Intrinsic Defects in Macrophage IL-12 Production Associated with Immune Dysfunction in the MRL/++ and New Zealand Black/White F₁ Lupus-Prone Mice and the Leishmania major-Susceptible BALB/c Strain¹

It is widely held that self-reactive lymphocytes are a normal component of the immune system, and it is the inability to limit the growth and function of these autoreactive cells that characterizes both organ-specific and systemic autoimmune diseases (1). In particular, systemic lupus erythematosus (SLE)⁴ is characterized by excessive B cell activity and abnormally high serum autoantibody levels. This leads to the deposition of Ab and immune complex in many tissues, and ultimately, to severe organ or tissue pathology, including vasculitis and glomerulonephritis (2). Unlike organ-specific B cell-mediated autoimmune diseases like myesthenia gravis, or Graves’ disease, the breadth of the Ab repertoire that develops during lupus suggests that the nature of the defects in immune function that trigger disease are likely to have global implications (e.g., involving regulatory molecules such as cytokines) rather than being finely focused (e.g., dependent upon selected B or T cell receptors or MHC polymorphisms).

Fundamental to understanding the initiation of autoimmune disease is the determination of why the control of autoreactive lymphocyte function is not established. One critical issue to address is the extent to which lymphocyte defects are truly intrinsic, or, alternatively, result secondarily from defects in the function of APCs or other regulatory cell populations. Evidence exists for both of these positions. Evidence supporting intrinsic B cell defects has come primarily from studies using MRL/lpr mice (3, 4). Because these mice display aberrant, nonfunctional Fas proteins, all cell populations in MRL/lpr mice whose growth and function are regulated by Fas-induced apoptosis are likely to be affected. The extent to which mice bearing the lpr mutation provide insight into the basis of multigenic human autoimmunity remains to be determined. The demonstration of B cell hyperactivity in young New Zealand Black/White (NZB/W) F₁ and BxSB lupus-prone mice, or the transfer of T cell-depleted bone marrow from these mice into SCID mice (5, 6), has suggested that B cells from these multigenic lupus models may also have intrinsic defects in function. While such experiments emphasize the early development of aberrant B cell function in lupus, they do not substantiate that defective function is intrinsically programmed within the B cell, and the importance of accessory cells in permitting expression of defective B cell function has been demonstrated (7).

Studies focusing on the function of regulatory cells, such as macrophages (Mφ) (8, 9, 10, 11, 12, 13, 14, 15), from the best-studied murine lupus models, MRL/++ and NZB/W F₁, have revealed dramatic defects in production of the proinflammatory cytokines TNF-α (12, 15), IL-1 (8, 9, 10, 15), and IL-6 (15), but not the anti-inflammatory cytokine IL-10 (15). Of these, aberrant IL-1 expression has been shown to be truly intrinsic, i.e., independent of the contribution of other cell types (9, 10). It is noteworthy that the onset of defective production of the individual proinflammatory cytokines occurs sequentially, and moreover, that the reduced autocrine stimulation caused by the TNF-α defect appears to be responsible for the subsequent IL-1 and IL-6 defects (15). Nevertheless, each of these events is triggered in and controlled by Mφ alone and is thus intrinsic to the Mφ. These studies have led us to question the potential functional contribution of Mφ to B cell hyperactivity in these mice. This contribution could be both direct, on the B cell itself, and indirect, via modulation of T cell function. To address these issues, we first extended our characterization of Mφ from MRL/++ and NZB/W F₁ mice to include the immunomodulatory cytokine, IL-12, which is mainly produced by Mφ and is critical for directing development of the Th1 responses (16) that may be defective in lupus-prone mice (17). Our results show that production of IL-12 is dramatically reduced in Mφ from both MRL/++ and NZB/W F₁ mice, which appears to be similar in nature to that of TNF-α but not the subsequent IL-1 and IL-6 defects. In addition, Mφ IL-12 production in response to the Th1-derived cytokine, IFN-γ, was normal in these lupus-prone strains, suggesting a defect in the initiation, but not the propagation, of Th1 responses in the disease process. This study provides additional insights into the nature of the cytokine defects that uniquely characterize the two principal models of multigenic lupus and suggests that the cytokine-dependent regulation of the Th1 subset by the cells of the innate immune system may be an important event in the establishment of lupus.

