In prior studies, we show that naturally occurring IgM anti-leukocyte autoantibodies (IgM-ALA) bind to CD3, CD4, CCR5, and CXCR4 receptors. These observations prompted us to determine whether IgM-ALA have a role in inhibiting HIV-1 infectivity by inhibiting viral entry into cells. We show that purified IgM, but not IgG, from individual sera of both normal and HIV-1 infected individuals is highly inhibitory (>95%) to HIV-1 viral infectivity both in vitro using PHA plus IL-2 activated PBL and in vivo using the human PBL-SCID mouse. Inhibition was observed with physiological doses of purified serum IgM and even after IgM was added 3 days postinfection in the in vitro assays. Absorbing purified serum IgM either with leukocytes or immobilized recombinant CD4 significantly decreased (>80%) the inhibitory effect on HIV-1 infectivity. IgM inhibited by >90% syncytia formation with the X4-IIIB infected SupT-1 cells indicating therefore that IgM inhibits viral attachment to core-receptors. IgM mediated anti-HIV-1 activity was highly specific as only certain IgM-ALA, obtained from human B cell clones inhibited HIV-1. IgM from certain HIV-1 infected individuals were not inhibitory to some R5-HIV-1 viral strains indicating that certain HIV-IgM may lack Abs reactive to strain specific coreceptor epitopes. These data indicate that an innate immune mechanism which is present from birth i.e., IgM-ALA, has a role in inhibiting HIV-1 viral entry into cells. Validation of this data with other in vivo models will be needed to determine whether in vivo administration or enhancement of IgM-ALA, e.g., through a vaccine, could prolong the asymptomatic state in HIV-1 infected individuals.
Naturally occurring IgM anti-leukocyte autoantibodies (IgM-ALA)3 belong to the innate immune system and are produced at birth in the absence of deliberate immunization or exposure to foreign Ags. The full repertoire of these Abs develops by early childhood and is present throughout life. IgM-ALA increases during various inflammatory and infectious disorders, including patients with end-stage renal disease (ESRD) and HIV-1 infections (1). We show that polyclonal IgM, purified from sera of normal patients and patients with ESRD and HIV-1 infections, has IgM-ALA that 1) binds to leukocyte receptors in a specific manner, even though they are polyreactive, 2) immunoprecipitates CD3, CD4, CCR5 and CXCR4 from whole cell lysates, 3) inhibits T cell activation as well as proliferation in response to alloantigens (MLR) or anti-CD3 (OKT3), 4) down-regulates both extracellular and intracellular CD4 and CD2 receptors, and 5) inhibits chemokine binding to their receptors as well as chemokine-induced chemotaxis and receptor down-regulation. However, the inhibitory effects of polyclonal IgM, purified from ESRD and HIV-1 sera, were more inhibitory on T cell proliferation and chemotaxis compared with normal IgM. Absorbing the IgM with leukocyte cell lines removed the inhibitory effects of polyclonal IgM on activation of T cells and on chemotaxis.
Prior studies in HIV-1 patients clearly showed that IgM –ALA increases after HIV-1 infection in 30% to 50% of patients. The increased IgM-ALA bound predominantly to CD4+ T cells (2, 3, 4, 5), or to both CD4+ and CD8+ T cells (6, 7). However, the increased levels of IgM-ALA did not correlate with the decrease in absolute CD4+ T cells or with disease progression and development of opportunistic infections, thus indicating that IgM-ALA did not contribute to the pathogenesis or progression of HIV-1 infection (2, 6, 7, 8). This lack of correlation could in part be explained on the finding that IgM-ALA are a heterogeneous group of Abs with specificities for different membrane receptors and a general increase in IgM-ALA in different individuals with the same disease may not necessarily reflect an increase in Abs having similar specificities, e.g., Abs with specificities for HIV-1 coreceptors. Furthermore, none of these studies examined the anti-HIV-1 activity of IgM from normal or HIV-1 sera.
The finding that IgM-ALA had binding to CD4, CCR5, andCXCR4, and that IgM also down-regulated CD4 prompted us to re-examine whether IgM-ALA was involved in the pathogenesis of HIV-1 infection. Our findings indicate that IgM-ALA inhibit HIV-1 cell entry, and more importantly, we show that the increase in IgM-ALA in different HIV-1 individuals can vary with regard to the specificity of IgM-ALA, and in particular with IgM having anti-HIV-1 activity.
Materials and Methods
Many of the techniques including purification of IgM from serum, activation of PBL by alloantigens (MLR), anti-CD3 or PHA plus IL-2, chemotaxis assays, quantitation of cytokines in culture supernatants, immunoprecipitation techniques and Western blots to detect IgM binding to solubilized cell membrane receptors, and absorption of purified IgM with cell lines to remove IgM with anti-leukocyte reactivity have been described in detail in an accompanying manuscript (1). Similarly, the different cell lines used are also described in the accompanying manuscript. Other experimental procedures are presented below.
Anti-CCR5 murine IgG monoclonals (clones 2D7, CTC-5, 45502, 45523, and 45549) and anti-CXCR4 (CTC-5, 12G5, 44708, 44717, and 44716) were obtained from R&D Systems. Clone 4G10, a murine IgG monoclonal that binds to the N-terminal region of CXCR4 was a gift from Dr. Chris Broder (9). The 2G12 human anti-gp120 Ab, that recognizes a conserved epitope, was obtained from the AIDS reagent program at National Institutes of Health. The following Abs were used as primary Abs in the Western blot procedure: polyclonal rabbit IgG Abs to CD3 (Santa Cruz Biotechnology, CA), CD4 (R&D Systems), or CXCR4 (Biochain); monoclonal mouse IgG Abs to CCR5 (clone CTC, N-terminal) and CXCR4 (clone 4G10 N-terminal).
HIV-1 viral isolates
R5 isolates (i.e., 8442, 8397, and 8658) were a gift from Dr. Homayoon Garadegan (Johns Hopkins University). X4 isolates (HT/92/599, IIIB), R5/X4 isolates (92US723, 92US076, and 92US077) were obtained from the AIDS reagent program at National Institutes of Health. All viral isolates are clade B. Viral isolates, except for IIIB, were previously propagated in PBL. Upon receipt of these primary isolates, we made several aliquots of these viruses from a single stock culture in PHA plus IL-2 activated PBL of a single individual. X4 isolate IIIB was grown in the T cell line (SupT-1) after which several aliquots of a single culture were made and frozen at −70°C.
