongly suggestive of a reactive process. In contrast, the individuals who were at low risk or HIV-1 seronegative in our earlier study had neither evidence of lymph node enlargement nor preexisting conditions that would have been anticipated to result in inflammation in their lymph nodes. The lower state of activation of the lymph nodes in the HIV-1-seronegative group in the previous study likely accounts for the substantial differences in α-defensin expression between seronegative subjects in that study and the present one. The strong direct relationship between lymph node weight, a well-accepted marker of lymph node reactivity, and α-defensin expression in seronegative subjects in the present study further bolsters the argument that expression of α-defensins 1, 2, and 3 increases with inflammation in HIV-1-seronegative lymph nodes. The lack of correlation of lymph node weight with α-defensin expression in seropositive subjects may have been due to the confounding effect of lymphocyte depletion, which occurs variably in lymph nodes of HIV-1-infected individuals during the course of the disease.14,15 Thus, the present study confirms our previous observation that expression of α-defensins is prevalent in lymphoid tissues of HIV-1-infected individuals and further suggests that this condition is not restricted to HIV-1 infection, but it also occurs in the lymph nodes of HIV-1-seronegative individuals with inflammatory processes.
Two previous studies suggested that α-defensins 1, 2, and 3 in breast milk20 or peripheral blood and cervical tissue13 may play a role in reducing transmission of HIV-1. This is the first study to our knowledge to evaluate the relationship between lymphoid tissue α-defensin expression and plasma virus concentration, which reflects virus replication in vivo. No statistically significant correlation was found between α-defensin expression and plasma HIV-1 RNA concentration. A limitation of our approach was that α-defensin expression was assessed as the percentage of lymphoid tissue area that stained positively, which may not accurately reflect the true amount of α-defensins present. More importantly, potentially confounding factors, such as CD4+ T-cell count, virus fitness, or CCR5 genotype, could not be controlled for because of the small number of subjects in the study. Thus, a relationship between α-defensin expression and plasma HIV-1 RNA concentration is possible but was not detected in this study. These data, nevertheless, establish the presence of α-defensins in human lymphoid tissue, the major site of HIV-1 replication, and thereby suggest that they could play a critical role in control of HIV-1.
Conflicting results concerning the identity of α-defensin-expressing cells in HIV-1-infected individuals have been reported in the past. Although Zhang et al5 initially reported that α-defensins were expressed by peripheral blood CD8+ T cells from HIV-1-infected individuals who had a long-term nonprogressor phenotype, they subsequently reported that this was an artifact of coculturing CD8+ cells with allogeneic-irradiated peripheral blood mononuclear cells (PBMCs).7,8 They concluded that α-defensins released by neutrophils were taken up by CD8+ cells during the permeabilization process of staining. A subsequent study also reported expression of α-defensins by CD8+ cells from PBMCs and cervical biopsies in both HIV-1-infected and uninfected individuals.13 However, this study as well cultured CD8+ cells with allogeneic PBMCs and therefore may have produced the same artifact. Mackewicz et al9 found no evidence that purified CD8+ cells from HIV-1-infected or HIV-1-uninfected individuals' peripheral blood produced α-defensin proteins or expressed α-defensin mRNA. Instead, they reported that monocytes harbored mRNA for α-defensins. Zaharatos et al8 demonstrated that α-defensin expression by numerous different cell types in PBMCs correlated with the fraction of neutrophils in the preparation and was largely abrogated by depletion of granulocytes. They concluded that most reports of α-defensins in peripheral blood cells, aside from neutrophils, were likely caused by neutrophil contamination, although they could not exclude another small source of α-defensins because of occasional detection of α-defensin proteins in granulocyte-depleted cell populations.
The present study is the first to evaluate the phenotype of cells that express α-defensins 1, 2, and 3 in lymphoid tissue, the primary site of HIV-1 replication. A strength of the present study was that lymphoid tissues were neither cultured nor extensively manipulated before staining, which rendered them less susceptible to staining artifacts caused by the release of α-defensins by neutrophils. Furthermore, the fact that neutrophils were a minority population (<3%) of lymph node cells, as compared with the majority population in peripheral blood, likely also diminished the chances for contamination by neutrophils. Colocalization of α-defensins with cell markers was difficult to determine definitively within tissue sections because α-defensins not only colocalized with cells, but also appeared to diffuse within the tissues. A previous study suggested that spillage of α-defensins by neutrophils is a consequence of fixation and staining.8 Whether the diffusion of α-defensins within lymphoid tissue was a consequence of the staining process or whether it reflects the natural state of α-defensins within lymphoid tissue is an open question. Physiologically, however, it makes sense that α-defensins would be released by neutrophils and diffuse through lymphoid tissues in vivo as they are known to exert their antimicrobial effects extracellularly.
