Background

Wegener's granulomatosis is a systemic inflammatory disease of unknown etiology. Many studies suggest that autoimmune reactions are involved, and there is good evidence for the participation of immunocompetent cells. In that context, we examined the activation of polymorphonuclear neutrophils (PMNs) of patients with Wegener's granulomatosis.

Methods

In a prospective study, the expression on the surface of PMNs of CD64 and of the major histocompatibility class II (MHC II) antigen was measured by cytofluorometry in whole blood. The expression of those antigens was correlated to disease activity.

Results

Up to 15% of the peripheral PMNs of patients with active disease expressed MHC II. Follow-up studies showed that expression correlated closely with disease activity and that it decreased rapidly under immunosuppressive therapy. Expression of CD64 was seen in approximately 50% of the patients, regardless of disease activity.

Conclusion

MHC II expression on PMNs might serve as a novel diagnostic marker for active disease and appears to be suitable for monitoring immunotherapy. Moreover, our data provide evidence that PMNs, which are normally MHC II negative, acquire MHC II antigens in the course of disease and may be an unrecognized function within the afferent limb of the immune response.

METHODS

Patients

After having obtained informed consent, patients with Wegener's granulomatosis and other forms of vasculitis attending the renal unit of the Heidelberg University Hospital were included in a prospective study analyzing the expression of MHC II and of CD64 on PMNs of the peripheral blood.

Wegener's granulomatosis, microscopic polyangiitis, and Churg–Strauss syndrome were diagnosed according to the definition of the Chapel Hill conference¹⁷ and to the American College of Rheumatology criteria¹⁸. Disease activity was determined with the "Birmingham vasculitis activity score" (BVAS)¹⁹. In all patients, anti-neutrophil cytoplasmic antibody (ANCA) titers, PR3 ANCA, and myeloperoxidase (MPO) ANCA were measured as reported elsewhere^20,21. Laboratory tests were performed by routine methods (Hitachi autoanalyzer, blood cell counts by Coulter counter). Urine analysis was done by test strips (Combur 8 kit; Boehringer Mannheim, Mannheim, Germany), Addis Count, and measurement of 24-hour protein excretion were done with Biuret, and urinary sediment was analyzed by phase contrast microscopy.

Patients with vasculitis and additional complications, including acute infections, as detected by x-ray, blood cultures, antiviral IgM titers, or measurement of procalcitonin levels of more than 1 ng/ml²², were excluded from the study, as were patients with chronic inflammatory disease (cholecystolithiasis, inflammatory bowel disease, bronchitis, osteomyelitis), diabetes, or patients with kidney transplants.

The first patients entered the study in September 1996, and the study was closed in April 1998. A number of patients were seen repeatedly, usually in 8- to 10-week intervals or when the disease became active again or complications arose. According to the first clinical presentation, three groups of patients were formed: (a) patients with active disease (BVAS of more than 1), (b) patients with inactive disease (BVAS 1 or 0) under immunosuppressive therapy, and (c) patients with inactive disease and no therapy.

Patients with systemic bacterial infections.

Twenty-four patients with systemic bacterial infections were included in the study. Diagnosis was based on bacterial culture, high plasma levels of C reactive protein, procalcitonin levels higher then 1 ng/ml, and fever.

Patients with rheumatoid arthritis.

Twenty-four patients with rheumatoid arthritis, diagnosed according to the American Rheumatism Association criteria²³, were also included in the study. The group was heterogenous with regard to activity or severity of disease according to a house score. Most patients with active disease received low-dose corticoids.

Healthy individuals.

For comparison, PMNs of healthy individuals (N = 54) of the same age and with the same sex distribution as the patients of groups A and B were analyzed.

Determination of surface expression on polymorphonuclear neutrophils by cytofluorometry

The respective surface molecules were detected by double labeling with fluorescein (FITC)-conjugated antibody to CD66b as marker for PMNs and phycoerythrin (PE)-conjugated antibodies to MHC II (anti-DP, -DQ, -DR). The antibodies were purchased from Dianova (Hamburg, Germany). For comparison, isotype-matched mouse IgGs (Dianova) were used in comparable protein concentrations. To avoid changes in surface expression caused by handling procedures, antibody labeling was performed in whole blood within two hours after withdrawal as follows: to 100 l whole blood, 5 l of the respective antibodies were given. After incubation for 30 minutes at room temperature, the erythrocytes were lyzed by FACS lysing solution (Becton Dickinson, Mountain View, CA, USA) and the residual cells were centrifuged and resuspended in phosphate-buffered saline containing 1% bovine serum albumin (BSA) and 0.1% sodium azide and 1% paraformaldehyde. Fluorescence was measured by FACSCalibur (Becton Dickinson) and was analyzed by CellQuest. Results are expressed as a percentage of gated cells in the respective quadrant. CD64 expression on CD66b-positive PMNs was measured with the same method. A monoclonal antibody to CD64 was obtained from Dianova.