In vitro infection of human PBL
24 to 48 h PHA activated PBL (0.5 × 106 cells/0.5 ml) were washed in culture medium and infected with 103 TCID50 of the HIV-1 viral strain in presence of IL-2 (40 U/ml). In certain experiments, cells were initially exposed for 1 h to purified IgM (1–20 μg/ml), heat inactivated (56°C) serum (25–100 μl/ml), CCL5 (RANTES) or CXCL12 (SDF-1α) (500 ng/ml), soluble CD4 −183 (20 μg/ml) or murine IgG anti-CXCR4 (12G5), anti-CCR5 (2D7) monoclonals (10 μg/ml) before adding the HIV-1 virus to the cells. Soluble CD4 at 5–10 μg/ml and 12G5/2D7 at <5 μg/ml had minimal inhibitory effect on these HIV-1 viral strains. PHA activated PBL were cultured in RPMI 1640 containing 10% FCS and cell cultures were set up in triplicate (0.5 × 106cells/0.5 ml) in a 48-well flat-bottom Falcon culture plate in presence of IL-2 (40 μl/ml). In these assays, the virus was not washed off the cells at 6 h as in our studies we detected no significant difference in the data when virus was washed or not washed off the cells at 6 h and avoiding the wash step allowed us to conserve scarce reagents, e.g., IgM. On day 6 or 7, cultures were replenished with 0.25 × 106 PHA activated cells in 0.25 ml medium and IL-2 and where indicated with half the quantity of purified IgM Ab, serum, or soluble CD4 or murine IgG anti-CXCR4/CCR5 monoclonals. CCL5 or CXCL12 was re-added to certain wells every 48 h. Cell-free supernatants were quantitated for p24 levels on day 11 or 12 of culture using an ELISA kit (Zepto Metrix).
HIV-1 infection of GHOST cells
In these studies we infected the GHOST CCR5 and GHOST CXCR4 transfectant cell lines which were obtained from the AIDS Reagent Program at National Institutes of Health. GHOST cells are derived from HOS cells stably transfected with CD4 and either CCR5 or CXCR4 genes and stably cotransfected with the HIV-2 LTR driving hGFP construct which emits a green fluorescence when the HIV-1 DNA integrates with the cell DNA. Single-cycle viral replication can be detected in <48 h in the GHOST infection assay. 2 × 104 GHOST cells were initially cultured for 12 h in 1 ml of RPMI 1640 medium with 10% FCS in a 12-well plate. Different amounts of IgM were then added to adherent GHOST CCR5 or CXCR4 cells 30 min before adding either R5 or X4 HIV-1 viruses. Virus and Ab were present throughout the 48-h culture period. No polybrene was used to enhance viral entry into cells. After 48 h incubation, cells were harvested, fixed in formalin and infected cells emitting green fluorescence were enumerated with flow cytometry. There was <10% variability in infection rates when performed in triplicate. Similar data were obtained when GHOST cells, exposed to virus and IgM Ab (or sera), were washed after 4 h.
IgM mediated inhibition of pseudotyped HIV-1 entry
IgM mediated inhibition was tested using a single-round viral entry assay. Pseudotyped HIV-1 expressing the firefly luciferase gene and containing various HIV-1 envelope glycoproteins was produced using previously published techniques (10). The envelope glycoproteins used to construct pseudovirus includes JR-FL and ADA, which use CCR5 as the coreceptor for entry; HXBc2, which uses CXCR4 as the coreceptor, and the glycoprotein of vesicular stomatitis virus (VSV-G), which is believed to use phospholipids as the receptor for entry. The target human PBL were preactivated with PHA and IL-2 for 48 h. 1.2 × 105 PBL in triplicate were incubated with or without 5 mg IgM before the addition of pseudotyped HIV-1. The mixture was incubated at 37°C in 5% CO2 for 2 days, after which the medium was aspirated and the luciferase activity in the target cells was measured using the GloMax luminometer (Promega).
Inhibition of syncytia induced by X4-IIIB
The T cell line (SupT-1), when infected with the X4-IIIB virus, spontaneously forms multinucleated giant cells termed “syncytia”. Syncytia are formed when the viral envelope, expressed on the cell-membrane, binds to CD4 and CXCR4 of an adjacent cell thus leading to fusion of cells. In this assay, infected SupT-1 cells (forming 1 to 2 syncytia per HPF × 400 magnification) were cocultured with un-infected SupT-1 cells at a ratio of 1 infected to 10 uninfected cells. Formation of new syncytia was evaluated at 24 to 48 h. In the inhibition assays, IgM (10μg/ml) or soluble CD4 (20μ/ml) was initially added to uninfected cells at 37°C for 1 h following which cocultures were set up in 24-well plates with 1 × 106 cells/ml.
In vivo HIV-1 infection of human PBL/SCID mice
We used (with modifications) the procedure developed by Mosier (11). CB17 SCID mice (7- to 8-wk-old female), purchased from Harlan Sprague-Dawley and having <1 μg/ml of mouse IgM in their plasma were injected i.p. with freshly isolated 25–35 × 106 PBL in 1 ml of RPMI 1640 containing 10% FCS, penicillin, streptomycin, fungizone, and ciprofloxacin (RPMI 1640 culture medium). Two hours later, mice were re-injected i.p. with 105 TCID50 HIV-1 virus in 1 ml RPMI 1640 culture medium. One milliliter of purified IgM at 1 mg/ml, obtained from the same PBL donor (to more closely mimic the in vivo situation), was injected i.p., either immediately after the HIV-1 injection or 48 h later. The same dose of IgM was injected every 5 days until day of sacrifice as kinetic studies revealed that human IgM in mouse plasma attained peak levels of 40–50 μg by day 2 and 8–10 μg per ml by day 5 after the i.p. dose. Mice were sacrificed three weeks after the human PBL injection. Blood and spleen were obtained from the mice on the day of sacrifice. Percent human CD45+ T lymphocytes expressing CD3 and CD4 in spleen cells were quantitated with three color immunofluorescence using flow cytometric techniques. Secondly, spleen cells were cocultured in vitro with IL-2-activated autologous PBL to quantitate HIV-1 in spleen cells. In coculture studies 2 × 106 spleen leukocytes in 1 ml of RPMI 1640 culture medium were cocultured with 2 × 106 IL-2 activated (1–2 days old) human PBL in 1 ml of RPMI 1640 culture medium containing human IL-2 (30 U/ml). Cocultures were fed at weekly intervals with 1 × 106 IL-2-activated autologous PBL. p24 Ag in cell-free coculture supernatants was quantitated after three weeks of coculture using an ELISA kit (Zepto Metrix). Mice were anesthetized with metofane inhaler before i.p. injections and were euthanized after an i.p. mixture of ketamine Xylocaine and acepromazine followed by cervical dislocation. Studies on SCID mice were approved by our Institutions Animal Care and Use Committee.