In lymph node tissue sections, the majority of cells that expressed α-defensins were granulocytes, as they coexpressed the CD15 marker. However, to definitively identify the cells that expressed α-defensins and particularly to evaluate whether CD68+ cells or CD8+ cells also expressed α-defensins, disaggregated lymph node cells were assessed for colocalization of markers. Unlike the lymphoid tissue sections, there was little question as to the cell of origin of α-defensins in these studies. Virtually all cells that expressed α-defensins also expressed the granulocyte marker, CD15. Only a few rare CD68+ cells were found to express α-defensins, and no CD8+ cells that expressed α-defensins were detected. It is unclear whether these rare CD68+ cells were macrophages or some other cell type, as CD68 is expressed on numerous different types of cells. In addition, it is not possible to exclude the possibility that neutrophil contamination may have produced these rare findings. Given the infrequency of this event, however, it was not possible to further investigate either the phenotype of these cells or the possibility that their presence was caused by contamination. Further evidence that α-defensin-staining cells were primarily granulocytes comes from observations that distribution patterns of CD15 and α-defensin staining within adjacent lymph node tissue sections closely paralleled each other, and the proportions of tissue area that stained positively for both markers were strongly correlated within individuals. Importantly, the majority of α-defensin-expressing cells were not within blood vessels, as defined by factor VIII staining, demonstrating that this finding was not an epiphenomenon because of an increased blood vessel supply in reactive nodes, but that the granulocytes were within the lymph node parenchyma. The finding that granulocytes are the major producers of α-defensins in human lymphoid tissue is consistent with previous studies in humans that have shown that neutrophils are the major producers of α-defensins 1, 2, and 31 as well as a more recent study of peripheral blood expression of α-defensins in HIV-1-infected humans.8
α-Defensins were observed primarily in the T-cell region of lymph nodes, which is where naive T cells come into contact with antigen-presenting cells and develop into antigen-specific effectors. The presence of α-defensins at this site is consistent with their known role as chemoattractants for naive CD4+21 and CD8+ T cells.22 It seems logical that a chemoattractant for naive T cells would also possess antimicrobial activity to prevent infection of effector cells at the site that they are generated. Interestingly, α-defensins are yet another T-cell chemoattractant, in addition to the β-chemokines23 and SDF-1α,24 with antiretroviral activity.
This study provides persuasive evidence that α-defensins are present in lymphoid tissues, which are the major site of HIV-1 replication, and harbored primarily by granulocytes. Further work is needed to determine the role of α-defensins in control of HIV-1 replication and the mechanisms by which they may exert antiretroviral activity in lymphoid tissue. Although serum has been shown to neutralize direct antiretroviral activity by α-defensins,11,25, lymph is thought to contain less protein than serum. Thus, it is possible that α-defensins within lymphoid tissue may exert significantly more antiretroviral activity than they do in peripheral blood.11 Identification and evaluation of polymorphisms within genes of α-defensins,26 as well as factors that regulate their synthesis and release,27 could potentially provide insights into their role in HIV-1 infection, analogous to the insights provided by identification of polymorphisms in genes encoding for β-chemokines and their receptors.28 A better understanding of the role of α-defensins in HIV-1-infected individuals could be critical to understanding the immunopathogenesis of this illness and developing immunotherapeutic strategies to combat multiple infectious diseases, including HIV-1.
The authors thank the subjects who participated in this study. They also thank Alden Harken for surgical assistance in obtaining lymph node specimens, Karen Whalen for nursing assistance during surgical procedures, Shirley Nelson for performing the assays for plasma HIV-1 RNA, Leica Corporation for the loan of the microscope and quantitative image analysis equipment, the University of Colorado Center for AIDS Research Immunology Core for facilitation of the fluorescent microscopy studies, Kevin Kisich for advice and thoughtful discussions regarding α-defensins, and Michael Grodesky, Robert Schooley, Susan Valone, Bev Putnam, Larry Sharkey, Cheryl McDonald, Ruth Berggren, Wheaton Williams, M. Graham Ray, Julie Subiadur, Ron Schimmel, Eileen Dunne, Steven Johnson, and Alex Kallen for assistance in recruiting subjects to this study.
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