Determination of surface expression on T-cells

The CD3, CD4/CD8 ratio, and expression of CD25 as activation marker on T cells were measured by cytofluorometry using the respective antibodies in a Simultest kit (all obtained from Becton Dickinson).

Statistical analysis

The results for MHC II and CD64 for the three groups of patients and for the healthy donors as the fourth group are presented as median with first and third quartiles. Nonparametric tests (Kruskall–Wallis test and Wilcoxon test) were used to compare the results. As pair-wise comparisons were intended, a closure testing procedure was used to test first the global null hypothesis of no difference between the four groups, followed by three group comparison and finally the pair-wise comparison in case the previous null hypothesis could be rejected. P values have to be interpreted descriptively. All analyses were performed with SAS, version 6.12.

RESULTS

Polymorphonuclear neutrophils of healthy donors expressed little or no MHC II antigens (median 0.95%, interquartile range 0.48 to 1.99%, MHC II-positive PMNs). In patients with active Wegener's granulomatosis, the expression of MHC II was found on PMNs. In some patients, up to 15% of the PMNs were positive for MHC II Figure 1.

Figure 1.

Expression of MHC II or of CD64 on PMNs of patients with Wegener's granulomatosis. By cytofluorometry using a FITC-labeled antibody to CD66b as marker for PMNs and a PE-labeled antibody to HLA-DR, a shift of the PMN population toward higher fluorescence intensity was noted with cells of a patients with active disease (upper right, B). PMNs of a healthy donor remain in the lower quadrant, indicating that PMNs are positive for CD66b, but not for MHC II (A). The lower panels show data for CD64 expression on PMNs. A FITC-labeled antibody to CD66b was used together with a PE-labeled anti-CD64. A patient with active disease is shown in (D), and a patient with inactive disease is shown in (C).

Full figure and legend (51K)

To assess whether MHC II expression on PMNs was linked to disease activity, a prospective study with 58 patients with the diagnosis Wegener's granulomatosis was performed. When entering the study, 16 patients had active disease (BVAS of more than 1). Forty-two patients with known disease were in remission either under immunosuppressive therapy (N = 21) or without therapy (N = 21). Of the 16 patients with active disease, 13 expressed MHC II on the peripheral PMNs (median 6.5%; Figure 2). Of the patients who entered the study when under immunosuppressive therapy (BVAS 1 or 0), only 2 of 21 had MHC II-positive PMNs. Of the patients with inactive disease and no therapy (N = 21), five patients showed low expression of MHC II on PMNs. The others were negative Figure 2.

Figure 2.

Expression of MHC II antigens on PMNs. The data obtained with PMNs of patients with Wegener's granulomatosis, patients with sepsis (N = 24), and rheumatoid arthritis (RA; N = 24), and of healthy donors (N = 54) are summarized as box charts. Patients with active disease when entering the study (N = 16) showed higher MHC II expression than patients with inactive disease under immunosuppressive therapy (N = 21) or patients with inactive disease and no therapy (N = 21) or the patients with sepsis or RA, respectively.

Full figure and legend (14K)

All of the 16 patients who had active disease when entering the study went into remission. Twelve remained clinically inactive, but four developed a relapse, which again was associated with the expression of MHC II antigens on PMNs Table 1.

Table 1 - MHC class II expression and expression of CD64 on PMN of patients with Wegener's disease.

Full table

Within the observation time (20 months), a majority of the patients came repeatedly to the hospital for a routine check-up (6- to 8-week intervals) or when complications arose. At each time, disease activity and MHC II expression were determined, allowing correlation of these two parameters in the same patient.

The follow-up studies (in some patients up to 18 months) showed that MHC II expression remained low or was unmeasurable when the disease remained inactive.

Of the patients who had entered the study with inactive disease (N = 21 under immunosuppressive therapy; 21 with no therapy), eight suffered a relapse, which in six was associated with MHC II expression on PMNs Table 1. One patient had two episodes of disease activity, both occurring concomitantly with enhanced MHC II expression Figure 3.

Figure 3.

A follow-up study of a patient with Wegener's granulomatosis. The patient entered the study with inactive disease. He developed a relapse accompanied by a surface expression of MHC II on PMNs and a rise in ANCA titer. Under immunosuppressive therapy, MHC II expression declined, as did the ANCA titer. In December 1997, the patient discontinued the immunosuppressive therapy. He developed a second relapse, again associated with MHC II expression and a rise in ANCA titer.