Absorbing out IgM anti-CD4 from purified IgM
Purified IgM (200 μg) in PBS containing Ca2+ and 0.1% BSA was absorbed with recombinant CD4, immobilized in a 96-well immulon plate where each well was coated with 500 ng of recombinant soluble CD4 using the Immuno-Tek ELISA construction kit (Zepto Matrix). Each absorption required IgM to be incubated with immobilized CD4 for 45 min at room temperature. It usually took three to five CD4-coated plates to remove >80% of IgM anti-CD4 as quantitated by an ELISA technique as previously described (1). IgM (30–35%) was recovered after the absorption procedure.
Sources of IgM and sera
IgM and sera were obtained from normal individuals, patients on chronic hemodialysis (ESRD) and patients with HIV-1 infection. The clinical characteristics of HIV-1 infected patients are presented in Table I. As controls, we used human IgG isolated from normal and patient sera, and IgM from a single patient with Waldenstrom’s macroglobulinemia, having in his serum large quantities of a monoclonal IgM reactive to an undefined receptor on leukocytes. We could not use a normal IgM control as none of the purified IgM from >30 normals lacked IgM-ALA activity. We used ESRD IgM to compare whether IgM-ALA that develops as part of an inflammatory response in ESRD has similar inhibitory activity as HIV IgM-ALA.
|HIV Patient Identification Number .||Number of Years Since Infection .||Viral Load .||Absolute CD4 Level .||History of AIDS Defining Illness .||Anti-HIV Therapy .|
|HIV Patient Identification Number .||Number of Years Since Infection .||Viral Load .||Absolute CD4 Level .||History of AIDS Defining Illness .||Anti-HIV Therapy .|
PCP, pneumocystis pneumonia.
MAC, mycobacterium avium.
HIV IgM binds to CD4, CXCR4, and CCR5: there are individual differences in receptor binding
Initial studies were aimed at determining whether there were differences in IgM binding to CD4, CXCR4 and CCR5 when using purified IgM from different HIV-1 infected individuals. The clinical profile of patients studied are depicted in Table I. In an accompanying manuscript, data are provided to show that IgM from all these HIV-1 infected individuals had detectable IgM anti-CD4 with both the immunoprecipitation or ELISA technique (1). Binding of IgM to CD4 was however particularly more pronounced when IgM was purified (see Fig. 1B in Ref. 1) perhaps because IgM in serum is in the form of a receptor/IgM complex, which is formed when IgM binds to cells. Additionally, adding purified IgM to autologous serum did not decrease the binding of purified IgM to CD4 thus supporting the concept that the observed low binding with serum IgM is not a result of an inhibitory serum factor. Of note, most of the individual HIV IgM had either undetectable or minimal binding to CCR5 receptor (see Fig. 3 in Ref. 1). Increasing the amount of lysate or IgM did not significantly enhance immunoprecipitation of CCR5 with HIV IgM. In contrast, the individual HIV IgM immunoprecipitated several fold more CXCR4 receptors when compared with individual normal IgM. Hence these data would indicate that most HIV-infected patients have a significant increase in IgM Abs reactive to CXCR4 but very minimal or no Abs reactive to CCR5. Conversely, a few HIV-1 patients, e.g., HIV no. 5 have increased IgM anti-CCR5 but no detectable Ab to CXCR4 (Fig. 3 in Ref. 1).
With the few HIV-1 patients we examined, we could find no obvious correlation between IgM-ALA with certain receptor specificities (e.g., CXCR4, CCR5, or CD4) and propensity for disease progression (see Table 1). For example, both nonprogressors (HIV nos. 20, 12, 21, 9) and patients with AIDS defining illness (HIV nos. 18, 15, 5) had no significant difference in IgM with specificities for CXCR4, CCR5, or CD4, indicating that the functional impact of each patients IgM-ALA on their autologous viral strain may be a more important determining factor.
Such findings provide added proof that IgM-ALA, even though polyreactive, bind to leukocyte receptors in a specific manner. More importantly, these findings could provide a mechanism to explain the preponderance of infection with the R5 virus in most HIV-1 infected patients. Studies on more patients are clearly needed to better define the IgM anti-chemokine receptor profile in these patients.