Four-week-old BALB/c, A/J, C57BL/6, C57BL/10, DBA/2, C3H/OuJ, MRL/++, and NZB/W F₁ male mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained for 1 wk after arrival in a pressurized (one-way flow) room.

Recombinant murine (rMu)TNF-α (10⁷ U/mg; cat. no. 19321T) was purchased from PharMingen (San Diego, CA), rMuIFN-γ (10⁷ U/mg) was kindly provided by Genentech (South San Francisco, CA), and rMuIL-12 (3 × 10⁶ U/mg) was a gift from Dr. Victor H. Van Cleave (Genetics Institute, Cambridge, MA). LPS (Escherichia coli: 0111:B4; Sigma, St. Louis, MO) was stored at −70°C (2 mg/ml in H₂O) and diluted immediately before use. RPMI 1640 medium with glutamine (cat. no. 12-702F) was supplemented with 0.5% HEPES (cat. no. 17-737), 1% penicillin/streptomycin solution (cat. no. 17-602E), and 5% FBS (all components of medium from BioWhittaker, Walkersville, MD) and was used for culturing Mφ. HBSS (BioWhittaker) was used for cell washing.

Peritoneal exudate Mφ were obtained by peritoneal lavage with cold HBSS 4 days after a 2 ml i.p. injection of 4% thioglycollate broth (Remel, Windsor, CT; cat. no. 07178). Cells were pooled from at least three mice per strain, washed, resuspended in fresh medium, and seeded at 10⁵ cells in 100 μl per well of 96-well flat bottom tissue culture-treated plates (Costar, Cambridge, MA; cat. no. 3596). Cells were incubated for 2 h at 37°C, 5% CO₂, in a humidified chamber to allow Mφ to adhere and spread. Nonadherent cells were removed by adding 200 μl of warmed HBSS to each well, resuspending nonadherent cells by moderately tapping the plate, and flicking the plate to discard the nonadherent cells. This washing was performed three times, after which 50 μl of medium was immediately added to each well. Remaining cells were >98% Mφ, as assessed by morphologic examination and nonspecific esterase staining. Although ∼85% of the total exudate cell population were adherent Mφ within each strain, to ensure that equal numbers of adherent Mφ among strains remained after washing, nonadherent cells from washes of single wells from each strain were routinely counted and showed no significant differences among strains. Cytokines, Abs, LPS, or medium were added to each well to yield a final volume of 200 μl. Conditioned medium was collected in a sequential fashion (e.g., 0–16, 16–24, 24–36 h), with change of medium and fresh LPS along with other factors at each time point, and stored at −20°C for assessment of cytokines. This method of conditioned medium collection has the advantage of permitting measurement of cytokine levels independent of levels produced during an earlier period, a feature that contributes to accurate assessment of changes in the kinetic pattern of cytokine production. The three culturing periods used for the kinetic assessment of cytokine correlate to an early, intermediate, and late period that collectively span the entire time during which most of the cytokine is produced. For example, >90% of the total amount of IL-12 is produced within 24 h, and the rest is produced within 24–36 h (see Fig. 2; see Ref. 15 for kinetics of other cytokines).

