INTRODUCTION

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The immune system of higher vertebrates consists of innate and adaptive components. Innate immunity exhibits immediate recognition and response without prior sensitization. Cells of the innate immune system (i.e., monocytes/macrophages, natural killer [NK] cells, and polymorphonuclear leukocytes) recognize pathogen-associated molecular patterns and activate events such as phagocytosis, induction of the synthesis of antimicrobial peptides, expression of inflammatory and effector cytokines and chemokines, induction of nitric oxide synthase in macrophages, and expression of costimulatory molecules on antigen-presenting cells. The adaptive immune system uses somatically generated antigen receptors that are clonally distributed on T and B lymphocytes. Generally, adaptive immune recognition in the absence of innate immune recognition results in inactivation of lymphocytes that express receptors involved in the identification events (20). Thus, innate immune responses have critical consequences in adaptive immune responses.

Little is known of the contribution of the innate immune system during infection with the human immunodeficiency virus (HIV). Based on similarities of biologic and genetic features, simian immunodeficiency virus (SIV) infection of rhesus macaques provides the best animal model of HIV infection and AIDS. Accordingly, this animal model is critical for the elucidation of mechanisms of pathogenesis and for the development of vaccines and antiviral therapies (12). As with almost all viral infections, the innate immune system is thought to be the first component of the immune system that recognizes SIV infection. However, few studies have methodically analyzed the changes induced in cell phenotype and cytokine levels by SIV infection. Recent studies have demonstrated that SIV infection results in a generalized increase in lymphocyte turnover (23) and that the primary site for viral replication is activated memory CD4⁺ T cells that are present in the intestinal lamina propia (46). Although cellular changes are not that dramatic at this early stage in peripheral lymphoid tissue, peripheral blood (PB) and lymph nodes (LN) still reflect the pathologic changes induced by the viral infection and are readily available for longitudinal studies.

To analyze changes in the activation state of cells from the innate and adaptive immune system after SIV infection, we evaluated NK activity, cytokine levels in plasma, and changes in activation markers on lymphoid cells of rhesus macaques after infection with pathogenic SIVmac251. We found the sequential appearance in plasma of interferon-/ (IFN-/) interleukin-18 (IL-18) and IL-12, whereas IL-4, IFN- and granulocyte-macrophage colony-stimulating factor (GM-CSF) remained undetectable. We also found transient activation of NK cells during the peak of viral replication, and this activation was not predictive of disease progression. Finally, we observed that after SIV infection, both CD4⁺ and CD8⁺ T cells became activated in the absence of markers for proliferation, suggesting that the increased turnover of these cells reflects activation-induced cell death rather than differential compartmentalization.

	MATERIALS AND METHODS

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Infection of rhesus macaques. Four colony-bred, weight- and age-matched adult male rhesus macaques (Macaca mulatta) seronegative for simian type D retroviruses, simian T-cell leukemia virus, and SIV were used in this experiment. The animals were used and cared for in accordance with the American Association for Accreditation of Laboratory Animal Care Guidelines. The macaques were inoculated intravenously with 1 ml of RPMI 1640 containing 100 50% tissue culture infective doses (TCID₅₀) of SIVmac251. The animals were euthanized when they showed three or more of the following clinical symptoms: (i) weight loss greater than 10% in 2 weeks or 30% in 2 months; (ii) chronic diarrhea that was unresponsive to treatment; (iii) infections that were unresponsive to antibiotic treatment; (iv) inability to maintain body heat or fluids without supplementation; (v) persistent, marked hematologic abnormalities including lymphopenia, anemia, thrombocytopenia, or neutropenia, and (vi) persistent, marked splenomegaly or hepatomegaly.

Cells and viruses. CEM-x-174 cells, rhesus peripheral blood mononuclear cells (PBMCs), and LN cells were used for SIV isolation and propagation. These cells and the NK-sensitive human erythroblastoid cell line K562 were maintained in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, 0.1 mg of streptomycin (Cellgro Mediatech, Herndon, Va.) per ml, and 100 U of penicillin (Cellgro) per ml. Human A549 cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum and antibiotics.