Full figure and legend (41K)

For statistical analysis, two situations were considered. First, based on the clinical presentation when individuals entered the study, four groups were formed: patients with active disease (N = 16), patients with inactive disease and immunosuppressive therapy (N = 21), and patients with inactive disease and no therapy (N = 21), and healthy controls (N = 54). Each patient was considered only once, namely, when entering the study. The data obtained during the follow-up studies were disregarded. Under these premises, MHC II expression was statistically different in the four groups (P < 0.001) and also in the three-group comparison (largest P value 0.01). By pair-wise comparison, MHC II expression of the patients' PMNs was different from MHC II expression of the control group (largest P value 0.01), and MHC II expression of patients with active disease differed markedly from that of both groups of patients with inactive disease (P < 0.01). There was, however, no difference between patients with inactive disease and immunosuppressive therapy and patients with inactive disease without therapy and healthy controls (P = 0.93).

In the second analysis, whether or not a change in disease activity coincided with a change in MHC II expression in the same patient was calculated. In 14 of 19 events (74%, 95% CI, 0.49 to 0.91) the changes were simultaneous.

Eleven of the 16 patients (69%) who at the beginning of the study presented with active disease had ANCA (titers ranging from 1:40 to 1:640) and autoantibodies to PR3 (ranging from 11.5 to 200), as had 7 of the 12 patients with a relapse (75%; Table 1). MHC II expression, however, was not linked to the presence of ANCA or the PR3 antibody. Of the patients negative for MHC class II (N = 5), two had a high autoantibody titer. One had only a low level of ANCA (1:40), and two had no autoantibodies at all. On the other hand, patients who never had any ANCA were MHC II positive. In patients having both autoantibody and MHC II, the respective levels did not correlate with each other; moreover, disease activity measured as BVAS did not correlate with the autoantibody titer nor with the extent of MHC II expression.

Under immunosuppressive therapy, the portion of the MHC II-positive PMNs decreased within days. The MHC II reduction paralleled the reduction of CD25 expression on peripheral T cells and, as expected, preceded the decline of the ANCA titers Figure 4.

Figure 4.

A follow-up study of a patients with active Wegener' granulomatosis. The patient entered the study with active disease, MHC II on PMNs, ANCA titer of 1:640 and 78% of interleukin 2 receptor (CD25)-positive T-cells. Under immunosuppressive therapy, the number of MHC II-positive cells declined, as did the ANCA titer and the percentage of CD25-positive T cells.

Full figure and legend (33K)

As a further parameter for leukocyte activation, the absolute leukocyte count was considered. The median values, 6.1 for patients with active disease, 6.7 for patients with inactive disease and immunosuppressive therapy, and 5.95 for patients with inactive disease and no therapy, did not differ. Moreover, there was no correlation between leukocyte count and MHC II expression (r = 0.31 for the group of patients with active disease).

As another indicator of PMN activation, the expression of CD64, the inducible high-affinity receptor for IgG (FcRI) on PMNs, was measured in all patients as well as in the healthy donors. PMNs of healthy donors either expressed no CD64 or very little CD64 on only a low percentage of PMNs. In approximately 50% of the patients with active Wegener's disease, a high expression of CD64 was seen in up to 80% of the PMNs Figure 1. There was no difference between patients with active disease and patients taking immunosuppressive therapy or patients with inactive disease and no therapy Figure 5.

Figure 5.

Expression of CD64 on PMN of patients with Wegener's granulomatosis (WG). PMNs of normal donors did not express CD64, but approximately 50% of patients with WG have CD64-positive PMN. The median values obtained from patients with active disease or patients with inactive disease were not different from each other but differed from values obtained from healthy controls (P < 0.05).

Full figure and legend (12K)

The expression of CD64 was not linked to the expression of MHC II. Of the patients with active disease and MHC II-positive PMNs (N = 24), only 11 were also positive for CD64.

Major histocompatibility class II expression appears not to be restricted to Wegener's disease, but was also seen in three of nine patients with active microscopic polyangiitis. Also, PMNs of one patient with a newly diagnosed Churg–Strauss syndrome showed a high expression of MHC II (27.9% MHC II-positive PMNs), whereas two other patients with inactive Churg–Strauss syndrome had PMNs negative for MHC II.

To determine whether or not MHC II expression was also associated with other forms of inflammatory disease, PMNs of patients with various systemic bacterial infections were tested for MHC II expression. Only 2 of 24 patients had PMNs expressing a low amount of MHC II in 3 to 4% of the cells. The median value (1.17) was not different from that seen for healthy controls Figure 2.