IL-12 levels in Mφ-conditioned medium were assessed by a MuIL-12-specific ELISA, a gift from Dr. Victor H. Van Cleave. The general cytokine ELISA protocol developed by PharMingen was used. Briefly, two rabbit polyclonal anti-MuIL-12 Abs preparations, R03B03 (3 μg/ml) and B03B02 (biotinylated, 1:500 dilution), that were prepared by immunizing rabbits with the IL-12 heterodimer (p70) were used for capture and detection, respectively, along with avidin-labeled peroxidase (Sigma, cat. no. A-3151) and 2,2′-azino-bis (3-ethyl benzthiazoline-6-sulfonic acid) solution (Sigma, cat. no. A-1888) for development. One hundred microliters of undiluted Mφ-derived conditioned medium was added to the ELISA, and rMuIL-12 was used to generate a standard curve. IL-6 and IL-10 levels in Mφ supernatants were assessed by ELISA (mAb from PharMingen; anti-IL-6 mAb, cat. nos. 18071D and 18082D, anti-IL-10 mAb, cat. nos. 18141D and 18152D). IL-1 bioactivity in Mφ lysates was measured in the D10.G4 bioassay as previously described (15, 18), and TNF-α levels in Mφ conditioned medium were assessed using the WEHI-164 bioassay as previously described (15, 19). One unit of cytokine activity in bioassay is defined as the amount of cytokine that caused half maximum cell viability as assessed with Alamar Blue solution (AccuMed International, Westlake, OH; cat. no. 00–100).

All means and SE in figures were calculated from triplicate values and the Student’s t test was used to compare mean values.

To evaluate one possible mechanism for aberrant Mφ cytokine production contributing to the dysregulation of lymphocyte function in lupus, we investigated whether Mφ from lupus-prone strains aberrantly produced IL-12. This cytokine is known to play a dominant role in the appropriate development of Th1 cells, in establishing the balance of Th1 and Th2 populations in normal individuals, and in triggering the divergence from this balance that appears to be a critical component of the development of autoimmune disease (16, 17). Peritoneal Mφ were obtained from young (4-wk-old), prediseased, male MRL/++ and NZB/W F₁ mice and from five age-matched control murine strains, BALB/c, A/J, C57BL/6, DBA/2, and C3H/OUJ. Mφ were activated with LPS for 16 h, and culture-conditioned medium was assessed for IL-12 levels by ELISA (Fig. 1). The data reveal that Mφ from MRL/++ and NZB/W F₁ strains are dramatically impaired and produce 10- to 20-fold less IL-12 than all control strains, except BALB/c. Activation of Mφ with other doses of LPS (i.e., 10 ng/ml and 1000 ng/ml) resulted in defects of similar magnitude in Mφ from the lupus-prone strains (data not shown). Interestingly, BALB/c Mφ produce notably less IL-12 (∼4-fold less) than Mφ from the other control strains. Thus, the BALB/c strain, which is known to have defective development of a protective Th1 type response during some parasitic infections (i.e., Leishmania major; reviewed in 20 , is here shown to have substantially impaired production of the Th1-inducing cytokine, IL-12. The association in BALB/c mice of low IL-12 levels with restrictions in selected Th2-dependent immune responsiveness suggests that the similar, if not more pronounced, IL-12 defect in the MRL/++ and NZB/W F₁ strains (in nine experiments, MRL/++ and NZB/W F₁ Mφ produced IL-12 levels at an average of 62% ± 10% (p < 0.01) and 70% ± 12% (p < 0.01), respectively, of those of the BALB/c Mφ during the first period) may well have physiological relevance to the immune dysregulation in these strains.

To characterize the kinetics of cytokine production, IL-12 levels were next assessed in conditioned medium collected during three sequential intervals that represent early (0–16 h), intermediate (16–24 h), and late (24–36 h) periods of IL-12 production, as described in Materials and Methods. Mφ from C57BL/6 mice were selected as a representative control strain because they routinely produced levels of IL-12 close to the median value for normal strains. Mφ from MRL/++ and NZB/W F₁ mice consistently displayed at least a 10-fold reduction in IL-12 production relative to C57BL/6, regardless of the period of evaluation (Fig. 2 A). BALB/c Mφ, as noted earlier, produced substantially less IL-12 than Mφ from any other normal strain. Defective IL-12 expression by Mφ from lupus-prone strains is not a consequence of a reduction in either viability or metabolic activity and does not reflect a broad dysregulation of Mφ function inasmuch as viability, protein synthesis, and induction of LFA-1 and IL-10 all do not significantly vary between Mφ from control and lupus-prone strains (Refs. 15 and 21, and data not shown). These results show that the defective Mφ cytokine production profile in the MRL/++ and NZB/W F₁ strains, originally defined as encompassing IL-1, IL-6, and TNF-α, but not IL-10 (15), can now be extended to include IL-12 as well.