Measurements in plasma. Plasma p27 antigenemia was measured by a commercial SIV core antigen capture enzyme-linked immunosorbent assay (ELISA) (Coulter Corp., Hialeah, Fla.) as instructed by the manufacturer (sensitivity of 50 pg/ml). The levels of GM-CSF, IL-4, IL-12, and IFN- were determined with commercially available ELISA kits (Immunotech for GM-CSF and IL-4, and Cytoscreen Monkey IFN- and IL-12 from BioSource, Camarillo, Calif.). The limits of detection were 5 pg/ml for GM-CSF and IL-4 and 4 pg/ml for IFN- and IL-12. The levels of IL-18 in plasma were determined with an ELISA kit kindly provided by H. Okamura (Hyogo College of Medicine and Hayashibara Corp.) as described previously (41). The sensitivity of this assay was 10 pg/ml.

LN biopsies. Peripheral LN (axillary and/or inguinal) were obtained by transcutaneous biopsy under ketamine HCl anesthesia (10 mg/kg, injected intramuscularly) (Parke-Davis, Morris Plains, N.J.) before and at 2, 4, 12, and 24 weeks postinfection (p.i.). The LN were divided aseptically into two fractions for single-cell preparation and pathologic analysis. Lymphocyte suspensions were obtained by mechanical teasing of tissues. The fractions for pathological analysis were fixed in 10% neutral buffered formalin, processed conventionally, cut at 5 µm, and stained with hematoxylin and eosin (H+E) for routine histologic examination. Tissues were examined blinded for changes in lymphoid architecture and cellularity.

Cell-associated viral loads. Cell-associated virus, latent or productive, was measured by a limiting-dilution assay of LN cells with CEM-x-174 cells in 24-well plates (45). Twice weekly, culture media were assayed for the presence of the SIV major core protein (p27) by ELISA (19). Cultures were recorded as positive for virus when p27 antigen was detected at two consecutive time points. End-point cultures were maintained and tested for 4 weeks before being scored as negative. Virus levels were calculated by the method of Reed and Muench (29) and expressed as TCID₅₀ per 10⁶ cells.

Lymphocyte phenotyping. Phenotypic characterization of lymphocytes in PB and LN was performed by flow cytometry using three-color direct immunofluorescence. Surface staining was performed by incubating whole blood or LN cells for 30 min at room temperature with monoclonal antibodies (MAbs) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), or Tricolor (PE-Cy5). Anti-monkey CD3 (clone FN-18)-FITC was obtained from BioSource. CD4 (clone OKT4)-PE was from Ortho Diagnostic Systems (Raritan, N.J.). CD8 (clone 3B5)-Tricolor, CD14 (clone Tuk4)-FITC, CD69 (clone CH/4)-Tricolor, HLA-DR (clone BU63)-FITC, and HLA-DR-Tricolor were from Caltag (Burlingame, Calif.). CD16 (clone 3G8)-PE was from Pharmingen (San Diego, Calif.). CD154 (CD40L, clone TRAP1)-PE, CD40 (Clone MAb89)-PE, and CD8 (clone 2St8.5H 7)-PE were from Immunotech (Westbrook, Maine). CD20 (clone B1)-FITC and CD20-PE were from Coulter Corp. Samples were acquired in a FACScan flow cytometer (Becton Dickinson, San Jose, Calif.), and data were analyzed with CellQuest software (Becton Dickinson Immunocytometry Systems). Lymphocytes were gated based on their characteristic forward-scatter versus side-scatter pattern, and a second gate was established using CD3 fluorescence. CD16⁺ NK cells and CD20⁺ B cells were determined in the CD3 lymphoid population, whereas the activation markers CD25, CD69, and CD154 were determined in the CD4 and CD8 CD3⁺ lymphoid cells. Absolute values for cells in whole blood were obtained by combining the percentages obtained by flow cytometry with the values of total white blood cell count per microliter and the differential formula for each animal at each time point.