Purified IgM from normal, HIV, ESRD sera inhibit in vitro infectivity of X4 and R5 HIV-1 viruses
Since IgM binds to CD4, CCR5 and CXCR4, it became important to determine whether IgM inhibits HIV-1 infectivity of PBL. In these studies PHA plus IL-2 activated PBL were infected in vitro with different HIV-1 strains in the presence of purified IgM obtained from sera of normal, HIV and ESRD patients. IgM and virus was present throughout the culture period. As can be seen from Fig. 1, physiological doses of IgM inhibited HIV-1 infectivity by >95%, in a dose-dependent manner. The X4 viruses (IIIB, HT/92/599), R5/X4 viruses (92US076, 92US077) and the R5-8658 virus were highly sensitive (>90% inhibition with 10 μg IgM/ml) to all individual purified IgM obtained from normals, HIV infected individuals or ESRD patients but not Waldenstrom IgM. However, the R5-8397 and the R5/X4 92US723 were less sensitive and had variable sensitivity to purified IgM from different individuals. Fig. 1,B exemplifies the sensitivity pattern with the R5-8397 virus. IgM from normal no. 1 and the HIV no. 5 inhibited viral infectivity of the R5-8397 by ∼80% using IgM at a dose of 10 μg/ml while HIVno. 18 had no inhibitory effect on the virus even at doses of 30 μg/ml. The inhibitory effect of IgM on HIV-1 infectivity was removed after using IgM that was absorbed with Jurkat and U937 cell lines thus clearly demonstrating that the inhibitory effect of IgM resides in IgM with anti-leukocyte reactivity i.e., IgM-ALA (Fig. 1, C and D). Additionally, IgM had an inhibitory effect on HIV-1 infectivity, even when added 48 h after PBL were infected with HIV-1 (Fig. 1,E), presumably by preventing infection of un-infected cells following the initial cycle of replication. The pentameric form of IgM is not essential for the inhibitory effect of IgM on HIV-1 as monomeric IgM was as effective (Fig. 1 F) indicating therefore that the inhibitory effect of the pentameric IgM on HIV-1 binding to coreceptors is not a result of nonspecific stearic hindrance.
IgM inhibition of HIV-1 is highly specific and anti-HIV activity is dependent on the specificity of IgM-ALA
Further studies were performed with IgM obtained from EBV-transformed human B cell clones, to more conclusively exclude the possibility of nonspecific inhibition of HIV-1 by IgM and to determine whether all IgM-ALA were inhibitory to HIV-1. Details on development, isolation, and characterization of these B cell clones are described in an accompanying manuscript (1). In these studies we compared IgM with and without IgM-ALA activity.
As depicted in Fig. 2, not all IgM is inhibitory to HIV-1. In particular, IgM without ALA activity is clearly non-inhibitory, furthermore, the data would indicate that IgM-ALA with anti-CD4 specificity has more anti-HIV activity. Clearly, studies with more well characterized monoclonal IgM would better define the specificities of IgM-ALA that are most inhibitory to HIV-1, nonetheless, these data clearly demonstrate that IgM inhibition of HIV-1 is highly specific.
Inhibitory effect of IgM is dependent on IgM source and viral strain: importance of IgM anti-CD4
Data in Fig. 3,A provide two other observations which indicate that IgM mediated inhibition of HIV-1 is complex and that there are differences in the inhibitory capacity of individual IgM, especially IgM obtained from HIV-1 patients. For example, HIV IgMs (i.e., no. 18 and no. 20) failed to inhibit the R5 viral strain 8397, even though these same HIV IgMs (i.e., no. 18 and no. 20) clearly inhibited two other R5 viral strains i.e., 8658 (Fig. 3,A) and 8442 (data not shown). Similar lack of inhibitory activity with the 8397 virus was also noted with IgM from four of seven normals and 2 of 8 ESRD patients (data not shown) even though these same IgM-ALA consistently inhibited the 8658 and 8442 viral strains, thus raising the possibility that certain IgM may lack IgM-ALA Abs reactive to certain HIV-1 coreceptors or receptor epitopes specific for the binding site of the R5-8397 virus. Previous investigators have clearly shown that different HIV-1 viral strains can use different binding epitopes on coreceptors (12, 13, 14). To further evaluate whether there were differences in receptor binding epitopes between the resistant R5-8397 virus and the sensitive R5-8658 virus, we used two approaches. Firstly, we wanted to determine whether both the R5 viruses were equally inhibited by agents known to inhibit R5 viral entry. As can be seen in Fig. 3 B, R5-8658 and R5-8397 were both inhibited by higher doses of soluble CD4 (20–40 μg/ml) and 2D7 (anti-CCR5). Of note, lower doses of soluble CD4 (10 μg/ml) had no inhibitory effect on both these primary isolates, and a similar lack of sensitivity to lower doses of soluble CD4 by other primary isolates has been previously noted (15). However, unlike R5-8658, the R5-8397 was resistant to CCL5 (RANTES) thus indicating that the R5-8397 and R5-8658 binds to different epitopes on the CCR5 receptor. Hence these findings could explain the resistance of the virus to HIV no. 18 IgM, which lacks IgM anti-CCR5 (see Fig. 3 in Ref. 1). Furthermore, differences in CCR5 epitope binding between HIV no. 20 IgM and R5-8397 could also explain resistance of R5-8397 to HIV no. 20 IgM that does not lack IgM anti-CCR5 (see Fig. 3 in Ref. 1). However the above findings do not explain the marked inhibitory effect of HIV no. 1 IgM on R5-8397 especially because HIV no. 1, like HIV no. 18, lacks IgM anti-CCR5 (see Fig. 3 in Ref. 1).
Another more important possibility to explain resistance of R5-8397 to HIV no. 18 and no. 20 IgM and marked sensitivity of the X4-IIIB virus to HIV no. 5 and no. 18 IgM which lacked IgM anti-CXCR4 (see Fig. 3 in Ref. 1), is the interaction of IgM with the CD4 receptor. The finding that R5-8397 and X4-IIIB viruses were significantly inhibited (∼85%) by soluble CD4 would indicate that both these viruses are highly CD4 dependent (Fig. 3,B). Hence IgM anti-CD4 that is present in the purified IgM could provide a more significant mechanism for HIV-1 viral inhibition and this possibility was explored by absorbing out IgM anti-CD4 from purified IgM with immobilized soluble CD4. As depicted in Fig. 3 C, IgM depleted of IgM anti-CD4 significantly decreased the inhibitory effect of IgM on the three viral isolates by >80%. These findings would therefore support the concept that IgM anti-CD4 has a significant role in inhibiting the X4-IIIB virus and R5-8658 virus but fails to explain the resistance of R5-8397 virus to HIV IgM no. 18 and no. 20 especially because both IgM preparations had IgM anti-CD4 levels that were comparable to other HIV IgMs (see Figs. 1 and 3 in Ref. 1). Hence it is possible that resistance of R5-8397 to HIV IgM no. 18 and no. 20 is more likely due to a lack of IgM anti-CD4 with specificity for the binding site of R5-8397 on the CD4 molecule.