Defective TNF-α production by Mφ from lupus-prone mice is manifested soon after stimulation (0–12 h), and clearly before defective expression of IL-6 (12–24 h) or IL-1 (48 h) (15). In fact, compromised autocrine stimulation by TNF-α in MRL/++ and NZB/W F₁ Mφ appears to cause the subsequent defects in IL-1 and IL-6 production (15). Therefore, we addressed whether the early TNF-α defect might contribute in a similar manner to the dramatically reduced IL-12 expression in these Mφ or, conversely, whether defective IL-12 might regulate TNF-α expression. Mφ from control and lupus-prone mice were activated with LPS in the presence or absence of a physiological concentration of TNF-α (1000 U/ml, a level routinely produced by control Mφ under these conditions (15)). Conditioned medium was collected at 16 h and assessed for IL-12 (Fig. 2, A and B). TNF-α did not stimulate LPS-activated IL-12 production by Mφ from any strain during any interval, and, thus, it is unlikely that the defective TNF-α production characteristic of Mφ from lupus-prone strains contributes to their IL-12 defect (Fig. 2,B). Similarly, the addition of a physiologically high concentration (10 ng/ml; see Figs. 1 and 2) of IL-12 to LPS-activated Mφ had no substantial effect on TNF-α expression, and, therefore, IL-12 is unlikely to be responsible for the defective TNF-α production by MRL/++ and NZB/W F₁ Mφ (Fig. 3). In fact, IL-12 treatment tended to modestly down-regulate TNF-α production in Mφ from all strains tested, except A/J, where inhibition of Mφ IL-12 production was routinely ∼50–60% (Fig. 3).

Additionally, IL-12 treatment had no substantial effect on IL-6 production by C57BL/6 or MRL/++ Mφ, nor did it affect the dramatic ability of TNF-α to correct the progressive MRL/++ Mφ defect in IL-6 production that occurs after 24 h (Fig. 4). Similar results were obtained with Mφ from other control strains and from NZB/W F₁ mice (data not shown). IL-12 also had no effect on either normal Mφ IL-1 expression, or on the defective kinetic production of IL-1 by Mφ from lupus-prone strains (data not shown). These findings further demonstrate that IL-12 does not substantially modulate expression of these proinflammatory cytokines, and more importantly, that defective IL-12 is not involved in dysregulated expression of the other proinflammatory cytokines by Mφ from lupus-prone strains. The unresponsiveness of Mφ to IL-12 is further demonstrated by the observation that TNF-α production by Mφ from C57BL/6 mice, a strain that produces low levels of TNF-α (15) and substantial levels of IL-12 (see Figs. 1 and 2), is not enhanced by the Ab-mediated neutralization of endogenous IL-12 production (453 ± 50 U/ml with LPS alone and 457 ± 44 U/ml with LPS plus 3 μg/ml anti-IL-12 mAb; IgG1 isotype control was similarly without effect on TNF-α expression). Thus, defective expression of IL-1 and IL-6 (15), but not IL-12, arises as a consequence of the early defect in TNF-α expression, and, moreover, defective IL-12 expression does not contribute to defective TNF-α, IL-1, and IL-6 expression.