Measurement of NK activity by the calcein release assay. Calcein-AM (Molecular Probes, Eugene, Oreg.) is a nonfluorogenic substrate that readily enters cells. Cytoplasmic esterase activity of viable cells cleaves calcein-AM to the intensely fluorescent calcein (49). NK activity was assayed on K562 cells that were labeled with calcein-AM. Briefly, cells were seeded to a concentration of 5 × 10⁵ cells/ml 24 h prior to testing to ensure that they were in log phase and not in a metabolically toxic environment. The cells were washed once with phenol red-free RPMI 1640 supplemented with 2.5% heat-inactivated fetal calf serum (cRPMI-2.5). K562 target cells were resuspended in cRPMI-2.5 at 2 × 10⁶ cells/ml and labeled with 10 µM calcein-AM for 30 min at 37°C in room air. The cells were washed four times in culture medium and adjusted to a concentration of 5 × 10⁴ cells/ml. Rhesus PBMC were washed twice with and resuspended in cRPMI-2.5. Cytotoxicity assays were carried out in round-bottom microculture plates (Costar, Cambridge, Mass.). Effector cells were prepared in triplicate by serial dilutions of the stock preparation to give effector-to-target cell ratios of 45:1, 15:1, 5:1, and 1.7:1. Target cells were added, and the plates were incubated for 4 h at 37°C in an atmosphere of 95% air-5% CO₂. Target cells were also incubated in medium alone and with 2% Triton (Sigma) for estimations of spontaneous and maximum release. Aliquots of 110 µl of supernatant were removed from each well and transferred to 96-well flat-bottom microtiter plates (Microfluor-Black; Dynex Technologies, Chantilly, Va.) for reading calcein fluorescence in each well using an automated fluorescence measurement system (HTS 7000 Bio Assay reader; Perkin-Elmer, Branchburg, N.J.) with an excitation filter setting of 482/20 nm and an emission filter setting of 530/25 nm. The value of the background fluorescence was subtracted from the values of the maximum, spontaneous, and experimental fluorescence, and the percentage of killing for each condition was calculated as (experimental fluorescence  spontaneous fluorescence) × 100/(maximum fluorescence  spontaneous fluorescence).

Analysis of the humoral immune response of rhesus macaques. Plasma samples were analyzed for the presence of antibodies reactive to SIV envelope glycoproteins and p27 core protein. Antigens for ELISA plates were obtained from a viral preparation of SIVmac239 concentrated by 20% sucrose cushion centrifugation. The protein content of the viral preparation was determined with the protein quantification kit (Bio-Rad, Hercules, Calif.).

Determination of anti-SIVgp160 antibody avidity. The antibody avidity index values of plasma antibodies to the SIVmac239 envelope glycoproteins were determined by using 8 M urea in the ConA ELISA. This method, as previously described (8), measures the resistance of antibody-envelope glycoprotein immune complexes. Briefly, plasma samples were diluted to produce an OD₄₅₀ of 1 to 1.5 in the ConA ELISA procedure. Following plasma incubation, the plates were treated three times for 5 min each with PBS (pH 7.4) or a solution of 8 M urea in PBS. This treatment was followed by incubation with peroxidase-conjugated anti-monkey immunoglobulin G (1:5,000). After color development, the reaction was stopped with 50 µl of 2 N sulfuric acid and the OD₄₅₀ was measured in an automated plate reader. The avidity index was then calculated from the ratio of the absorbance obtained with urea treatment to that with PBS and then multiplied by 100.

Statistical analyses. Correlation analysis was performed by using the Pearson product moment correlation coefficient. Baseline and follow-up data were compared using the paired t test or Wilcoxon matched-pairs test, according to the type of distribution of the variables.