Taken together, these data also support the possibility that IgM anti-CCR5 could have an additional inhibitory effect on entry of some R5 viruses by binding to CCR5 as studies using murine IgG anti-CCR5 (2D7) clearly showed that anti-CCR5 Abs and CCL5 can (without soluble CD4) partially inhibit entry of some R5 viruses, e.g., R5-8658 and 8442 (Fig. 3,B). With the few cases we studied, we could find no obvious correlation between binding of IgM to CCR5 and HIV inhibition (Fig. 3 A). These findings clearly demonstrate that more studies are needed to better define differences in the IgM-ALA and their interactions with core receptors important for viral entry.
Normal IgM inhibits in vivo HIV-1 infection in a human PBL-SCID mouse model
We used this well described in vivo model to confirm observations with the in vitro PHA plus IL-2 activated PBL assay (11). The PBL in this model are not preactivated with mitogens before viral infection and hence the previously described inhibitory effect of IgM on T cell activation can also play a role in controlling viral replication (1). In these studies normal IgM was used as it was difficult to obtain large amounts of sera from patients. IgM was introduced i.p. within 15 to 30 min after i.p. HIV-1 infection and the dose of IgM was repeated every 5 days. Data using two different HIV-1 strains are depicted in Table II. These data bring out two observations. Firstly, 30% of HIV-1 infected mice can spontaneously become noninfected because of CD4+ human T cell depletion, and this observation was also noted by Mosier (11). Hence at 3 wk, ∼60–70% of mice remain infected. However normal IgM reduced the number of infected mice to 22% and this difference was statistically significant (p < 0.01) using the Fishers exact test. This decrease in HIV-1 infection of human-PBL-SCID mice in the presence of human IgM was not due to IgM mediated depletion of human PBL as by three color flowcytometry we could not detect significant changes in the splenic human T cell population (CD45+, CD3+, CD4+) between SCID mice treated with IgM and HIV vs SCID mice treated with only HIV (data not shown). The in vivo findings with the IIIB virus may need to be viewed cautiously, as with repeated passaging this virus could have developed increased sensitivity to inhibitors (15), however, increased in vitro sensitivity of IIIB virus has been shown to not always operate in vivo (17).
|Experimental Group .||Number of Mice Infected at 3 wk .|
|PBL + HIV||13/19 (68%)|
|PBL + HIV + IgM||4/18 (22%)b|
|Experimental Group .||Number of Mice Infected at 3 wk .|
|PBL + HIV||13/19 (68%)|
|PBL + HIV + IgM||4/18 (22%)b|
20 × 106 human PBL in 0.75 ml of medium were injected i.p. in SCID mice (indicated as PBL), and 2 h later, 105 TCID50 HIV-1 virus in 1 ml (15 mice R5-8658, 4 mice X4-IIIB) was re-injected i.p. (indicated as PBL + HIV). In some mice, pooled normal IgM (1 mg in 1 ml) was also introduced into the peritoneum within 15–30 min after HIV-1 infection (indicated as PBL + HIV + IgM) (11 mice R5–8658, 7 mice X4-IIIB). Pooled normal IgM (1 mg in 1 ml) was injected i.p. every 5 days. Peak IgM in mouse plasma varied from 40 to 50 μg, and by day 5 levels varied from 7 to 10 μg/ml. Mice were euthanized at 3 wk, spleens co-cultured with mitogen-activated PBL (supplemented each week with fresh mitogen-activated PBL), and supernatants of coculture analyzed at weekly intervals for p24 core Ag. Mice were defined as uninfected if spleen p24 levels were <30 pg/ml at 1, 2, and 3 wk of coculture. In infected mice, levels varied from 4,000 to 13,800 pg/ml.
The decrease in infection rate after introducing IgM is statistically significant (p<0.01) using the Fisher’s exact test.
Potential mechanisms to explain IgM mediated inhibition of HIV-1 infectivity
The loss of inhibitory activity after absorbing IgM with leukocytes and more importantly with immobilized CD4 (Fig. 3,C) provided the initial clues supporting the concept that IgM inhibits HIV-1 infectivity by binding to leukocyte receptors, especially CD4, which is involved in HIV-1 cell entry. Secondly, the finding that IgM immunoprecipitated HIV-1 coreceptors such as CD4, CCR5, and CXCR4 together with the inhibitory kinetics of IgM (see Fig. 1 E) added further evidence to support such a concept. Importantly, in these studies, the inhibitory effect of IgM on HIV-1 infectivity could not be attributed to the inhibitory effect of IgM on T cell activation as in our viral inhibitory assays, we used PBL that were preactivated for 24 to 48 h with IL-2 plus PHA and under these conditions IgM has no inhibitory effect on the activation state of PBL. Three approaches were used to further investigate whether IgM inhibits HIV-1 viral entry:
IgM inhibits HIV-1 infectivity of GHOST.
CCR5 and CXCR4 GHOST-CCR5 and CXCR4 transfectant cell lines are stably cotransfected with the HIV-2 LTR driving hGFP construct, which emits a green fluorescence upon integration of HIV-1 viral genome into the cell DNA. Hence one can measure entry efficiency of the virus especially if cells are harvested in 36 to 48 h, which allows for a single cycle of viral replication. As depicted in Fig. 4 A, in the presence of normal IgM, there is a marked reduction in integration of HIV-1 viral genome into the cell DNA which most likely is a result of IgM mediated inhibition of HIV-1 viral entry into the cell, although, one cannot completely exclude the possibility that IgM, may in addition, have inhibited events leading to integration of viral genome into the cell DNA.
IgM inhibits X4-IIIB-induced syncytia.
We used the syncytia assay to more directly determine whether the inhibitory effect of IgM on HIV-1 infectivity is a result of IgM inhibiting attachment of HIV-1 gp120 onto coreceptors. Multinucleated giant cells termed “syncytia” are formed when the HIV-1 viral envelope, expressed on the cell membrane of an infected cell, binds to coreceptors of an adjacent cell thus leading to fusion of cells. As depicted in Fig. 4 B soluble CD4 and HIV IgM (but not Waldenstrom IgM) clearly inhibited syncytia formation. Conversely, absorption of IgM anti-CD4 from HIV IgM removed the inhibitory effect on syncytia formation. These findings provide additional evidence confirming that IgM-ALA (especially IgM anti-CD4) inhibit attachment of virus to their coreceptors.