The compromised ability of lupus-prone Mφ to produce sufficient levels of IL-12 induced with mitogen alone suggests that these Mφ would produce suboptimal levels of IL-12 during the initiation of a Th1 response. IL-12 is required for the initial production of the Th1-derived cytokine, IFN-γ, which in turn amplifies the development of Th1-responsive cells by up-regulating Mφ IL-12 production; therefore, we determined whether lupus-prone Mφ were appropriately responsive to IFN-γ in their IL-12 production, and thus, would be capable of promoting an ongoing Th1 response. To determine the degree of responsiveness to IFN-γ by Mφ from lupus-prone strains, Mφ from control and lupus-prone mice were activated with LPS in the presence or absence of IFN-γ, and conditioned medium was then assessed for IL-12 (Fig. 5,A). The greatest augmentation (roughly 20-fold) of IL-12 production by IFN-γ treatment occurred in Mφ from the two lupus-prone strains and the normal BALB/c strain. Strikingly, IFN-γ up-regulated IL-12 production by LPS-activated Mφ from all control strains in a strictly-defined manner that was inversely proportional to IL-12 levels induced by LPS alone (Fig. 5,B). Additionally, this response is not simply due to a shift in kinetics because Mφ from lupus-prone mice continued to demonstrate an enhanced responsiveness to IFN-γ during the intermediate and late intervals, and in response to other doses (0.1 or 10 ng/ml) of IFN-γ as well (data not shown). Interestingly, the low levels of IL-12 produced by the BALB/c strain were restored to normal levels by IFN-γ treatment, but unlike those of lupus-prone strains, were enhanced to precisely the extent predicted by the relative differences among the other control strains (Fig. 5 B). A likely interpretation of these data is that IFN-γ-signaling is intact in both lupus-prone and BALB/c mice, but the BALB/c strain, although a low IL-12 producer upon stimulation with LPS alone, defines the lower limit of the normal cytokine production spectrum because IL-12 levels were efficiently restored by IFN-γ to those of all other normal strains. However, lupus-prone strains display a truly “defective” pattern of IL-12 expression stimulated with LPS alone because their response to IFN-γ, although intact and operating maximally, did not restore IL-12 levels to those of normal strains. Nevertheless, the dramatic response to IFN-γ by lupus-prone (and by BALB/c) Mφ, which led to IL-12 levels that approached those of normal strains, suggests that these Mφ would be capable of propagating, but not initiating, a Th1 response.

The IL-12 and TNF-α defects may arise from common mechanisms inasmuch as they show similar kinetics (i.e., early expression, see Figs. 1–3, and 15 , expression of one cytokine does not appear to modulate expression of the other, and the extent of their defects is dramatically reduced by IFN-γ-treatment (see Ref. 15 for IFN-γ regulation of defective TNF-α expression). Conversely, the diminished autocrine stimulation attributable to early defective TNF-α expression (15), and not, as shown here, to the early defective IL-12 production (see Figs. 3 and 4), appears responsible for the downstream IL-1 and IL-6 expression defects in MRL/++ and NZB/W F₁ Mφ. Thus, while at least part of the mechanism leading to the downstream IL-1 and IL-6 defects has been revealed, the underlying aberrant events responsible for defective TNF-α and IL-12 expression remained to be determined. Moreover, the regulation of these cytokines by IFN-γ also distinguishes two classes of cytokine defects (Table I). Production of TNF-α and IL-12 is strongly stimulated by IFN-γ (see Fig. 5 and Ref. 15, respectively), whereas IL-1 and IL-6 production are either suppressed or not substantially modulated, respectively, by IFN-γ treatment (Table I). Other doses of IFN-γ (i.e., 0.1 and 10 ng/ml) demonstrated similar modulation of IL-1 and IL-6 production in a dose-dependent fashion (data not shown). These data demonstrate that the two classes of cytokine defects, 1) the early onset defects of TNF-α and IL-12, and 2) the later onset, or progressive, defects of IL-1 and IL-6, can be further distinguished by their initial response to IFN-γ in that the earliest aberrant functional events in Mφ from lupus-prone strains appear to compromise the expression of cytokines that can be strongly stimulated by IFN-γ. We next tested, as a corollary to this finding, whether cytokines that are not dysregulated in lupus-prone Mφ might also be refractory to IFN-γ-mediated stimulation. IFN-γ treatment did not stimulate and, in fact, suppressed IL-10 production by LPS-stimulated Mφ from control and lupus-prone strains (Table I), supporting the contention that only cytokines stimulated during early periods by IFN-γ (i.e., IL-12 and TNF-α) show early defective expression.