	RESULTS

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Four adult rhesus macaques (identification numbers 863, 868, 876, and 880) were inoculated intravenously with 1 ml of RPMI 1640 containing 100 TCID₅₀ of the pathogenic isolate SIVmac251. Blood samples and peripheral LN were obtained periodically before and after infection. All animals became infected, and virus was isolated from PBMC by 1 week p.i. (data not shown). SIV infection of rhesus 880 progressed rapidly, and the animal was euthanized at 20 weeks p.i. due to severe immunodeficiency. Postmortem examination of this animal showed profound lymphoid depletion in the LN, spleen, and intestinal tract, adenovirus pancreatitis and gastritis with protozoal colonization, and necrotizing hepatitis. Rhesus 876 also developed immunodeficiency and died at 32 weeks p.i. Rhesus 863 and 868 became chronically infected and had moderate lymphadenopathy but did not show signs of immunodeficiency throughout the course of this experiment (32 weeks). Serial histological analysis of peripheral LN obtained from the rapid progressors 876 and 880 showed a rapid onset of lymphadenopathy, characterized by destruction of the LN architecture, absence of germinal centers, and profound lymphoid depletion. The results of pathologic analysis of LN from the slow progressors 863 and 868 were not remarkably different from those found for the rapid progressors during the first 4 weeks p.i. However, LN obtained from these animals at later time points showed the concomitant presence of areas of hyperplasia and active germinal centers and zones of moderate lymphoid depletion (data not shown).

Changes associated with innate immunity. Plasma samples were analyzed for the presence of IL-12, IL-18, SIVp27, IFN-, IL-4, and GM-CSF by specific ELISA and for IFN-induced antiviral activity by a biological assay. We determined that IL-18 is usually detected at low levels (200 to 400 pg/ml) in uninfected animals. However, after infection with SIV, increments in the level of IL-18 in plasma were observed in all animals during the first 2 to 3 weeks p.i., with those in two macaques reaching concentrations of 3,000 pg/ml or higher (Fig. 1A, 868 and 880). In contrast to IL-18, the levels of IL-12 did not change immediately after infection (Fig. 1B). For rhesus 863, 868, and 876, the IL-12 levels reached a maximum by 3 to 4 weeks p.i. and then gradually declined. Rhesus 880 had unusually high levels of IL-12 in plasma before challenge, and these levels dropped continuously until the time of death. The extent of viral replication, as measured by the concentration of SIV p27 in plasma, coincided with that of IL-18, reaching a peak by 2 weeks p.i. (Fig. 1C). However, there was no correlation between IL-18 and SIV p27 levels for individual animals. Rhesus 880 had the highest level of SIV p27 during the peak of viral replication; antigen was always detectable after that, and it increased to higher levels at the onset of AIDS. The level of antiviral activity in plasma, measured as the ability of plasma to prevent EMCV-mediated cytopathic effects on A459 cells, is an indicator of the presence of IFN- and/or IFN-/ (Fig. 1D). This antiviral activity was undetectable in all macaques before infection. However, all animals showed the transient appearance of antiviral activity by 1 week p.i., which was no longer detectable in three macaques by 4 weeks. The exception was rhesus 880, which had continuously increasing values of antiviral activity until the time of necropsy. The other rapid progressor, rhesus 876, became positive again after 8 weeks p.i. Interestingly, the levels of IL-4, IFN-, and GM-CSF in plasma remained below the limit of detection of the respective assays at all time points (data not shown). The inability to detect IFN- in the same samples by a very sensitive ELISA, combined with the lack of reduction in antiviral activity after combining plasma with a neutralizing antibody to IFN-, points to IFN-/ as being responsible for the antiviral activity found in plasma.