IgM mediated inhibition of pseudotyped HIV-1 entry.
To further validate that IgM inhibits HIV-1 entry, we used a single round infection assay using pseudotyped HIV-1 expressing the firefly luciferase gene (10). As can be seen from Fig. 4C, IgM inhibited the binding and entry of pseudotyped HIV-1 prepared from both R5 and X4 viral strains. In contrast, the entry of the glycoprotein from vesicular stomatitis virus (VSV-G), which is believed to use phospholipids for entry, was enhanced by more than two-fold.
IgG from HIV-1 sera has no IgG-ALA
The second protein peak from the column chromatography (to obtain IgM) was highly enriched in IgG. Later fractions of this peak had no contaminating IgM and were used in these studies. Ten of the 23 purified IgG obtained from HIV-1 sera had binding activity to recombinant CD4 with the ELISA. However, none of these IgG anti-CD4 Abs bound to CD4 expressing cell lines or had binding to leukocytes. Additionally, only one of the 10 IgG Abs with IgG-anti CD4 reactivity (detected by ELISA) inhibited HIV-1 (R5-8658, X4-IIIB) infectivity of activated PBL, when IgG was used at concentrations of 5 μg/ml. However, 6 of the 10 IgG purified from HIV-1 sera, but no normal IgG, inhibited infectivity of HIV isolates by 70–90% when used at concentrations of >100 μg/ml and this inhibitory activity was dependent on the HIV-1 viral isolate (data not shown). These findings clearly indicate that HIV IgG lacks IgG-ALA and that IgM mediated inhibition of HIV-1 is not mediated by contaminating IgG in the IgM preparation.
Some HIV-1 sera but not normal sera inhibit HIV-1
Since normal and HIV-1 IgM-ALA (but not HIV-IgG) inhibited HIV-1 infectivity, it became necessary to determine whether human sera could also inhibit HIV-1 infectivity of cells. In these studies, we used 8 HIV-1 sera obtained from patients not on anti-retroviral agents and 9 sera from normal individuals. Five of the eight HIV-1 sera inhibited infection of activated PBL by the X4-IIIB and R5-8658 viruses as depicted in Fig. 5. However, none of the nine normal sera inhibited HIV-1 infectivity of activated PBL even though purified IgM from all these normal sera inhibited HIV-1 infectivity in the same assay system. The finding that normal sera had no HIV-1 inhibitory activity was not unexpected as the majority of normal sera, unlike HIV-1 sera, lack IgM-ALA reactivity. In an accompanying paper we show that purified normal IgM has significantly higher IgM-ALA and IgM-anti-CD4 reactivity when compared with IgM in serum (see Fig. 1 in Ref. 1). Additionally we show that autologous normal serum does not inhibit IgM-ALA reactivity of purified IgM. Data in Fig. 6 also show that normal sera does not inhibit the inhibitory effect of purified IgM on HIV-1 infectivity. However normal sera inhibits the inhibitory effect of soluble CD4 as well as that of the human anti-gp120 Ab, 2G12 (Fig. 6). The latter finding might provide another explanation as to why the 2G12 Ab (which binds to a conserved epitope on gp120) and hyperimmune IVIG isolated from asymptomatic HIV-1 infected individuals were not more effective in vivo even though these Abs were very inhibitory to HIV-1 in vitro (16, 17).
The current data provide evidence to show that purified IgM, from sera of normal individuals, HIV-1 infected patients and ESRD patients will, in physiological doses, inhibit HIV-1 from infecting activated PBL in vitro and human PBL in an in vivo human PBL-SCID mouse model. The inhibitory activity resides in IgM that binds to leukocytes (IgM-ALA) as IgM, preabsorbed with leukocytes, fails to inhibit HIV-1 infectivity. These findings, together with the findings in the accompanying manuscript (1), provide several mechanisms to explain the inhibitory effect of IgM-ALA on HIV-1 infectivity. Potential mechanisms include the following 1) inhibiting HIV-1 viral cell entry by binding of IgM to CD4, CXCR4 and CCR5 thus inhibiting viral attachment to these coreceptors; 2) decreasing membrane expression of the CD4 receptor by downmodulation of the CD4 receptor; and 3) inhibiting T cell activation, which is important for viral replication. The current studies do not however explore the impact of IgM mediated inhibition of T cell activation on HIV-1 infectivity as the PBL used in these in vitro studies were already activated with PHA (before adding IgM) and in this setting IgM fails to inhibit T cells that are already in an activated state. Hence the inhibitory effects mediated by IgM, in the current in vitro studies, mostly involve the first two mechanisms.
We do not believe that the IgM mediated effects is due to a direct effect of IgM in neutralizing the virus as absorbing IgM with leukocytes or immobilized recombinant CD4, removed the inhibitory effect of IgM on HIV-1. Additionally, studies by other investigators clearly demonstrated that fresh normal human serum cannot lyse or inactivate the virus (18, 19). Similarly, the current findings cannot be explained on IgM with anti-Tat or anti-gp120 activity present in human serum as previous studies have shown that such IgM does not have HIV-1 neutralizing activity and does not inhibit viral entry into cells (20, 21). Finally, our findings cannot be explained by contaminating chemokines or soluble CD4 in the IgM preparations as by Western blot and ELISA techniques, we could not detect CCL5, CXCL12 or soluble CD4 in the purified IgM preparation.