Determination of genetically programmed (i.e., intrinsic) functional defects is critical to our understanding the origins of autoimmunity. One approach lies in genetic analysis, and such studies have identified linkage groups that confer sensitivity to disease (22, 23, 24). Another approach is to identify defects in immune cell function that appear early in development, before the onset of any disease signs, and thus have the potential to initiate as well as propagate disease. In the latter category, it has been difficult to demonstrate substantial lymphocyte-associated intrinsic defects that are shared among the lupus-prone strains, such as MRL/++ and NZB/W F₁, whose disease is multigenic in origin (25, 26). However, we have reported that these strains are characterized by conserved defects in Mφ production of at least three proinflammatory cytokines, TNF-α, IL-1, and IL-6, that may be fundamental to the disease process (9, 10, 15). In addition, Mφ from both strains are also characterized by normal IL-10 expression, and by the hierarchy of TNF-α that, by virtue of its under-expression, causes the subsequent defects in IL-1 and IL-6 (15). That the defects are readily apparent in Mφ obtained from young, prediseased mice, are common to both lupus-prone strains, and are intrinsic in nature, suggest that aberrant expression of these immunomodulatory cytokines may play a critical role in the initiation and development of the disease process. Indeed, the critical contribution of these defects in Mφ-derived cytokines has been demonstrated by the therapeutic effect of administration of TNF-α or IL-1 to young, lupus-prone, NZB/W F₁ mice before the onset of disease (12, 27, 28). Here, we demonstrate a novel and dramatic defect in MRL/++ and NZB/W F₁ Mφ production of IL-12, a cytokine that is critical in the development of Th1 responses (16). This IL-12 defect was manifested in a manner similar to that of TNF-α (15) in that both defects developed rapidly after activation with LPS and could be substantially normalized by treatment with exogenous IFN-γ. However, defective expression of IL-12, unlike that of TNF-α (15), did not contribute to the subsequent IL-1 and IL-6 defects in Mφ from lupus-prone mice because addition of rMuIL-12 (or the addition of anti-IL-12 mAb) did not substantially regulate IL-1 or IL-6 production. Furthermore, the IL-12 and TNF-α defects did not contribute to each other because addition of either cytokine had no substantial regulation of expression of the other. These findings strengthen the premise that cytokine dysregulation defines a conserved and perhaps dominant pathway leading to systemic autoimmunity in those strains whose disease, like human lupus, is multigenic in origin.

Earlier we had reported evidence for two distinct classes of cytokine defects expressed in both MRL/++ and NZB/W F₁ Mφ (15). These are defined operationally by their time of expression after Mφ stimulation: expression of the TNF-α defect preceded expression of the defects in IL-1 and IL-6, and, moreover, caused the latter defects. The IL-12 defect reported here falls into the former category based on kinetics, yet is distinct from TNF-α in that it neither precipitated nor modulated the ensuing defects in IL-1 and IL-6. The two classes of cytokine defects defined above can be further distinguished by their responsiveness to IFN-γ, an NK and T cell-derived cytokine known to enhance Mφ cytokine production (29). IFN-γ treatment dramatically stimulated LPS-induced IL-12 and TNF-α production (15) by Mφ from all strains, and the response was noted to be inversely proportional to the basal level of cytokine produced upon stimulation with LPS alone. Conversely, IFN-γ suppressed the early (16 h) production of IL-1 and did not substantially regulate IL-6 expression, these targets being representative of the second class of cytokine defects. This selective inhibitory activity of IFN-γ on IL-1 (as well as on IL-10), and its lack of regulation of IL-6 production, have also been reported by others (30, 31). It is reasonable that the cytokine defects of the first class (i.e., TNF-α and IL-12) are caused by one or more underlying aberrant events that can be overcome or normalized by IFN-γ-induced signals that normally stimulate those cytokines. These stimulatory signals arising from the IFN-γ receptor may include both secondary messenger molecules, such as JAK 1 and 2, and STAT1α molecules, and several transcription factors (32) and may provide clues as to the aberrant molecular events that arise in these strains.