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Analysis of plasma from SIV-infected rhesus macaques. Blood was collected from rhesus macaques before and after challenge with 100 TCID50 of SIVmac251. Plasma was separated, and IL-18 (A), IL-12 (B), SIV p27 (C), and IFN-/ (D) were measured as described in Materials and Methods.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Analysis of rhesus macaque NK cell activity after infection with SIV. NK cell activity was determined in PBMC isolated from blood of rhesus macaques before and after challenge with 100 TCID50 of SIVmac251. (A) NK cytotoxic activity was measured on K562 cells by a nonradioactive 4-h killing assay as described in Materials and Methods. (B) Activation of NK cells. PBMC were stained with anti-CD3-FITC, anti-CD16-PE and anti-CD69-Tricolor. Lymphocytes were gated based on their side- versus forward-scatter characteristics, and a second gate was established on the CD3-negative population. This population was plotted in a CD16-PE versus CD69-PE-Cy5 graph, and a region was drawn on the CD16+ cells. Finally, a second region on the CD16+ CD69+ cells allowed for the determination of the percentage of activated NK cells. (C) Correlation between NK cytotoxic activity and activation for the first 12 weeks p.i.

Changes in activation markers of T cells after SIV infection. Lymphocytes from PB and lymphoid organs were analyzed by cell surface staining and flow cytometry for cell subset composition and for expression levels of CD25, CD69, and CD154. The absolute number of B cells in the PB dropped during the first 2 to 3 weeks of infection, remained generally at lower levels than the values before infection, and increased dramatically at the time of AIDS for the rapid progressors (rhesus 876 and 880). In LN, however, the proportion of B cells increased at 2 weeks p.i. and then returned to preinfection levels, except for rhesus 880, which had escalating values of B cells (Fig. 3, top panels). For CD4⁺ T cells, the absolute numbers in PB increased immediately after infection for most animals and remained relatively constant. Macaque 880 showed a progressive decline in the percentage of CD4⁺ T cells (data not shown), but there was a remarkable lymphocytosis by 16 and 20 weeks p.i. that resulted in increased absolute numbers of CD4⁺ T cells and B cells. In LN, the percentage of CD4⁺ T cells declined abruptly by 2 weeks p.i. and continued to decrease more slowly afterward for all animals (Fig. 3, second row). For CD8⁺ T cells, SIV infection resulted in an increase in the absolute number of PB that varied considerably from animal to animal, whereas this increase was discrete in LN. Macaque 880 had a sharp decline in cellularity and the content of CD8⁺ T cells in LN at the time of death (Fig. 3, third row), which may explain the sharp increase in the percentage of B cells. Finally, the proportion of NK cells slightly increased after infection and varied considerably from animal to animal and for each animal individually (Fig. 3, bottom row, left panel). The percentage of total T cells (CD4⁺ and CD8⁺) in LN decreased over the course of infection. For the rapid progressor 880, T cells, which comprised 90% of all lymphoid cells in the LN before infection, declined to 30% at the time of death (Fig. 3, bottom row, right panel).

View larger version (31K): [in this window] [in a new window]   FIG. 3.   Changes lymphoid cells from rhesus macaques in PB (total cell number) and LN (percentages of lymphoid cells) after infection with SIVmac251.

View larger version (31K): [in this window] [in a new window]   FIG. 4.   Changes in the level of expression of activation markers on CD4 T lymphocytes from rhesus macaques in PB (total cell number) and LN (percentages of the CD4 T-cell population) after infection with SIVmac251.

View larger version (36K): [in this window] [in a new window]   FIG. 5.   Changes in the level of expression of activation markers on CD8 T lymphocytes from rhesus macaques in PB (total cell number) and LN (percentage of the CD8 T-cell population) after infection with SIVmac251.