As an example of chronic inflammatory disease, patients with rheumatoid arthritis were tested. Here, a median value of MHC II expression of 1.7% was determined. None of these patients had a MHC II expression above 3% Figure 2.

An ongoing study with patients suffering from lupus erythematosus suggests that active disease is not correlated to MHC II expression on PMNs.

Because recent publications point to the participation of T cells in Wegener's disease^7,8,9, we measured in our patients' expression of CD25 as an indicator of T-cell activation, as well as the ratio of CD4-positive to CD8-positive T cells. As already shown by others in almost all patients, whether with active or inactive disease, the proportion of CD8-positive cells was enhanced relative to CD4-positive cells⁷. Moreover, CD25-positive T cells ranging from 20 to 60% were found in all patients, again irrespective of disease activity Table 2. In follow-up studies, a decrease in CD25-positive T cells was noted in those undergoing immunosuppressive therapy Figure 4.

Table 2 - Analysis of peripheral T-cells of patients with Wegener's granulomatosus.

Full table

DISCUSSION

Constitutive MHC II expression is restricted to professional antigen-presenting cells, including dendritic cells, B cells, and cell of the monocyte/macrophage lineage. PMNs of healthy individuals normally do not express MHC II antigens. Expression, however, can be induced by culturing the isolated cells in the presence of interferon or granulocyte CSF¹⁶. We have now also found MHC II-positive PMNs in vivo. PMNs of the peripheral blood of patients with Wegener's granulomatosis expressed MHC II antigens as detected by cytofluorometry. MHC II expression was closely associated with active disease and declined rapidly under immunosuppressive therapy.

Thus, in addition to autoantibody titers^24,25,26,27, MHC II expression on PMNs might be a useful marker for active disease, particularly for the recognition of a relapse. MHC II expression of PMNs coincided more frequently with disease activity than the rise in PR3/ANCA titers, most probably because of the rapid up-regulation of MHC II and the rapid turnover of PMNs.

Major histocompatibility class II expression is not up-regulated in patients with severe bacterial infection, suggesting that MHC II expression is not imperatively linked to PMN activation as it occurs in infectious disease. Thus, MHC II expression of PMNs might be helpful to discriminate between relapses and acute infection. Furthermore, in patients suffering from rheumatoid arthritis, no MHC II expression was seen, indicating that up-regulation of MHC II on PMNs is not necessarily linked to all chronic inflammatory processes.

Although not yet studied systematically, MHC II expression does not appear to be restricted to Wegener's granulomatosis, but also occurs in other forms of vasculitis, including microscopic polyangiitis or Churg–Strauss syndrome. The number of those patients, however, was too low to assess an association with disease activity.

How MHC II is up-regulated on PMNs in vivo has not been studied yet. Induction of de novo protein synthesis by interferon or CSF was shown before for isolated PMNs of healthy donors¹⁶. Because activated T cells are found in vasculitic disease (unpublished observations)⁷, it is feasible that T-cell–derived cytokines such as interferon induce expression of MHC II on PMNs. The assumption is supported by the observation that MHC II expression decreases rapidly after immunosuppressive therapy in parallel with the decline of CD25-positive T cells. In that MHC II expression on PMNs might be interpreted as an indicator of an ongoing immune response, the interpretation is also in line with the finding that renewed increase of MHC II coincided with relapse.

In approximately 50% of the patients with active disease, aside from MHC II, expression of CD64 was also found on PMNs. Expression, however, was not restricted to patients with active disease but was seen in 53% of patients with inactive disease. Although not ruling out a role for the high-affinity Fc receptor in the course of the disease, the expression of CD64 does not appear to be a suitable marker for the diagnosis of active disease or relapse. Apparently, the induction of CD64 expression is regulated independently of MHC II.

The consequences of MHC II expression on PMNs are still a matter of speculation. So far, the only known function of MHC II molecules is the presentation of antigenic peptides to T lymphocytes. Whether or not MHC II-positive PMN might fulfill such a function is under investigation. There are data showing that PMN are able to function as accessory cells for staphylococcus enterotoxin-dependent T-cell proliferation²⁸, an observation that is particularly interesting with regards to a possible involvement of staphylococcus enterotoxins in the pathogenesis of vasculitis (abstract; Cohen Tervaert et al, J Am Soc Nephrol 8:533A, 1997)²⁹.

In conclusion, (a) MHC II expression on PMNs might serve as a novel diagnostic marker for active vasculitic disease and appears to be suitable for monitoring the efficiency of immunotherapy. Extended long-term observations might tell whether MHC II expression precedes relapse or progression of the disease². (b) MHC II on PMNs may result from T-cell activation occurring in active vasculitic disease, and points to an as yet unrecognized function of PMNs within the afferent limb of the immune response.

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