Although IFN-γ dramatically suppressed LPS-induced production of the autocrine inhibitory cytokine IL-10, it is unlikely that IFN-γ reduced the extent of defective TNF-α and IL-12 expression in MRL/++ and NZB/W F₁ Mφ solely by suppressing IL-10 for the following reasons: 1) The autocrine inhibitory activity of IL-10 does not appear to be involved in defective TNF-α (15) or IL-12 production (our unpublished observation) because Ab-mediated neutralization of IL-10 did not reduce the extent of these defects. 2) LPS-activated Mφ from MRL/++ and NZB/W F₁ strains do not over-express IL-10 (15). 3) The IFN-γ-induced augmentation of TNF-α (15) and IL-12 production among control Mφ is proportional to their IL-10 levels (15), suggesting that IFN-γ may normalize TNF-α and IL-12 production by inhibiting IL-10; however, clearly the TNF-α and IL-12 response to IFN-γ by Mφ from lupus-prone mice is not proportional to their IL-10 levels (i.e., MRL/++ have lower IL-10 and NZB/W F₁ have higher IL-10 levels than BALB/c (15)). Therefore, IFN-γ appears to normalize TNF-α and IL-12 expression in MRL/++ and NZB/W F₁ Mφ by an IL-10-independent mechanism.

These findings suggest the possibility that the aberrant expression of TNF-α and IL-12 could develop as a consequence of an up-stream defect in IFN-γ expression by these LPS-activated Mφ. There is evidence that LPS-activated murine peritoneal Mφ express minute levels of IFN-γ mRNA along with intracellular, but not extracellular, expression of translated IFN-γ (33). Although it was suggested that intracellular IFN-γ may cooperate with LPS to induce TNF-α expression (33), and while we have confirmed Mφ IFN-γ production by RT-PCR and Southern blot analysis (our unpublished observation), we did not find defective expression of LPS-activated IFN-γ mRNA levels in Mφ from lupus-prone strains by these procedures (our unpublished observation), nor could we detect secreted IFN-γ by ELISA, or modulate TNF-α or IL-12 production with anti-IFN-γ mAb treatment (our unpublished observation). These results suggest that the mechanisms underlying dysregulated TNF-α and IL-12 production do not include dysregulated endogenous IFN-γ production.

Defective expression of IL-12, in cooperation with defective TNF-α and IL-1, by Mφ from lupus-prone mice may have an impact on disease both during and after initiation of the disease process. The initial polyclonal hyperexpansion of ubiquitous autoreactive lymphocytes that appears as the first stage of disease (1, 2) may be linked to defective TNF-α-induced apoptosis in addition to diminished suppressor activity by IFN-γ, an NK- and T cell-derived cytokine normally stimulated by the combination of TNF-α, IL-1, and IL-12 (34, 35, 36), all of which we have found to be defectively produced by MRL/++ and NZB/W F₁ Mφ (17). Following this polyclonal hyperexpansion of autoreactive lymphocytes, defective IL-12 expression may also permit the development of the strong, Ag-specific Th2-mediated humoral responses, such as those directed toward chromatin (24), that characterize later stages of lupus. Indeed, administration of IL-12 blocks pathogenic autoantibody production and end-stage renal pathology in lupus-like, mercury-induced autoimmune disease in mice (37). Moreover, that IL-12 administration to cultures of PBMC from human SLE patients directly suppresses the excessive spontaneous production of Ig and anti-DNA IgG Ab (38), along with the reduced IL-12 and IFN-γ levels produced by these cultures (39, 40), suggests that defective IL-12 levels in human SLE leads to hypergammaglobulinemia and the generation of pathogenic Ab. Furthermore, it has recently been reported, using a T cell receptor transgenic mouse, that Mφ-derived TNF-α and IL-1 are required for IL-12 to induce development of a Th1 population from naive precursors in vitro (41). Thus, the defective TNF-α and IL-1 expression by Mφ from lupus-prone strains, in concert with defective IL-12 production, could play a fundamental role in lupus by fostering the polyclonal hyperexpansion of autoreactive lymphocytes and the subsequent establishment of a Th1-deficient environment, which, ultimately, would lead to enhanced production of pathogenic autoantibodies.