Humoral immune response to infection. The presence of antibodies directed at SIV gp160 and Gag in infected macaques was evaluated by antigen-specific ELISA. Purified virus was used as source of gp160 for plates coated with ConA and as a source of Gag for plates coated with a murine MAb specific for SIV p27. Antibodies to SIV gp160 were detected by 4 weeks p.i. in all animals. However, these antibodies were transient for rhesus 880 and were no longer detected by 8 weeks p.i. Macaques 863 and 868 mounted a strong humoral response, while rhesus 876 had a constant, low-titer anti-gp160 antibody production (Fig. 6A). The humoral immune response to SIV Gag had a different outcome. The rapid progressor, rhesus 880, failed to make antibodies, whereas rhesus 876 had a transient low-titer humoral response that became undetectable by 20 weeks p.i. Rhesus 863 and 868 mounted strong anti-Gag humoral responses, with titers for 863 being more than 1 order of magnitude higher than the ones for 868 (Fig. 6B). The avidity of the anti-SIV gp160 antibodies was analyzed with samples obtained 16 and 20 weeks p.i. Although higher in anti-SIV gp160 ELISA titer at 20 weeks p.i., the avidity for rhesus 868 antibodies was slightly lower than that observed for macaque 863. Coincidental with its low titer, the gp160 avidity of rhesus 876 antibodies was very poor at both time points (Table 1).

View larger version (15K): [in this window] [in a new window]   FIG. 6.   Humoral immune response of rhesus macaques infected with SIVmac251. (A) Anti-gp160 antibodies titers were determined on ELISA plates treated with ConA and detergent-disrupted SIVmac239. (B) Anti-Gag antibodies titers were determined on ELISA plates treated with an anti-SIVp27 murine MAb and detergent-disrupted SIVmac239.

	DISCUSSION

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Innate immune responses of rhesus macaques to SIV infection. The innate immune system reacts to microbial invaders with the production of cytokines and the activation of its cellular components. Cytokines that are produced by cells of the innate immune system include IFN-/, transforming growth factor , tumor necrosis factor, IL-1, IL-6, IL-10, IL-12, IL-15, and IL-18. A characteristic innate cytokine response to viral infections is the early production of IFN-/ (4). As shown in this study, infection with SIV also results in early production of IFN-/, which precedes the detection of SIVp27 in plasma and usually becomes undetectable after the peak of viremia (Fig. 1). In general, we have observed that high levels of IFN-/ correlate with high viral loads and that persistent presence of IFN-/ in plasma is associated with rapid disease progression or onset of AIDS (reference 14 and this study). The triggering event for the release of IFN-/ appears to be the interaction between the mannose receptor of PB dendritic cells and the glycosylated viral envelope protein (22). It has been shown that IFN- has a potent in vitro antiviral effect on SIV by blocking steps between attachment and reverse transcription (42). However, as we show in this study, IFN- does not seem to be sufficient to control SIV infection in vivo in the absence of other antiviral immune mechanisms. For example, the constant high levels of IFN-/ in rhesus 880 were not effective in limiting SIV replication.

Adaptive immune responses of rhesus macaques to SIV infection. Dramatic changes in the number and phenotype of the cells that constitute the adaptive immune system can be seen early after infection with SIV. Whether these reductions in cell numbers represent actual disappearance of cells or redistribution to other body compartments is still a matter of debate (33). Initially, the virus replicates preferentially in activated memory CD4⁺ T cells that are present in the intestinal lamina propia, which becomes rapidly depleted of these cells (46). Our sequential analysis of SIV-infected macaques shows that by 2 weeks p.i. a similar type of CD4⁺-T-cell depletion is noticeable in peripheral lymphoid tissue but not in PB, even though depletion of peripheral CD4 T cells and B cells has been associated with rapid disease progression in SIV- and SHIV-infected macaques (13, 40). We observed a slight reduction in levels of circulating B cells, but we did not find a correlation between this decline and disease progression or antibody production. Interestingly, the percentage of B cells in LN did not change drastically with infection. For the rapid progressors, the combined pathological examination and analysis of the cell composition of the LNs showed a loss of LN architecture, T-cell depletion, and internal redistribution of B cells from germinal centers to other areas of the LN.