The data also indicate that IgM-ALA are a heterogeneous group of autoantibodies and that IgM from different individuals, in particular, HIV-1 infected individuals, can vary in their receptor specificities. For example the majority of HIV-1 infected patients had levels of IgM anti-CXCR4 that were several fold higher when compared with normal IgM (see Fig. 3 in Ref. 1). Only a small subset of HIV patients, e.g., HIV no. 5, who had AIDS defining illnesses, had low or undetectable IgM anti-CXCR4. Conversely, the majority of HIV-1 patients had low or undetectable IgM anti-CCR5. These findings could provide an explanation for the preponderance of infection with R5 HIV-1 viruses in the majority of HIV-1 infected individuals. Additionally, the data would suggest that IgM from different HIV-1 infected individuals may not bind to the same epitope on a given receptor as exemplified by IgM from HIV no. 18, no. 20, no. 3, and no. 1. Both HIV no. 18 and no. 20 have high levels of IgM anti-CD4 and inhibited the 8658 and the 8442 R5 viral strains but not the R5-8397 viral isolate. Conversely, HIV no. 3 and no. 1 inhibited all three viral isolates including the more resistant R5-8397 virus (Fig. 3,A). However, depletion of IgM anti-CD4 from HIV no. 3 IgM removed the inhibitory effect of IgM on these three isolates, thus clearly indicating that these three isolates are dependent on CD4 binding for viral cell entry (Fig. 3,C). A potential explanation for these findings is that the more resistant R5-8397 and the sensitive R5-8658, which are both very dependent on CD4 for cell entry (Fig. 3 B), bind to different epitopes on CD4 and that IgM from HIV no. 18 and no. 20 (but not HIV no. 1 and no. 3) may lack IgM anti-CD4 that has specificity for the CD4 binding site of the R5-8397 virus. This is a viable possibility as previous investigators have clearly demonstrated differences in CD4 epitope binding among different HIV-1 viral strains (22). Hence these findings clearly indicate that studies on the in vivo functional importance of IgM-ALA in a particular HIV-1 infected individual will have to rely on experiments using autologous PBL as well as the autologous HIV-1 viral strain.
These studies clearly demonstrate that the majority of normal human sera, unlike some HIV-1 sera, fails to inhibit HIV-1 infectivity even though purified normal IgM has enhanced inhibitory effect on HIV-1 infectivity. In a previous paper we clearly show that normal serum, in general, lacks IgM-ALA that binds to leukocytes even though IgM purified from normal sera has high IgM-ALA activity (1). These findings can best be explained on basis that normal sera has low levels of IgM-ALA, most of which is complexed to cell receptors, rendering the IgM-ALA in sera less effective. Conversely, several observations clearly show that HIV-1 infected sera, in general, have high levels of IgM-ALA, which bind to leukocytes (2, 3, 4, 5, 6, 7). These findings, together with the possibility that HIV IgM-ALA from HIV-1 patients may also be enriched with higher levels of IgM Abs to core-receptors, could explain why HIV-1 sera is highly inhibitory to HIV-1 in our in vitro studies. Additional support for this concept (and for our in vitro observations) come from passive hyperimmune therapy, where plasma from asymptomatic HIV-1 infected individuals was more effective than normal plasma in temporarily reducing HIV-1 viral load to undetectable levels when administered to AIDS patients (23, 24, 25). We believe that HIV-1 viral neutralization resulted from high levels of IgM-ALA in HIV plasma and not from the IgG Abs in plasma especially because pooled hyperimmune IgG isolated from HIV-1 plasma, was ineffective in protecting against HIV infection even though the pooled hyperimmune IgG was highly inhibitory to the HIV-1 virus in vitro (17). The latter findings can be explained by a serum factor that inhibits human IgG anti-gp120 (see Fig. 6).
Our in vitro studies clearly show that soluble CD4, in the absence of human serum, strongly inhibits entry of all the HIV-1 viral strains we tested. Data in Fig. 6 clearly indicate that human serum inhibits the inhibitory effects of soluble CD4 in these in vitro assays. A potential explanation is that normal serum has naturally occurring IgM with anti-CD4 reactivity and hence IgM competes with the virus for the binding site on soluble CD4, thus rendering soluble CD4 less effective. These findings would suggest that one may have to use higher doses of soluble CD4 in vivo to combat the inhibitory effect of serum IgM-ALA on soluble CD4 (15, 26).
The naturally occurring IgM autoantibodies to chemokine receptors that we describe differ in many respects from autoantibodies to CCR5 that have previously been reported (27, 28, 29, 30). Firstly, the previously reported naturally occurring IgG anti-CCR5 autoantibodies differ from IgM anti-CCR5 autoantibodies (that we describe) in their ability to inhibit HIV-1 infectivity in vitro. Supraphysiological doses of purified pooled IgG anti-CCR5 Abs were needed to partially inhibit HIV-1 infectivity while in our studies, physiological levels of purified IgM were sufficient to inhibit (>90%) viral infectivity in vitro (27). Secondly, the autoantibody to CCR5 (Ab isotype unknown) detected in sera of HIV-exposed individuals who are HIV-seronegative, is not detected in normal sera (29). The naturally occurring IgM autoantibodies that we describe are present in normal uninfected individuals. Thirdly, the IgG anti-CCR5 autoantibody detected in macaques is not naturally occurring and develops after immunization with human T lymphocytes (30). Fourthly, the autoantibody to CCR5 (Ab isotype unknown) detected in a few CCR5 32 homozygous individuals is not naturally occurring and develops after exposure to cells bearing normal CCR5 (28).
Similarly the naturally occurring IgM autoantibodies to CD4 that we describe differ from the IgG anti-CD4 autoantibodies that have previously been reported (31). These IgG anti-CD4 autoantibodies were not detected in HIV seronegative normal individuals, but seen (a) in 34% of HIV seronegative individuals with reported exposures to HIV-1 and (b) in 44% neonates from HIV-positive mothers indicating therefore that IgG anti-CD4 may not be naturally occurring. In support of such a premise is the finding that IgG anti-CD4, unlike IgM anti-CD4 could not be detected in our studies in any of the umbilical cord B cell clone supernatants or umbilical cord serum from uninfected newborns (1). Additionally we could not detect IgG anti-CD4 Abs in normal sera but detected this Ab in 10 of 23 HIV patients using an ELISA. Such findings would indicate that presence of IgG anti-CD4 in a small subset of HIV-1 exposed or infected individuals could represent an autoimmune response induced by HIV-1. However, these IgG anti-CD4 Abs detected in HIV-1 patients with an ELISA technique failed to bind to CD4 positive T cell lines or PHA activated leukocytes. Only one of these 10 HIV IgG (containing anti-CD4 reactivity) inhibited HIV-1 infectivity of activated PBL, thus indicating that purified HIV-1 IgG may in general not be protective.