The physiological relevance of these findings will be determined, in part, by whether the extent of defective IL-12 expression noted in the lupus-prone strains is sufficient to alter the balance of T cell subsets or otherwise promote pathogenic Ab responses. In this light, it is noteworthy that the low IL-12 expression by Mφ from BALB/c mice shown here correlates with the sensitivity of this strain to infection by the parasite, L. major (20). BALB/c mice are unable to lodge a protective Th1 response against L. major infection, and this is associated with exuberant IL-4 production and a strong Th2 response (20). Conversely, C57BL/6 and C3H/OuJ strains, whose Mφ produce dramatically more IL-12 than do BALB/c Mφ, are resistant to infection and mount a strong Th1 response against this parasite. These findings thus suggest a genetic basis for BALB/c susceptibility to L. major infection linked to deficient IL-12 production. Indeed, it has been demonstrated, using L. major-resistant strains created from congenic back-crosses of BALB/c and C57BL/6 strains, that disease susceptibility of BALB/c mice segregated with a region of chromosome 11 that contains the IL-12 (p40) gene (42). If the low levels of IL-12 produced by BALB/c Mφ do contribute to enhanced IL-4 expression and L. major susceptibility in this strain, then the more dramatically reduced IL-12 levels in the MRL/++ and NZB/W F₁ strains are likely to be of similar, or even greater, physiological significance, and as such, may contribute to autoimmunity. All the cytokine defects that we have reported are found in both MRL/++ and NZB/W F₁ Mφ, and, moreover, are expressed before any detectable disease signs. Thus, it is tempting to speculate that the two defective pathways discussed earlier, 1) reduced TNF-α, leading to reduced IL-1 and IL-6, and 2) reduced IL-12, may both be important for establishing lupus of multigenic origin.

Our findings demonstrate that defective Mφ proinflammatory cytokine production has been conserved among at least two genetically distinct backgrounds prone to murine lupus, suggesting a common, intrinsic cellular aberrancy that might be diagnostic for this disease and, more importantly, may be involved in the initiation and propagation of the disease process. Clearly, the contribution of the Mφ defects described here must be viewed in the context of the gene mapping studies which have revealed multiple genetic linkage groups associated with specific aspects of murine lupus (24). In particular, the elegant genetic studies of Wakeland and coworkers (22, 24) and Kotzin and coworkers (23) have revealed loci which are associated with B cell hyperactivity. Although there are claims of intrinsic defects in B lymphocytes function from lupus-prone strains, the splenic B cell populations studied generally are the product of T cell depletion, and thus enriched both for B cells and Mφ (5, 6, 22). Neither percoll enrichment of B cells nor adherence-based elimination of Mφ are particularly helpful procedures due to the marked ability of spleen cells to generate Mφ from immature, nonadherent precursors in culture (our unpublished observations). While the use of purified long-term B cell lines transferred into SCID mice provides more compelling support for intrinsic B cell defects (6), these animals do not manifest all aspects of NZB/W F₁ B cell hyperactivity, in keeping with the concept that the complexity of lupus is most likely due to several mutations operating at different times in development or simultaneously in different cells types. Our findings here place the Mφ, among other cells of the innate immune system, in a central position regarding the initiation and development of autoimmunity. Moreover, they raise questions as to whether defects ascribed to B and T cells in these multigenic models of lupus (MRL/++, NZB/W F₁) are intrinsic to lymphocytes, or alternatively, reflect differences in lymphocyte development or regulation that arise as a consequence of aberrant control by accessory cells. It is anticipated that continued efforts to understand the control and function of cells regulating innate immunity will provide insight into the ways that dysregulated cytokine expression can contribute, not only as a possible effector mechanism of the end-stage inflammation of the disease process, but also to the fundamental immune dysregulation that initiates the development of lupus.

We thank Dr. Victor H. Van Cleave (Bioanalytical Sciences Department, Genetics Institute, Cambridge, MA) for generously providing rMuIL-12 and Ab for the IL-12 ELISA and IL-12 neutralization, and Genentech, Inc., (South San Francisco, CA) for providing rMuIFN-γ (10⁷ U/mg). Much gratitude is extended to Karen S. Alleva for her assistance in preparing the manuscript and in laboratory maintenance, and to Dr. John J. Alleva for his critical analysis of data presentation.