Previous investigators have suggested that IgM autoantibodies are a first line of defense against pathogens as these Abs cross-react with viral and bacterial Ags. Proposed mechanisms for IgM autoantibody-induced protection include enhanced sequestration of pathogens in the spleen, increased opsonization leading to phagocytosis, and binding of IgM to HIV-Tat protein, which can induce apoptosis of human T cells (20, 32, 33, 34). The current investigations suggest yet another mechanism for IgM autoantibody mediated protection against certain pathogens i.e., limiting HIV-1 infectivity of cells by binding to cell receptors important for viral entry. Such Abs are particularly suited to perform such functions as they are large pentameric molecules (900 kDa), and can bind to cells without having a cytolytic effect at 37°C. Furthermore, the in vivo studies with human PBL-SCID mice would suggest that IgM can enter extracapillary sites in infected or inflamed tissue, as after i.p. administration of IgM, infection of human PBL by HIV-1 was significantly inhibited in murine spleens (Table II).
Data in these studies clearly show that IgM, by inhibiting viral entry into cells and attenuating T cell activation could provide yet another mechanism for decreasing viral replication and prolonging the latency period after HIV-1 infection. In an accompanying manuscript we show that IgM, especially from certain patients, inhibit T cell proliferation in response to alloantigens or anti-CD3 Abs and inhibits production of certain cytokines and chemokines, e.g., TNF-α as well as inhibits proximal signaling events that are involved in T cell activation, e.g., phosphorylation of Zap 70 (1). Inhibition of TNF-α production may be particularly relevant as in vitro observations indicate that high levels of TNF-α can enhance HIV-1 viral replication (35). The mechanism behind IgM-ALA mediated attenuation of T cell activation remains to be clarified but potential mechanisms include binding of IgM to CD3 and CD4 and downmodulation of CD4 and CD2. However, there is a growing body of evidence to indicate that attenuation of T cell activation is directly associated with a slow disease progression after HIV-1 infection (reviewed in Ref. 36). Evidence to support this model derives from several observations: 1) studies showing that immune activation is a strong independent predictor of disease outcome in anti-retroviral untreated and treated individuals, 2) observations in certain patients with multiple drug resistant HIV-1 where high levels of virus can coexist with persistently high levels of CD4+ T cells, having low levels of activation and where in vitro the virus from the patients replicate as well as wild type in mitogen activated CD4+ T cells and 3) observations in Sooty Mangaby monkeys, who tolerate high levels of SIV viremia but fail to exhibit a clear increase in immune activation and where the lifespan of infected animals are not dramatically different from those of uninfected animals. These observations and the conceptual model of limiting T cell activation to preserve sustained high levels of CD4+ T cell counts have led investigators to seek new approaches to limit the deleterious effect of HIV-1 on immune activation. One such approach is to inhibit T cell activation, e.g., by Abs to costimulatory molecules or by using cyclosporin A together with anti-retroviral agents (37, 38). Data from our studies would indicate that naturally occurring IgM-ALA could provide another approach to limit or attenuate T cell activation as well as limit HIV-1 cell entry and hence strategies that increase the repertoire of IgM-ALA either before HIV-1 infection or in the early stage of HIV-1 infections could prove to be effective. One such strategy is a vaccine approach to increase naturally occurring IgM-ALA which could have a synergistic or additive effect on vaccines designed to increase neutralizing anti-viral Abs or virus specific CTLs.
Finally, how can one explain disease progression in the majority of patients, especially older individuals if there already exists a good level of IgM-ALA in normal individuals and during the asymptomatic state? Firstly, naturally occurring IgM autoantibodies decline with age and hence one would predict that protection would decline with age. Such a mechanism could provide an explanation for the more rapid progression of HIV-1 infection with advancing age (39, 40). Secondly, it is possible that the HIV-1 virus could infect and deplete certain B cell clones secreting IgM-ALA. For example, there is evidence to show that activated CD4+ B cells (present in lymphoid areas) are susceptible to HIV-1 infection and can undergo apoptosis following cross-linking of CD4 by gp120 (41, 42). AIDS patients are also reported to have a clonal deletion of a B cell subset expressing Ig VH3 gene products possibly as a results of gp120 binding to Ig VH3 gene products (43). Such a mechanism may apply to explain depletion of some naturally occurring IgM autoantibodies which are known to use nonmutated VH3 gene segments (44, 45). In this regard, Rodman et al. have reported depletion of some (but not all) naturally occurring IgM autoantibodies (e.g., anti-Tat and anti-protamine) with disease progression while our studies indicate that some HIV-1 infected patients can be depleted of IgM anti-CCR5 or anti-CXCR4 (46). Conversely, there are data clearly showing that certain naturally occurring IgM autoantibodies can increase with disease progression, e.g., IgM autoantibodies to glycolipids and phospholipids (reviewed in 1). Data from our studies also demonstrate that there is an increase in IgM anti-CXCR4 Abs in the majority of HIV-1 infected patients (see Fig, 3 in Ref. 1). Therefore understanding mechanisms that increase or decrease B cell clones secreting naturally occurring IgM autoantibodies, especially in HIV-1 infected patients, should have a beneficial effect on strategies that can enhance the protective effect of IgM-ALA Abs. Studying the repertoire of IgM-ALA in long term nonprogressors, who are not on an anti-retroviral agent, should also provide clues in this regard.
We thank the Ryan White HIV Clinic at University of Virginia for identifying patients willing to donate their blood. We thank Dr. David Rekosh and Dr. Marie-Louise Hammarskjold for their helpful suggestions and use of the Myeles Thaler Biosafety Level-3 Laboratory. We also thank Rhonda S. Richardson for her expert assistance in preparing this manuscript.
Patents are pending on IgM anti-leukocyte antibodies.
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.
This work was supported by National Institutes of Health, National Institute of Allergy and Infectious Diseases Grant 5R21AI052740-02 (to P.I.L.), Nephrology Division Research Development Funds, and the University of Virginia Research Development Funds.
Abbreviations used in this paper: IgM-ALA, IgM anti-leukocyte autoantibodies; ESRD, end-stage renal disease patients; MCF, mean (geometric) channel fluorescence.